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Nucleocytoplasmic transport of macromolecules through the nuclear pores of Xenopus oocytes

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Nucleocytoplasmic transport of macromolecules through the nuclear pores of Xenopus oocytes
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Dworetzky, Steven Ira, 1959-
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xiii, 130 leaves : ill. ; 29 cm.

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Cytoplasm ( jstor )
Molecules ( jstor )
Nuclear membrane ( jstor )
Nuclear pore ( jstor )
Oocytes ( jstor )
Proteins ( jstor )
RNA ( jstor )
Signals ( jstor )
Tracer bullets ( jstor )
Transfer RNA ( jstor )
Anatomical Sciences thesis Ph.D ( mesh )
Biological Transport ( mesh )
Dissertations, Academic -- Anatomical Sciences -- UF ( mesh )
Nucleoproteins ( mesh )
Xenopus ( mesh )
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bibliography ( marcgt )
theses ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1988.
Bibliography:
Includes bibliographical references (leaves 117-129).
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Also available online.
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Typescript.
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Vita.
Statement of Responsibility:
by Steven Ira Dworetzky.

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University of Florida
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NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES





By

STEVEN IRA DWORETZKY


A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY


UNIVERSITY OF FLORIDA


1988





























DEDICATED TO MY GRANDPARENTS, LEON AND FRANCES DWORETZKY















ACKNOWLEDGEMENTS

The studies presented in this dissertation could not have been

performed without the guidance and assistance of Dr. Carl Feldherr. He

served as an excellent role model for conducting scientific research of

the highest caliber. I would like to express my gratitude to him for

allowing me to work in his laboratory throughout the past three years.

Special thanks are extended to Dr. Robert Cohen for serving as a

committee member and providing insight to the biophysical aspects

associated with my research. I would like to thank Drs. C. West and G.

Stein for serving as members of my committee and providing critical

review of the manuscripts submitted for publication.

I am grateful to Dr. Robert Lanford at the Southwest Foundation

for Biomedical Research in San Antonio, Texas, for providing all of the

BSA-conjugates that were used to study the effects of signal number on

protein uptake.

The work presented in Chapter II is reproduced from the Journal of

Cell Biology, 1988, volume 106, number 3, pages 575-584 by copyright

permission of The Rockefeller University Press.

I would like to thank Denifield Player for advice concerning

techniques in electron microscopy and Linda Mobley for typing of

manuscripts and other pertinent material throughout my graduate career.








Finally, and most importantly, I wish to express my utmost

gratitude to Barbara Ludwig. She was a constant source of moral

support and encouragement throughout the trials and tribulations of my

graduate education. In addition, I would like to thank my parents for

their words of encouragement and steadfast support.















TABLE OF CONTENTS


Page


ACKNOWLEDGEMENTS . . . . . . . . . . . . iii

LIST OF FIGURES . . . . . . . . . . . . . vii

LIST OF TABLES . . . . . . . . . . . . .. viii


ABBREVIATIONS . . . . . . .

ABSTRACT . . . . . . . ..


CHAPTERS


I A REVIEW OF NUCLEAR PERMEABILITY:
TRANSPORT

Introduction . . . . ..
Morphology . . . . . ..
Nuclear Permeability Experiments.
Nuclear Efflux . . . . ..
Statement of Research . . .


DIFFUSION AND


II TRANSLOCATION OF RNA-COATED GOLD PARTICLES THROUGH THE
NUCLEAR PORES OF OOCYTES . . . . . . . ..

Introduction . . . . . . . . . . .
Materials and Methods . . . . . . . . .
Results . . . . . . . . . . . .
Discussion . . . . . . . . . . . .


Ill THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE OF
TARGETING SIGNALS ON NUCLEAR UPTAKE . . . . .

Introduction . . . . . . . . . . .
Materials and Methods . . . . . . . . .
Results . . . . . . . . . . . .
Discussion . . . . . . . . . . . .









IV SUMMARY AND, PROSPECTUS . . . . . . . . .. 103

Summary of Results . . . . . . . . . .. 103
Nuclear Envelope Selectivity . . . . . . .. 104
Dynamic Aspects of the Transport Channels . . . ... .107
Proposed Model . . . . . . . . . . .. 109
Future Trends . . . . . . . . . . .. . 111


APPENDIX . . . . . . . . . . . . . .. 116

REFERENCES . . . . . . . . . . . . . .. 117

BIOGRAPHICAL SKETCH . . . . . . . . . . . .. . 130
















LIST OF FIGURES




A schematic representation of regions 1 and 2


2. tRNA-gold, nuclear injection . . ..

3. 5S RNA-gold, nuclear injection . . ..

4. Poly(A)-gold, nuclear injection . . .

5. PVP-gold, nuclear injection . . . .

6. Double injection experiment . . . .

7. 5S RNA-gold, cytoplasmic injection. . .

8. BSA-WT11-gold . . . . . . .

9. BSA-cT7-gold . . . . . . . .

10. Nuclear particle distributions: BSA-WT8

11. Nuclear particle distributions: BSA-WT8,
nucleoplasmin . . . . . .

12. Accumulation of tracers along the nuclear

13. Coinjection of gold particles coated with
and nucleoplasmin . . . . ..


a


. . . . . 51

. . . . . 51

. . . . . 56

. . . . . 56

. . . . . 64

. . . . . 64

. . . . . 83

. . . . . 83

nd BSA-WTR . 88


BSA-WT11, and


envelope . .

BSA-conjugates


Figure

1.


Page

S. 47















LIST OF TABLES


Table Page

I Amounts of Coating Agent Required to Stabilize Gold Sols. 45

II Translocation of Gold Particles as a Function of the
Coating Agent . . . . . . . . . . .. 52

III Size Distribution of Gold Particles Present in the Nuclei
and Pores . . . . . . . . . . . .. 54

IV Translocation of Gold Particles Coated with Polyglutamic
Acid . . . . . . . . . . . .. 59

V Concentration Dependence of tRNA-gold Translocation . . 61

VI Protein Preparations Used as Coating Agents . . . 76

VII Volumes of Coating Agent (pl) Required to Stabilize 1 ml
of Gold Sol . . . . . . . . . . .. 78

VIII N/C ratios 1 h . . . . . . . . . . .. 85

IX Size Distribution of BSA-WT5- and WT8-coated Particles
in Injected Cells . . . . . . . . . .. 87

X Size Distribution of BSA-WT8-, WT11- and Nucleoplasmin-
coated Particles in Injected Cells . . . . .. 91
XI Size Distribution of BSA-WT -cT7- and Large T-Ag-coated
Particles in Injected Cells . . . . . . .. 92

XII Envelope-Associated Particles . . . . . . ... 96


viii















LIST OF ABBREVIATIONS


a Alpha

A Angstrom

ATP Adenosine-5'-triphosphate

Arg Arginine

B Beta

BSA Bovine serum albumin

cT Mutant large T-antigen

DMSO Dimethyl sulfoxide

DNA Deoxyribonucleic acid

G Guanosine

3H Tritiated

h Hour(s)

HEPES N-2-Hydroxyethylpiperazine-N'-2-

Ethanesulfuric acid
1251 lodinated

IgG Immunoglobulin

kd Kilodaltons

M Molar concentration

min Minute(s)

ml Milliliter(s)

mM Millimolar









mol wt

N

N/C

ng

nl

NTPase

Os04

P

PAGE

Poly(A)

Poly d(A)

Poly(I)

Poly(U)

Pro

PVP

RNA

hnRNA

mRNA

rRNA

tRNA

RNP

s

SDS

SV 40

T-antigen

U


Molecular weight

Normal concentration

Nuclear to Cytoplasmic

Nanogram

Nanoliter(s)

Nucleotide triphosphatase

Osmium tetroxide

Probability

Polyacrylamide gel electrophoresis

Polyadenylic acid

Poly-deoxy-adenylic acid

Polyinosinic acid

Polyuridylic acid

Proline

Polyvinylpyrrolidone

Ribonucleic acid

Heteronuclear ribonucleic acid

Messenger ribonucleic acid

Ribosomal ribonucleic acid

Transfer ribonucleic acid

Ribonucleoprotein

Seconds)

Sodium dodecyl sulfate

Simian virus 40

Tumor antigen

Uridine









P1 Microliter(s)

Pm Micron(s)

Val Valine

vol/vol Volume/Volume

WGA Wheat germ agglutinin

WT Wild type large T-antigen

wt/vol Weight/Volume















Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy



NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES


By

STEVEN IRA DWORETZKY

August 1988



Chairman: Carl M. Feldherr
Major Department: Anatomy and Cell Biology


To localize and characterize the pathways for RNA translocation to

the cytoplasm, gold particles coated with tRNA, 5S RNA, or poly(A) were

microinjected into the nuclei of Xenopus oocytes. At various times

after injection, cells were fixed and the particle distributions

analyzed by electron microscopy. Similar results were obtained with all

RNAs used. Particles ranging from 20-230 A in diameter were observed

within central channels of the nuclear pores and in the adjacent

cytoplasm. Particles larger than 90 A would not be expected to diffuse

readily through the pores, suggesting that mediated transport occurred.

Furthermore, approximately 97% of the pores analyzed were involved in

the translocation of gold particles. After nuclear injection, particles

coated with exogenous macromolecules were essentially excluded from the









pores and cytoplasm. These results indicate that translocation of RNA-

coated gold was due to the presence of RNA associated with the

particles.

To determine if the number of targeting signals affects the

transport of proteins into the nucleus, oocytes were injected with gold

particles coated with BSA cross-linked with various numbers of short

synthetic peptides containing the SV 40 large T-antigen nuclear

transport signal. Large T-antigen also was used as a coating agent.

Particles carrying active targeting signals entered the nucleus through

central channels within the nuclear pores. Analysis of the

intracellular distribution and size of the tracers entering the nucleus

indicated that the number of signals per molecule affects both the

uptake of particles and the functional size of the channels available

for translocation. In control experiments, particles coated with BSA or

BSA conjugated with inactive SV 40 transport signals were virtually

excluded from the nucleus. Nucleoplasmin-coated gold was transported

through the nuclear pores more effectively than the BSA conjugates. The

accumulation of tracers along the nuclear envelope suggests that

differences in the activity of the two targeting signals might be

related to their binding affinity for envelope receptors.

Double injection experiments demonstrated that individual pores are

capable of transporting both protein and RNA and they can also recognize

and transport proteins containing different nuclear targeting signals.


xiii















CHAPTER I
A REVIEW OF NUCLEAR ENVELOPE PERMEABILITY:
DIFFUSION AND TRANSPORT

Introduction

The formation of the nuclear envelope around the cell's genetic

material is one of the major evolutionary differences between

prokaryotes and eukaryotes. The nucleus, site of both RNA and DNA

synthesis, contains the genetic information needed for phenotypic

expression. The cytoplasm, on the other hand, is the site of protein

synthesis, which requires certain RNAs that are exported from the

nucleus. Furthermore, the nucleus is dependent on proteins synthesized

in the cytoplasm. Therefore, cell growth, division, differentiation,

and maintenance require a continual exchange of information between both

the cytoplasm and nucleus. To better understand cellular function, it

is important to determine the factors that control the rate of movement

between the two compartments and the types of molecules that are

exchanged. For this reason, knowledge of the nuclear envelope's

structure and permeability properties is essential in understanding the

interrelationships that exist between the nucleus and cytoplasm of the

eukaryotic cell.

In the first part of this chapter an ultrastructural description of

the nuclear envelope is provided, followed by a review of permeability

experiments which have helped to elucidate the role of the envelope in

regulating nucleocytoplasmic exchanges.









2

Morphology

The discovery of the nucleus is documented as early as 1833. Fifty

years later, light microscopists began speculating that a membrane

surrounded the nucleus. It was Chambers and Fell (1931) who

microdissected cells and demonstrated the existence of a nuclear

envelope. With the use of the electron microscope, Callan and Tomlin

(1950) performed ultrastructural studies on manually isolated oocyte

nuclei and confirmed the existence of an envelope surrounding the

nucleus. They described it as a double membrane structure, with a

porous outer membrane adjacent to the cytoplasm and a continuous inner

membrane adjacent to the nucleoplasm.

With advances in the resolution of the electron microscope and

improvements in fixation procedures, the nuclear envelope and pore

structure became the subject of intense morphological studies. The

envelope and associated structures can be visualized with the electron

microscope by a variety of techniques including i) in situ fixation

followed by ultrathin sectioning, ii) negative staining of isolated

envelopes, iii) freeze-etching, and iv) high-resolution scanning

microscopy. One of the first detailed studies utilizing transmission

electron microscopy was performed by Watson (1955, 1959). He analyzed

nuclei from a wide variety of cell types and found that the inner and

outer membranes fuse at regular intervals along the envelope resulting

in the formation of nonmembranous regions ("holes"), ranging from 800-

1200 A in diameter, referred to as the nuclear pores. These pores form

channels that represent potential sites of interaction between the

nucleoplasm and cytoplasm. Since then, laboratories have used the








3

procedures listed above to accumulate a vast amount of ultrastructural

information concerning both the nuclear envelope and pore structure from

a range of cell types, resulting in many interpretations of the pore's

three-dimensional architecture. Comprehensive reviews of the morphology

of the nuclear envelope and pore complexes have been published by Franke

(1974), Harris (1978), and Maul (1977). The following discussion will

be limited to a general morphological description of the nuclear

envelope and its associated pore complex, without describing some of the

finer substructures, since their existence remains controversial.

The nuclear envelope is considered to have four structural

components: the inner and outer membranes, the pore complexes, and the

nuclear lamina. The envelope, seen in thin sections perpendicular to

the nucleus, is composed of a double membrane system with the inner and

outer membrane parallel to one another until they approach the pore

complexes. In general, each membrane is approximately 75 A in diameter.

A homogenous region, which varies in width from 100-600 A depending on

the cell type, lies between the two membranes and is referred to as the

perinuclear cisterna (Wischnitzer, 1958). Often, the luminal space of

the perinuclear cisterna is observed to be in direct continuity with the

luminal cavity of the endoplasmic reticulum (Watson, 1955). The outer

and inner membranes are associated with different structures.

Polyribosomes are seen to be attached to the outer membrane, whereas,

the inner surface of the inner membrane appears to have an intimate

association with a distinct morphological layer, referred to as the

lamina. These observations suggest that the inner and outer membranes









4

are different, although they have not been analyzed by biochemical

methods.

As visualized by electron microscopy, the nuclear lamina is a

discrete layer of material, varying in thickness from 30-100 nm, lining

the entire inner surface of the nuclear envelope, excluding the pore

complexes. A lamina enriched fraction is obtained by non-ionic

detergent and high salt extraction of isolated envelopes (Aaronsen and

Blobel, 1975; Dwyer and Blobel, 1976). Protein analysis of the residual

fraction shows the presence of three predominant proteins, referred to

as lamins A, B, and C. They comprise approximately 40% of this

fraction. During interphase, these polypeptides have been localized, by

immunocytochemistry, exclusively to the lamina at the nuclear periphery

(Gerace et al., 1978). Gerace and Blobel (1982) have shown that the

lamina forms a fibrous network that is a component of the nuclear

skeleton. The lamina has been implicated to function as a structural

framework for the nuclear envelope and an anchoring site at the nuclear

periphery for chromatin. In addition to its structural role, the lamina

is thought to be involved in reversible nuclear envelope assembly and

disassembly during mitosis (Gerace et al., 1984).

The pores are filled with diffuse electron-opaque material that

extends a short distance into both the nucleus and cytoplasm. In

tangential sections, this electron-opaque material has a ring-like

appearance and has been referred to as the annular material (Watson,

1955; Wischnitzer, 1958). The pore with its associated annulus is

called the pore complex.









5

Discrete subunits, 100-250 A in diameter, are distributed evenly

around both the cytoplasmic and nuclear margins of the pore (Watson,

1955; Wischnitzer, 1958). Franke (1966) using a multiple rotational

exposure technique on negative-stained preparations of isolated

envelopes, describes the arrangement of annular globules (granules) to

be in eight-point radial symmetry. Furthermore, the subunits lying on

the pore margin within the nucleus overlap the same sites as subunits on

the cytoplasmic pore margin.

Within the centers of some pores, an electron-dense granule with

diameters ranging from 40-350 A can be observed. This central granule

has been visualized with different preparative techniques; however, the

observed frequency of its presence is highly variable (Franke and

Scheer, 1970). It has been suggested that the granule is actually

material in transit, e.g. ribonucleoprotein (RNP), and that a

correlation exists between the frequency of the central granule and the

activity of RNA synthesis (Scheer, 1970). Cytochemical studies

performed by Franke and Falk (1970) support this view. Even with this

data it still remains controversial whether the granule is a permanent

structural component of the pore complex or material passing through the

pore.

Thin filaments, 25-50 A in diameter, have also been observed in the

interior of the pore. These filaments radiate laterally from the pore

margins and appear to connect with the central granule (Franke, 1974).

The filaments have a spokelike radial appearance and exhibit eightfold

symmetry similar to that of the annular globules.









6

Unwin and Milligan (1982) have used thin section electron

microscopy coupled with Fourier averaging methods to describe the pore's

three-dimensional organization to a resolution of 90 A. They found the

pores, in Xenopus oocytes, to occupy 20-30 percent of the envelope

surface and are composed of several discrete components: plugs, spokes,

particles, and rings. The rings have internal and external diameters of

800 A and 1200 A, respectively. Spokes extend radially from the margins

of the channel toward a plug (30-350 A in diameter) located in the

center of the pore. Particles are seen on the cytoplasmic surface of

the pores but do not appear to be integral components, as they can be

released with high salt treatments. This model, with its high

resolution of analysis, confirms many of the components put forth in the

model described by Franke (1974).

Until recently, little was known about the molecular composition of

the pore complex. Gerace et al. (1982) have identified a 190 kd

intrinsic membrane glycoprotein that is specifically localized to the

nuclear pores in liver nuclei. After detergent treatment, which

solubilizes most nuclear envelope intrinsic proteins, the 190 kd

glycoprotein remains associated with the pore complex-nuclear lamina

fraction. Based on this and its biochemical properties, it is suggested

that the 190 kd glycoprotein is involved in anchoring the pore complex

to the envelope. Davis and Blobel (1986), using monoclonal antibodies

and immunoelectron microscopy, have identified a 62 kd protein that

localizes within the annuli of rat liver nuclei. Snow et al. (1987)

have described a group of eight glycoproteins, with common epitopes,

that are localized exclusively to the nuclear pore complex. The group









7

of eight glycoproteins exist in multiple copies per pore complex and

their function is unknown at this time.

Compiling the data available on the components of the pore complex,

many investigators have suggested models or schematic representations of

the nuclear envelope architecture. These models, in conjunction with

the biochemical data, provide a framework for investigating how the

nuclear pores might be involved in regulating nucleocytoplasmic

exchanges. The appendix presents a schematic that is based on Franke's

(1974) and Unwin and Milligan's (1982) data.



Nuclear Permeability Experiments

It is clear from morphological evidence that the nuclear envelope

with its associated pore complexes is different from the plasma membrane

and other intracellular membranes. Since cellular processes both within

the nucleus and cytoplasm are dependent on a mutual exchange of

material, an important question arises as to the permeability properties

of the nuclear envelope and to what extent the envelope might regulate

nucleocytoplasmic exchange.

Three general approaches are used to study intracellular exchanges

across the nuclear envelope. First, microinjection of labelled tracers

(fluorescent, radioactive or electron-dense) is employed extensively to

study the permeability of the nuclear envelope to molecules within

intact cells. Second, permeability experiments can be performed on

isolated nuclei; however, these results must be carefully interpreted as

the permeability characteristics can change as a result of the isolation

(e.g., Paine et al., 1983; Peters, 1986). Third, recombinant DNA









8

methodology has been used to probe structural features of proteins that

are specifically targeted to the nucleus.



Ion permeability

Ions are known to interact with chromatin and activate specific

genes. During mitosis, meiosis, DNA synthesis, and hormonal

stimulation, ionic changes are occurring within the cell; thus, it is

important to understand intracellular ion distributions and how the

envelope might regulate their exchange between the nucleus and

cytoplasm.

Earlier permeability studies were qualitative in nature and based

on the structural or colloidal changes that occurred within isolated

nuclei following incubation in different salt solutions. It was

concluded from these studies that both cations and anions can rapidly

penetrate the nuclear envelope.

The use of radioactive isotopes as tracer molecules provided a new

method to give precise data on the permeability of the envelope.

Abelson and Duryee (1949) incubated individual oocytes in 24Na+ and

later studied the intracellular ion distribution by quick freezing and

radioautography. The results demonstrated that the nuclear envelope in

amphibian oocytes was readily permeable to 24Na+. In addition to these

findings, the nucleus, at equilibrium, contained twice as much

radioactive tracer per unit volume as the cytoplasm. The measurement of

water content in both compartments indicated the nucleus to have a

higher percentage of water than the cytoplasm, which could account for

the difference in Na+ concentrations. Other molecules, 42K+, 32P04=,









9

35SO4, leucine-14C, and alanine-14C were shown by Naora et al. (1962)

to pass readily across the nuclear envelope.

To measure precisely the intracellular distribution of Na+, Horowitz

and Fenichel (1970) utilized ultra-low temperature autoradiographic

techniques. They described at least three intracellular Na+ fractions:

a cytoplasmic fast fraction, a cytoplasmic slow fraction, and a nuclear

fast fraction. The fast fractions were thought to represent freely

diffusible Na+, whereas the slow fraction is interpreted to be Na+ bound

to nondiffusible elements within the cytoplasm. This slow diffusion

fraction could account for the higher concentration of the Na+ ion found

within the cytoplasm. Therefore, the asymmetric ion concentrations can

be accounted for by two factors: first, the differences in water

content between the nucleus and cytoplasm, and second, possible binding

in the cytoplasm.

Another method for studying the permeability of the nuclear

membrane to ions is by measuring the envelope's electrical resistance.

Inserting intracellular microelectrodes into the nucleus and cytoplasm

of amphibian oocytes, Kanno and Loewenstein (1963) found no appreciable

resistance, indicating that ions are able to diffuse freely across the

envelope. Comparing the results from oocytes, the flux measurements and

electrical resistance data are in good agreement.

The results presented above suggest that the nuclear envelope is

not a barrier against ion diffusion. Since the nuclear envelope does

not impede ion passage and ion transport by the nuclear envelope is not

indicated (Century et al., 1970), it is likely that ions pass through

the aqueous channel formed by the nuclear pores.











Small molecules

Permeability of the nuclear envelope to small molecules was studied

by microinjecting sucrose (Horowitz, 1972), inulin (Horowitz and Moore,

1974), and the non-metabolizable amino acid a-aminoisobutyric acid (AIB)

(Frank and Horowitz, 1975) into oocytes. All of these small molecules

rapidly crossed the envelope such that standard flux measurements could

not accurately define the rate of movement, suggesting that the nuclear

envelope is of equivalent permeability to an equal thickness of

cytoplasm. To better define the uptake kinetics of small molecules,

Kohen et al. (1971) used high resolution microfluorimetry to quantitate

rapid movement across the envelope. They investigated the permeability

of glycolytic intermediates. Their results demonstrate that the

metabolites move across the nuclear envelope with a 35 millisecond

delay, indicating that for small molecules the nuclear envelope does not

constitute a permeability barrier. Thus, similar to ions, it is likely

that small molecules migrate into the nucleus through the nuclear pores.



Exogenous macromolecules

The nuclear uptake of radio-labelled or fluorescein-labelled

exogenous molecules has been investigated by microinjecting these

tracers into the cytoplasm of cells and following their intracellular

distribution with time. Colloidal gold particles and ferritin, both of

which can be visualized with the electron microscope, have also been

used to study nuclear permeability. Microinjection of these exogenous

tracers provides a method to better understand some of the physical

characteristics of the nuclear envelope with respect to diffusion.









11

The pores, from a morphological standpoint, would appear to be a

likely pathway for movement into and out of the nucleus. In support of

this view, Feldherr (1962) injected colloidal gold particles coated with

polyvinylpyrrolidone (PVP) into the cytoplasm of the amoebae Chaos chaos

and localized the distribution of the tracers by electron microscopy.

After 1-2 minutes, gold particles were observed in the centers of the

pores and 24 hours after injection, gold particles were found to

accumulate in the nucleus. Injection of different size gold fractions,

20-50 A and 20-160 A in diameter, showed that small gold particles

localized within the nucleus after 3 min, in contrast to the larger gold

fraction which had negligible uptake after 10 min. These results

indicate that the rate of movement across the nuclear envelope is

inversely proportional to the molecular size of the tracer. After

longer time intervals, it was determined for Amoeba proteus that PVP-

coated gold particles up to 120 A can penetrate the pores of the nuclear

envelope, whereas in Chaos chaos, particles as large as 140 A can enter

the nucleus (Feldherr, 1965). It is important to note that the maximum

size of the particles able to penetrate the envelope is smaller than the

800 A internal diameter of the pores. Overall, it was concluded from

these studies that pores can serve as pathways for nucleocytoplasmic

exchange but can restrict the movement of large molecules since the

entire area of the pore is not available for free exchange.

Gurdon (1970) studied permeability properties of the nuclear

envelope by injecting 1251-labelled histone (10,000-20,000 kd) and

bovine serum albumin (67,500 kd), into amphibian oocytes. After 50

minutes, it was demonstrated by autoradiographic analysis that the









12

nucleus accumulated 1251-histone to at least twice the concentration of

the cytoplasm. In contrast, the 1251-BSA was more concentrated in the

cytoplasm, even after 12 hours. It was clear from these results that

the nuclear envelope shows some selectivity for inhibiting passage of

large exogenous proteins.

The nucleocytoplasmic exchange of a range of exogenous

macromolecules was studied quantitatively by Paine and Feldherr (1972).

Well characterized macromolecules with respect to size, shape, and

electrical charge were used: ferritin (mol wt 465,000; pI 4.4), BSA

(mol wt 67,000; pI 4.71), ovalbumin (mol wt 45,000; pI 4.7), myoglobin

(mol wt 17,500; pI 6.9), lysozyme (mol wt 14,500; pI 11.8); and

cytochrome c (mol wt 13,000; pI 10.7). All the proteins were

fluorescently labelled, with the exception of ferritin which is

electron-opaque, and injected into the cytoplasm of oocytes from

Periplaneta americana. Their results showed that ferritin and BSA had

extremely low N/C ratios even after 5 h. Ovalbumin slowly entered the

nucleus; however, a higher concentration was present in the cytoplasm as

compared to the nucleus after 5 h. On the other hand, cytochrome c

entered the nucleus within 5 min, whereas lysozyme and myoglobin had N/C

ratios greater than 1.0 within 3 min. These results demonstrated that

neutrally or positively charged proteins, less than 20 A in diameter,

can readily enter the nucleus. Furthermore, the nuclear uptake of the

proteins, 20-95 A in diameter, was inversely related to size and not

simply based on charge. Similar conclusions were reached by Bonner

(1975a) who studied the nuclear uptake of the small basic proteins;









13

histones, lysozyme, and trypsin inhibitor, and the neutral protein,

myoglobin.

To determine if exogenous macromolecules exit the nucleus, Paine

(1975) injected ovalbumin, horseradish peroxidase, myoglobin, and

cytochrome c directly into the nucleoplasm of Chironomus salivary gland

cells. The results showed that the fluorescein-labelled molecules enter

and leave the nucleus with similar kinetics, indicating that

nucleocytoplasmic exchange is bidirectional and the rate of movement

across the envelope is dependent on 1) the restrictions imposed by the

diameter of the diffusion channel and 2) the size of the molecule.

To estimate the patent diameter of the nuclear pores in amphibian

oocytes, Paine et al. (1975) injected tritiated dextrans with known

hydrodynamic radii of 12.0, 23.3, and 35.5 A. Dextrans are uncharged

polymers of D-glycopyranose and available in a range of molecular sizes.

To determine the intracellular localization of the tracers, the cells

were quickly frozen at different time intervals, sectioned, and

autoradiographed. Comparison of the grain profiles for all three

tracers showed that the nuclear envelope is less permeable to larger

dextrans, supporting the view that the envelope can limit the rate of

entry of larger macromolecules. The relationship between the size of

the dextrans and their rates of entry suggested that the nuclear

envelope had a sieving effect due to its porous nature. Developing a

mathematical model from the rates of uptake, diffusional coefficients,

and the length of the channel, the authors estimated the patent pore

diameter to be 90 A.









14

Stacey and Allfrey (1984) also used well characterized proteins to

study the permeability of the nuclear envelope of cultured cells.

Thirteen proteins ranging from 13 kd to 669 kd with pI's from 4.4 to 11

were microinjected into the nucleus or cytoplasm of HeLa cells.

Proteins less than 40 kd entered almost immediately, in contrast to

proteins between 40 kd and 60 kd which entered more slowly. Many of the

proteins larger than 60 kd were excluded from the nucleus. The rates of

diffusion into and out of the nucleus were indistinguishable.

To estimate the patent pore diameter in cultured liver cells,

Peters (1984) used fluorescent microphotolysis to study the permeability

properties of the nuclear envelope. Fluorescein-labelled dextrans

ranging from 3 to 150 kd were injected into hepatocytes and allowed to

reach equilibrium. A pulse from an argon laser depleted the fluorescent

signal within a region of the nucleus and then microfluorimetry was used

to measure the influx of the tracer back into the depleted region. His

results showed the influx of the dextrans, from the cytoplasm to the

nucleus, to have an exclusion limit between 17 and 41 kd. Using similar

equations as Paine et al. (1975), Peters derived the functional pore

diameter to be 100-110 A with respect to diffusion.

All of the experiments involving the microinjection of exogenous

tracers support the view that the nuclear envelope has properties of a

molecular sieve. The entry of exogenous tracers into the nucleus occurs

by non-selective mechanisms, that is, diffusion through the pores. The

rate of influx is inversely related to size which reflects some of the

limitations imposed by the pore complex. The pore diameter has been

estimated for amphibian oocytes (90 A; Paine et al., 1975),









15

amoebae (120-140 A, Feldherr, 1965), and hepatocytes (100-110 A, Peters,

1984). These differences in size can markedly affect the exclusion

limits and flux rates of macromolecules. For example, it would take

approximately 1 h for a molecule, 50 A in diameter, to diffuse through a

pore with a diameter of 110 A. In contrast, it would take the same

molecule 8 h to pass through a pore with a diameter of 90 A (estimates

from Fig. 6; Paine et al., 1975). This can explain the 2-5 orders of

magnitude difference in dextran flux rates between oocytes and

hepatocytes.



Endogenous macromolecules

Subsequent nuclear permeability studies have focused on the

selective uptake of endogenous proteins. Labelled endogenous proteins

can serve as probes to investigate the normal molecular interactions and

behavior found within cells, in contrast to exogenous molecules, which

are unlikely to have intracellular interactions and solely used to

characterize the passive permeability properties of the nuclear

envelope. It soon became evident that the rates of nuclear accumulation

of endogenous molecules were much greater than exogenous molecules of

comparable sizes. The data demonstrated that exchanges of endogenous

proteins between the nucleus and cytoplasm were controlled by selective

processes, explaining in part, the observation that endogenous proteins

distribute differently than exogenous tracers.

There are two possible mechanisms that can account for the rapid

rate of endogenous protein accumulation within the nucleoplasm. First,

proteins can diffuse freely through the nuclear pores and then can be








16

selectively retained within the nucleus, by binding to a nondiffusible

substrate. Second, the exchange of some or all of the endogenous

proteins, through the pores, can occur by facilitated uptake. This mode

of exchange is governed by the intrinsic properties of the molecule.

Following translocation across the envelope, accumulation in the nucleus

can occur by selective binding or by another mechanism for irreversible

transport.

It has been reported that many proteins accumulate in the nucleus

against a concentration gradient--e.g., for example, histones (Gurdon,

1970) and N1/N2 (De Robertis et al., 1978)--attain nuclear to

cytoplasmic ratios of 115 and 120, respectively. Austerberry and Paine

(1982) used cryomicrodissection and two-dimensional electrophoresis to

measure quantitatively the intracellular distribution of 90 proteins in

the living cell. Their results demonstrated that at least 35 proteins

within the nucleus have nuclear to cytoplasmic ratios greater than 10,

which suggests some form of selective retention. Additional evidence to

support nuclear binding comes from the experiments performed by Feldherr

and Ogburn (1980). When the permeability properties of the envelope are

altered, as shown by its failure to exclude labelled BSA, most of the

endogenous nuclear proteins still accumulate within the nucleus (see

below).

Bonner (1975b) studied the migration of in vivo labelled endogenous

proteins into nuclei of amphibian oocytes. After injection of labelled

nuclear proteins into the cytoplasm of unlabelled oocytes, the

radioactivity was found to concentrate in the nucleus. If labelled

cytoplasmic proteins were injected into the cytoplasm of unlabelled









17

oocytes, radioactivity was concentrated in the cytoplasm although some

labelled protein entered the nucleus. From these results, Bonner

suggested the existence of three classes of proteins: N proteins, found

predominantly in the nucleus; C proteins, found predominantly in the

cytoplasm; and B proteins, found in equivalent amounts in both the

nucleus and cytoplasm. Feldherr (1975) measured the rate of nuclear

uptake for endogenous proteins that were labelled with tritiated amino

acids in vivo. His results demonstrated that newly synthesized nuclear

proteins, ranging from 94,000 to 150,000 kd, were three times more

concentrated within the nucleus as compared to the cytoplasm after 6 h.

The rates of accumulation for endogenous proteins of this size were

greater than expected in comparison to the results obtained for

exogenous proteins of similar size.

To better understand the role of the nuclear envelope in regulating

nucleocytoplasmic exchange of endogenous proteins, Feldherr and

Pomerantz (1978) altered the diffusion barrier by using glass needles to

puncture holes in the envelope and then measured the uptake of labelled

nuclear proteins. The results, as determined by one-dimensional gel

electrophoresis, showed no qualitative differences in the accumulation

of nuclear proteins in punctured nuclei as compared to nuclei from

nonpunctured control oocytes. Feldherr and Ogburn (1980) further

studied the mechanism of selecting nuclear proteins by first disrupting

the nuclear envelope and then using two-dimensional gel analysis,

fluorography, and double-labelling techniques to determine intracellular

distributions. Analysis of over 300 nuclear polypeptides showed less

than five percent of the proteins varied between experimentally









18

punctured oocytes and nonpunctured controls. It was concluded from

these results that nuclear binding plays a major role in regulating the

nucleocytoplasmic distribution of endogenous proteins, and that the

envelope does not function as a rate-limiting barrier.

It is apparent from the above studies that many of the nuclear

proteins accumulate in the nucleus at significantly higher rates than

exogenous molecules of similar size. As discussed previously, exogenous

proteins larger than 45 kd (ovalbumin) do not readily enter the nucleus;

however, endogenous proteins larger than 90 kd accumulate quite rapidly.

Evidence for facilitated transport was obtained by Feldherr et al.

(1983) for RN1, a 148 kd nuclear protein, in the oocytes of Rana

pipiens. After microinjection or in vivo labelling of RN1, the kinetic

results demonstrated rapid accumulation within the nucleoplasm.

Theoretical estimates of the diffusion rate of a 148 kd protein through

the 90 A channel indicated that the rate of uptake could not be

accounted for by simple diffusion through the pores and subsequent

binding. Thus, in this instance some form of mediated transport is

apparently required for rapid accumulation.

Recent research has shown that signal sequences of some nuclear

proteins are required to target macromolecules to the nucleus. Two

approaches have been used to study the polypeptide domains that contain

these sequences. The first approach is the partial digestion of a

protein followed by analysis of the fragments capable of accumulating in

the nucleus and the second approach employs DNA methodology.

The first approach has been successfully employed to study

nucleoplasmin, a 110 kd karyophilic protein in Xenopus oocytes, which











rapidly accumulates in the nucleus. Nucleoplasmin is a thermostable,

pentameric molecule present within the nucleus in a high concentration

(Mills et al., 1980). It appears in the electron microscope as a disc

with a molecular dimension of 74 A in diameter (Earnshaw et al., 1980).

Pepsin digestion selectively cleaves nucleoplasmin into two structural

domains, a protease-resistant core and five protease-sensitive tails

(Dingwall et al., 1982). Protease removal of the five tail domains

leaves a pentameric core that is excluded from the nucleus after

microinjection into the cytoplasm. In contrast, the tail domains, after

cytoplasmic injection, are capable of nuclear accumulation. When the

core is injected directly into the nucleus, it migrates throughout the

nucleoplasm but is still excluded from the cytoplasm, indicating that

the core has the ability to be retained by the nucleus but not the

ability to migrate into the nucleus. These results demonstrate that the

tail domain contains the necessary information to specify selective

entry. By reducing the time of digestion, a mixture of pentameric cores

that contain different numbers of tail regions can be prepared. A core

molecule with only one tail is capable of entering the nucleus, but at

one-tenth the rate of intact nucleoplasmin (Dingwall et al., 1982).

Amino acid analysis of the tail domain (C-terminus) indicates an

enrichment in lysine residues and a depletion in hydrophobic amino

acids.

The second approach, utilizing genetic engineering, entails the

construction of plasmids encoding proteins normally found in the

cytoplasm linked to putative nuclear signal sequences. The end product

is a hybrid protein, converting an otherwise cytoplasmic protein into a









20

protein localizing in the nucleus. After narrowing down a region of the

protein necessary for nuclear localization, oligonucleotide-directed

mutagenesis can be used to map out the essential amino acid sequence

necessary for targeting. Overall, these approaches have been used to

study an array of proteins that are targeted to specific organelles:

endoplasmic reticulum, mitochondria, nuclei, and peroxisomes.

The simian virus (SV) 40 large T-antigen targeting signal has been

the most extensively studied sequence that confers nuclear localization.

Large T-antigen, during lytic infection, is responsible for the

regulation of viral transcription and replication. The protein has 708

amino acids and in the monomeric form (can be tetrameric) has a

molecular weight of 94 kd. The size of this protein suggests that it

cannot enter the nucleus through the pores by simple diffusion, although

large T-antigen is found predominantly within the nucleoplasm. Butel et

al. (1969) described a large T-antigen from a mutant SV 40 hybrid virus

that was localized in the cytoplasm of an infected cell. Comparison of

the sequence analysis of wild type large T (WT) to the mutant large T-

antigen (cT) revealed the amino acid at the 128 position was changed

from lysine to asparagine (Lanford and Butel, 1984). The mutant large

T-antigen was found to be soluble in the cytoplasm and capable of

binding DNA (Lanford and Butel, 1980). Thus, the failure to accumulate

in the nucleus was not caused by its inability to either diffuse

throughout the cytoplasm or bind DNA, but caused by a mutation in a

critical region of the protein sequence that is required for nuclear

localization.








21

Kalderon et al. (1984a) used site-directed mutagenesis to identify

a seven amino acid sequence within large T-antigen that is required for

nuclear localization, with the lysine at position 128 being the critical

residue. A change in this lysine residue to asparagine, threonine,

isoleucine, leucine, methionine, or glutamine caused the large T-antigen

to become localized in the cytoplasm of the cells. Mutations within

the signal sequence on either side of the lysine influenced the rate of

uptake and qualitative distribution of large T-antigen, but did not

abolish transport activity. The minimum sequence required for nuclear

localization is Pro-Lys-Lys128-Lys-Arg-Lys-Val (Kalderon et al., 1984b).

One important aspect of this signal sequence is its ability to function

when removed from its normal environment and placed in a different

region of a protein. For example, if the signal is linked to the amino

terminus of either a mutated large T-antigen or chicken muscle pyruvate

kinase (a nonnuclear protein), it can still confer nuclear localization.

In addition, a synthetically synthesized signal peptide cross-linked to

a carrier protein has the ability to target the conjugate into the

nucleus of cultured cells (Lanford et al., 1986; Yoneda et al., 1987) as

well as oocytes (Goldfarb et al., 1986). Thus, the functional autonomy

of the targeting signal suggests that it does not require a fixed

secondary structure.

Recently, nucleoplasmin has been cloned and sequenced (Burglin and

De Robertis, 1987; Dingwall et al., 1987). The fifty amino acids from

the carboxy terminal tail domain have two regions similar in homology

with the nuclear localization signal from the SV 40 large T-antigen

(Dingwall et al., 1987). In addition, two other regions have been









22

identified to have weak homology with targeting signals from the yeast

MATa2 protein. It was postulated from these results that each tail

domain might have four targeting signals, with a total of 20 signals per

pentamer. Further experimentation on the tail domain has now

demonstrated that each tail contains only 1 signal sequence consisting

of approximately 14 amino acids and though it is similar to the SV 40 T-

antigen targeting signal, it is not homologous (Dingwall, personal

communication 1988).

The list of proteins containing nuclear targeting signals continues

to be expanded. In a search to find a conserved sequence, several

targeting signals have been compared to SV 40 large T-antigen. Protein

signals that have similar amino acid sequences include nucleoplasmin

(Dingwall et al., 1987), polyoma virus large T-antigen (Richardson et

al., 1986), SV 40 VP1 (Wychowski et al., 1986), and Xenopus laevis N1/N2

protein (Kleinschmidt et al., 1986). Nuclear localization sequences

from other proteins showing little homology to SV 40 large T-antigen

include the adenovirus Ela protein (Lyons et al., 1987), the yeast

proteins L3 (Moreland et al., 1985) and MATa2 (Hall et al., 1984), and

GAL4 (Silver et al., 1984).

The translocation of proteins directly across intracellular

membranes has been well documented for endoplasmic reticulum (Warren and

Dobberstein, 1978) and mitochondria (Schatz and Butow, 1983). Proteins

destined for either organelle contain a signal sequence, which is

cleaved after entry. Protein import into the endoplasmic reticulum

occurs by a cotranslational mechanism; however, this mechanism is

unlikely for nuclear uptake since cytoplasmically injected proteins,









23

extracted from the nucleus, can accumulate back into the nucleus.

Mitochondrial proteins have signal sequences that target them into

specific regions of the mitochondria and cross the membrane(s) by a

posttranslational mechanism. However, no evidence exists that nuclear

transport occurs by a posttranslational process directly across the

membrane, although it still remains a possibility.

An elegant approach to study the exchange sites of a known

transportable nuclear protein was performed by coating colloidal gold

particles with nucleoplasmin (Feldherr et al., 1984). Their results

demonstrate that nucleoplasmin-coated gold enter the nucleus through

channels located in the centers of the pores. Particles, ranging in

size from 50-200 A, were able to penetrate the pore region, thus

indicating that the functional size of the transport channel is

different than the size of the diffusion channel. The transported

particles are nondeformable and spherical, which argues against

structural deformation of nucleoplasmin prior to transport.

Furthermore, the particles are markedly larger than the diffusion

channel, which suggests the involvement of a selective uptake process.



In vitro uptake

The use of an in vitro assay system to investigate protein

transport has both advantages and disadvantages. The drawbacks are,

first, changes in the permeability characteristics of isolated nuclei.

Peters (1983) measured the permeability of single isolated liver nuclei

to dextrans using fluorescent microphotolysis and estimated the pore

diameter to be 112-118 A, which is 2-8 A larger than pores from in vivo









24

nuclei (see exogenous molecules section). According to the model of

Paine et al. (1975), a small change in pore diameter can have a marked

effect on flux rates. Second, after isolation of oocyte nuclei under

aqueous conditions, 95% of the proteins is lost with a half-time of

250 s (Paine et al., 1983). Third, assuming some nuclear proteins are

transported along cytoskeletal elements to the nuclear surface, the

isolation procedure removes the nucleus from its cytoplasmic anchorage,

thus removing a possible step in transport. In light of these

disadvantages, the results from the in vitro experiments must be

carefully interpreted.

An in vitro system is advantageous to study protein uptake since

the steps in the process can be easily dissected and experimentally

manipulated. In vitro assays can be used: i) to characterize

components needed for translocation, ii) to determine inhibitors of

protein transport, and iii) to determine if protein transport is ATP-

dependent.

Forbes et al. (1983) have shown that bacteriophage DNA

microinjected into Xenopus eggs can form nucleus-like structures.

Addition of DNA from either bacteriophage lambda or Xenopus to an

extract from Xenopus eggs results in the formation of synthetic nuclei

that have morphological similarities to intact nuclei (Newmeyer et al.,

1986). These synthetic nuclei have a double membrane envelope, however,

not all possess nuclear pore complexes. The reconstituted nuclei, in

conjunction with labelled nucleoplasmin, have been used to investigate

aspects of the protein transport process. Newmeyer et al. (1986)

demonstrated that nucleoplasmin uptake is ATP- and temperature-









25

dependent. In addition, the lectin wheat germ agglutinin (WGA)

effectively inhibits the transport of nucleoplasmin and the inhibitory

effects could be blocked with the addition of N-acetylglucosamine

(Finlay et al., 1987). Ferritin-labelled WGA was shown to be localized

to the cytoplasmic face of the nuclear pores. Furthermore, iodinated

WGA stains a glycoprotein, 63-65 kd, on nitrocellulose blots of rat

liver nuclei (Finlay et al., 1987). This glycoprotein is similar to the

one detected by Davis and Blobel (1986) and Snow et al. (1987). It has

been suggested that this glycoprotein is part of the pore complex and

might be involved in the nuclear transport of proteins.



Nuclear Efflux

Introduction

The nucleus synthesizes the RNAs required for protein synthesis;

thus, the efflux of messenger RNA (mRNA), ribosomal RNA (rRNA), and

transfer RNA (tNRA) is an obligatory step in the process of gene

expression. Heterogeneous nuclear RNA (hnRNA), small nuclear RNAs

(snRNA) and tRNA precursors are found normally within the nucleus.

Prior to mature mRNA export to the cytoplasm, hnRNA must undergo i)

processing, which involves removal of the introns and splicing together

of the exons, and ii) posttranscriptional modifications, which can

include polyadenylation and methylation. tRNA precursors must also be

processed before efflux and precursor rRNA is processed into 28S, 18S,

and 5.8S RNAs and are present in 40S and 60S RNP particles prior to

transport into the cytoplasm.








26

Goldstein and Plaut (1955) obtained direct evidence in Amoeba

proteus for the transfer of RNA to the cytoplasm by first labelling the

RNA and then transplanting the labelled nucleus to an unlabelled

recipient cell. As shown by autoradiography, the number of grains

within the cytoplasm increased with time. In addition, no radioactivity

was observed in the unlabelled recipient cell nucleus. Ribonuclease

digestion was performed to show that the radioactivity was associated

with the RNA. Due to the size of the RNA, it was postulated that simple

diffusion was not the mechanism involved in RNA efflux.

The movement of RNA from nucleus to cytoplasm is likely to be a

three-step process, involving 1) migration from its site of

transcription to the inner surface of the nuclear envelope, 2)

translocation across the envelope, presumably through the nuclear pores,

and 3) movement from the outer surface of the nuclear envelope to its

final destination within the cytoplasm. The first two steps have been

the focus of intense investigation in an attempt to understand the

mechanisms) involved in RNA transport. The final step of directing RNA

to its destination within the cytoplasm is not as well characterized as

steps 1 and 2. In addition, Agutter (1985a) has suggested that RNA

efflux occurs by a solid-state process; that is, movement occurs along

structural elements both within the nucleus and cytoplasm.



Transfer RNA

The structure of tRNA and its involvement in protein synthesis have

been the subject of many investigations. Transfer RNAs are small,

compact molecules. In the crystallized state, yeast tRNAPhe has a









27

length of 77 A and an average thickness of 20 A (Kim et al., 1973). A

Stokes's radius of 34 A was derived for tRNAPhe from its translational

diffusion coefficient in water (Peters, 1986). A molecule having these

dimensions will not readily diffuse through the nuclear pores.

Injection of the cloned tRNAtyr yeast gene into the nuclei of oocytes

produces a precursor transcript with a length of 108 nucleotides (Melton

et al., 1980). After processing, mature tRNA (78 nucleotides long) is

then exported into the cytoplasm. De Robertis et al. (1982) injected

labelled tRNA into the cytoplasm of oocytes and showed it to be excluded

from the nucleus, suggesting that tRNA efflux is vectorial

(unidirectional).

Zasloff (1983) studied tRNA transport in amphibian oocytes. This

is a convenient system since oocytes can transcribe genes that have been

injected into the nucleus. In addition, analysis is facilitated by the

fact that nucleus and cytoplasm can be readily separated. Two genes

were used in this study; the first was normal human tRNAmet and the

second was a variant human tRNAmet with a G-to-U substitution at

position 57 in the highly conserved loop IV region. Nuclei were

injected with either the genes encoding for the two tRNAs or prelabelled

tRNAs, and the cells were fractionated at various times thereafter. It

was found by electrophoretic analysis of both the nucleus and cytoplasm

that 80% of the normal tRNA entered the cytoplasm within 5 min; in

contrast, 90% of the variant tRNA remained intranuclear after 15 min.

Precise kinetic analysis, performed by liquid scintillometry, showed the

rate of transport of the variant tRNA to be 20-fold slower than normal

tRNA. In addition, normal tRNA transport is saturable and displays









28

temperature dependence. Furthermore, tRNAPhe can act as an effective

competitor of tRNAmet transport. These results strongly suggest that

tRNA transport into the cytoplasm involves a carrier-mediated process

rather than simple diffusion.

In an attempt to define the critical region of the tRNA molecule

required for transport, Tobian et al. (1985) used site-directed

mutagenesis to obtain 30 different mutants with single nucleotide

substitutions. Many of the mutations perturbed the rate of transport.

The most deleterious effects were in the highly conserved T stem-loop

regions; however, many of these tRNAs had impaired processing. It is

suggested from this study that transport, in addition to processing, is

dependent on maintenance of the secondary structure.

The nuclear pores can serve as diffusion pathways for exogenous

tracers and as a transport pathway for some karyophilic proteins. As

mentioned above, the diffusion of tRNA out of the nucleus seems unlikely

due to i) its size and ii) the involvement of a carrier-mediated

process. Zasloff (1983) has suggested that the pores serve as the

pathway for exchange and that peripheral ribosome-like particles on the

cytoplasmic surface of the nuclear envelope provide the motor mechanism

required for transport.



Small nuclear RNAs

The snRNAs comprise 0.1-1.0% of the total cellular RNA. They are

small (100-250 nucleotides), stable, uridylic-acid rich RNAs that are

localized in the nucleus and are transiently found in the cytoplasm. At

least eight snRNAs, U1-U8, have been identified, and all are associated











with a core of five common proteins. Functional roles that have been

identified for a few of the U-snRNAs include polyadenylation,

intranuclear transport, and splicing of hnRNA.

To investigate intracellular RNA transport within intact cells,

De Robertis et al. (1982) injected radiolabelled RNA from HeLa cells

into the cytoplasm of Xenopus oocytes. The results demonstrate that

snRNAs can migrate from the cytoplasm into the nucleus and concentrate

there 30- to 60-fold. It was also established that HeLa snRNAs complex

with oocyte proteins in the cytoplasm before their migration into the

nucleus. Zeller et al. (1983) analyzed the distribution of snRNAs and

their associated binding proteins during oogenesis and early

development. Mature Xenopus oocytes and embryos prior to gastrulation

are known to contain an excess of snRNA binding proteins (De Robertis et

al., 1982) and less Ul and U2 snRNAs than previtellogenic oocytes. In

embryos, the midblastula transition (Newport and Kirschner, 1982) marks

the onset of a burst in snRNA synthesis and the migration of snRNA

binding proteins into the nucleus. Injection of labelled snRNA into the

cytoplasm of mature oocytes can prematurely displace the cytoplasmic

binding proteins into the nucleus, suggesting that movement of the

proteins requires the formation of the snRNPs. To determine whether

nuclear accumulation occurs only when snRNA and its binding proteins are

completed together, Mattaj and De Robertis (1985) used site-directed

mutagenesis to delete the protein binding site on U2 snRNA. As a result

of the snRNA's inability to bind its cytoplasmic proteins, it becomes

unable to enter the nucleus. It is suggested that the formation of the

snRNP within the cytoplasm might unmask a cryptic targeting signal which









30

then allows for the accumulation within the nucleus. Whether the signal

is in the protein, the RNA, or possibly a combination of both, has yet

to be determined.



Ribosomal RNA

The ribosomal DNA transcript, located in the nucleoli, contains the

18S, 5.8S, and 28S genes. After transcription, the rRNA is completed

with proteins to form a 80S ribosomal ribonucleoprotein particle, which

is processed to the mature 40S and 60S particles prior to cytoplasmic

transport. The 5S RNA is transcribed, processed, and transported

separately. These rRNPs are the major components involved in ribosome

biogenesis.

Previtellogenic oocytes (stages I and II; Dumont, 1972) synthesize

and store 5S RNA which comprises 30-40% of the total cellular RNA.

TFIIIA is a transcription factor with a molecular weight of 39 kd that

binds to 5S genes. In addition, TFIIIA also binds to cytoplasmic 5S RNA

to form a 7S ribonucleoprotein particle. By employing immunocyto-

chemical procedures on sections of Xenopus ovaries, Mattaj et al. (1983)

demonstrated that TFIIIA is located predominantly in the cytoplasm. In

previtellogenic oocytes, TFIIIA is associated with the 5S RNA (Picard

and Wegnez, 1979); thus, the immunocytochemical staining patterns are

actually localizing 7S RNPs. Pelham and Brown (1980) studied the

in vitro transcription of the 5S RNA gene and showed that transcription

could be inhibited when 5S RNA, in excess of the 5S gene, competed for

TFIIIA binding. They suggested from these results that TFIIIA might be

involved in a feed-back mechanism to control 5S gene expression. It is









31

important to mention that TFIIIA alone can enter the nucleus to bind to

the 5S gene; however, when bound to 5S RNA, it remains in the cytoplasm.

Free 5S RNA also is capable of entering the nucleus and it accumulates

in the nucleoli (De Robertis et al., 1982). The 7S RNP is able to

diffuse throughout the cytoplasm of oocytes (Ford, 1971) but is excluded

from the nucleus. The mechanism of exclusion is not yet known; however,

it is possible that the formation of the 7S RNP in the cytoplasm masks a

transport signal, either on the RNA or the protein that is required for

nuclear entry.

Wunderlich and co-workers have studied 18S and 28S rRNA transport

from isolated Tetrahymena macronuclei. They reported that rRNP efflux

is ATP dependent and the amount of RNP transport is sensitive to a shift

in temperature (Giese and Wunderlich, 1983). It was also found that

rRNA is integrated into the nuclear matrix which might control the

transport process since removal of the nuclear envelope had no effect on

the temperature dependence of rRNP export (Wunderlich et al., 1983).

Although 28S and 18S RNA leave the nucleus completed with ribosomal

proteins, it is not clear whether the RNA or protein contains the efflux

signals.

The role of the nuclear envelope in regulating rRNA translocation

was investigated in vivo (Feldherr, 1980) by disrupting the nuclear

envelope with glass needles, and in vitro (Stuart et al., 1977) by using

membrane-denuded nuclei. The efflux of rRNA from the nucleus to the

cytoplasm was not affected by either experimental procedure suggesting

that rRNA is most likely bound to nondiffusible nuclear elements.









32

Overall, tRNA, 5S RNA, and rRNA are generally believed to

translocate into the cytoplasm through the nuclear pores. However, much

of the evidence is circumstantial and there are no definitive data to

indicate that the pores serve as the major pathways for the efflux of

these different classes of RNA.



Messenger RNA

Heterogeneous nuclear RNA is processed and undergoes extensive

posttranscriptional modifications before mature mRNA can be transported

into the cytoplasm. Unspliced mRNA is retained in the nucleus and only

mature mRNA appears in the efflux supernatant from isolated nuclei in

vitro or in the cytoplasm of intact cells, suggesting that processing is

one of the many requirements necessary for export. The movement of

mature mRNA, from its site(s) of transcription and processing, to the

nuclear envelope is the first step involved in export (see above).

Agutter (1985a) has suggested that mRNA might not be freely diffusible

throughout the cells. To determine whether RNA is preferentially

associated with the nuclear matrix, nuclei were treated with nonionic

detergents, high salt, EDTA, and urea. It was shown that greater than

90% of the nuclear RNA was tightly associated with the insoluble nuclear

matrix (Berezney, 1980; van Eekelen and van Venrooij, 1981). Release of

the mature ovalbumin mRNA but not the immature mRNA from the nuclear

matrix occurs in the presence of ATP and does not require hydrolysis of

the phosphodiester bond (Schroder et al., 1987). This selective

detachment from the matrix might be another level of control in

regulating the transport of mRNA. Overall, the migration of mature mRNA








33

to the envelope is likely to involve the processing of the precursor,

posttranscriptional modification, and release from the nuclear matrix.

Once at the inner surface of the nuclear envelope, the second step is

the translocation across the envelope, presumably through the pores.

Translocation of mRNA across the nuclear envelope appears to

involve a series of reactions requiring nucleoside triphosphatase

(NTPase), protein kinase, protein phosphohydrolase, and poly(A) binding

sites. Using isolated nuclei, Agutter et al. (1976) first reported a

correlation between RNA efflux and the presence of NTPase activity which

was localized in nuclear envelope fractions. Further studies on the

energy requirement of poly(A)+mRNA translocation were performed by

Clawson et al. (1978). These investigations determined that both ATP

and GTP could serve as energy sources.

Clawson et al. (1984) used photoaffinity labelling on rat liver

nuclear envelopes and localized the NTPase activity to a 46 kd protein.

Schroder et al. (1986a) subsequently purified and characterized a 40 kd

NTPase from rat liver nuclear envelopes which is thought to be the same

polypeptide identified by Clawson. In addition to the nuclear envelope,

NTPase activity was also found to be present in the matrix (Clawson et

al., 1984; Maul and Baglia, 1983). There are several experiments that

correlate the energy-dependent transport of poly(A)+mRNA with the NTPase

activity. First, an increase in the rate of RNA efflux by thioacetamide

treatment (Clawson et al., 1980), insulin administration (Purrello et

al., 1982), or tryptophan feeding (Murty et al., 1980), causes a

parallel increase in NTPase activity. Second, in a transport assay

using isolated nuclei, NTPase activity and mRNA efflux have similar









34

kinetic properties, substrate specificities, and sensitivities to

inhibitors (Agutter et al., 1976; Clawson et al., 1978, 1980). One

additional line of evidence that mRNA and NTPase are interrelated is the

inhibition of mRNA efflux by antibodies against pore complex-lamina

components that affect NTPase activity (Schroder et al.; unpublished

results).

The effects of poly(A) on NTPase activity were observed initially

by Agutter et al. (1977) on isolated, intact nuclear envelopes. They

showed that addition of poly(A) or poly(G) to the media could enhance

NTPase activity. Furthermore, Bernd et al. (1982) demonstrated the

NTPase activity to be markedly stimulated by either po1y(A)+mRNA or a

homopolymer of poly(A) at least 15-20 bases long. Additional evidence

comes from experiments by Riedel and Fasold (1987). They prepared

resealed nuclear-envelope vesicles that retain NTPase activity and found

that both poly(A) and mRNA can stimulate the NTPase activity associated

with these vesicles. It is suggested from these data that poly(A) can

modulate NTPase activity and possibly regulate the translocation of

mRNA.

It has been demonstrated that protein kinases and phosphohydrolases

are present in the nuclear envelope (Lam and Kasper, 1979; Smith and

Wells, 1983; Steer et al., 1979a, 1979b). McDonald and Agutter (1980)

have studied the effects of polyribonucleotide binding on the

phosphorylation and dephosphorylation of nuclear envelope proteins and

found that the same polyribonucleotides that stimulate NTPase, inhibit

protein kinase activity and stimulate one of the phosphohydrolases.

They also found the nuclear envelope to contain a population of poly(A)









35

binding sites that increase in affinity by a kinase-dependent

phosphorylation reaction. The actual role these components play in the

in vivo translocation of mRNA across the envelope has not yet been

clearly defined; however, poly(A)+mRNA efflux models have been proposed

by Agutter (1985a) and Schroder et al. (1988).

The model proposed by Agutter (1985a) integrates the components

involved in RNA translocation: NTPase, poly(A) binding sites, protein

kinases and phosphohydrolase, to explain nucleocytoplasmic transport of

poly(A)+mRNA. The following scheme is based on Agutter's model (1985a).

The NTPase exists in a complex with the poly(A) binding site. A protein

kinase uses MgATP as a substrate to phosphorylate the poly(A) binding

site. Binding of nuclear poly(A)+mRNA to the [phosphorylated binding

site-NTPase] complex stimulates dephosphorylation by the

phosphohydrolase. MgATP is also a substrate for NTPase, the binding of

MgATP to the NTPase of the [poly(A)+mRNA-poly(A) binding site-NTPase]

complex causes the displacement of poly(A)+mRNA, thus facilitating the

translocation of mRNA. The NTPase then hydrolyses ATP and the [NTPase-

poly(A) binding site] complex is available again to start another cycle.

At least two cytosolic proteins, 34 kd and 58 kd, with an affinity

for poly(A) can stimulate mRNA efflux in vitro (Moffett and Webb, 1981,

1983; Schroder et al., 1986b). The 34 kd protein, in the presence of

poly(A), stimulates the NTPase activity to a greater extent than poly(A)

by itself. In the absence of poly(A), p34 has no effect on kinase or

phosphohydrolase activity. The 34 kd protein is thought to enhance

NTPase activity by increasing the affinity of poly(A) to the poly(A)

binding site in the phosphorylated nuclear envelope. Schroder et al.









36

(1988) suggest that the 58 kd protein inhibits kinase activity by

promoting poly(A) binding to unphosphorylated envelopes, which prevents

the down-regulation of NTPase activity by the kinase.

The third step in the overall RNA transport is the involvement of

the cytoplasm. Although it is established that much of the mRNA is

bound to the cytoskeleton (Jeffery, 1982), it is not clear how the RNA

finds its final destination. The proteins associated with poly(A)+mRNA

in the cytoplasm are different from those that are bound to poly(A)

within the nucleus (Baer and Kornberg, 1983; Sachs and Kornberg, 1985;

Setyono and Greenberg, 1981). It is not known if these proteins are

involved in the transport of RNAs within the cytoplasm or serve as

anchors to cytoskeletal elements. In this regard, it is necessary to

further understand the functional role of the cytoplasmic proteins

associated with RNAs.

Ribonucleoproteins (RNPs) have been visualized exiting the nucleus

through the nuclear pores by electron microscopic cytochemical analysis

(Stevens and Swift, 1966). In a more recent study, Skoglund et al.

(1983) studied the formation and transport of an hnRNP particle, a

transcription product from the Balbiani ring genes in Chironomus. Their

results, by using the Miller spread technique, describe the growth of a

100 A wide filament into a large 400-500 A granule. Prior to export,

these mature RNP particles undergo conformational changes that result in

the formation of rod-shaped structures with an average length of 1350 A

and diameters of 250-300 A that pass through the centers of the nuclear

pores. Even though these RNPs undergo a shape change, they are still









37

too large to pass through the pores by passive diffusion, suggesting

that some facilitated mechanism is a requirement for export.


Statement of Research Topic

It has been demonstrated that there are selective mechanisms which

facilitate the passage of certain endogenous macromolecules through the

nuclear pores. The mechanism and selectivity of the pore by which

transport occurs is not known; for example, does translocation proceed

through the pores by a gated mechanism, or does transport occur through

a fixed channel? How does the nuclear targeting signal interact with

the pore region to initiate translocation? Do different nuclear

transport signals use the same pore for uptake? What effects do

variations in the number and sequence of targeting signals have on

nuclear uptake? In relation to RNA transport, what is the precise

location for the translocation of different classes of RNA to the

cytoplasm? Are there specific functional classes of pores that are

involved in RNA transport? or do all pores have the capability to

translocate both RNAs and proteins?

To better understand the role of the nuclear envelope in regulating

nucleocytoplasmic exchange, experiments were designed to elucidate

characteristics of the macromolecular transport process. The data

obtained will focus on the translocation of RNAs. More specifically,

analysis will include the proportion of functional pores, the precise

location and size of the transport channels, and the multifunctional

capability of individual pores (i.e., both protein uptake and RNA

efflux). In addition, data will be obtained for the effect of different









38

numbers of synthetic nuclear targeting signals on protein uptake and the

functional size of the transport channel. It will also be determined

whether individual pores have the capability to transport proteins

containing different nuclear targeting sequences.

Many of the previous nucleocytoplasmic exchange experiments were

based on quantitative and qualitative studies performed with

fluorescein- or radio-labelled molecules; however, there are certain

characteristics of the exchange sites that cannot be studied using these

type of tracer molecules. In this study, colloidal gold particles will

be used to identify and characterize the sites for macromolecular

exchange.

Colloidal gold tracers have some unique properties that make them

ideal for this type of experimentation. First, gold particles with

different diameters can be easily prepared. Second, the surface

properties of the particles can be modified by the adsorption of

different molecules and the particles will then acquire the

characteristics of their coating agents. Third, tracer particles can be

localized precisely to specific cell structures by electron microscopy.

The following study will focus primarily on nucleocytoplasmic

transport of macromolecules, especially with regard to the

characteristics of the exchange sites located within the nuclear pores.

The experiments are performed on amphibian oocytes and involve the

microinjection of either RNA- or protein-coated gold particles into the

nuclear or cytoplasmic compartment of the cells. The subsequent

intracellular distribution of the tracer particles and their relation to

the nuclear pores will be determined by electron microscopy.















CHAPTER II
TRANSLOCATION OF RNA-COATED GOLD PARTICLES
THROUGH THE NUCLEAR PORES OF OOCYTES


Introduction

The nuclear envelope in eukaryotic cells is the site of continual

macromolecular exchange between the nucleoplasm and the cytoplasm. Such

exchanges can occur either by passive diffusion or mediated transport

through the nuclear pores (Dingwall and Laskey, 1986). Microinjection

of various sized exogenous molecules has been used in previous studies

to determine the dimensions of the diffusion channels within the pores.

The channels were estimated to be 90 A in diameter in amphibian oocytes

(Paine et al., 1975), and are available for diffusion both into and out

of the nucleus (Paine, 1975). Evidence for protein transport across the

envelope was initially obtained for RN1 (Feldherr et al., 1983) and

nucleoplasmin (Dingwall et al., 1982), both of which are major

karyophilic proteins found in amphibian oocytes. The specific sites of

transport for nucleoplasmin have been identified by microinjecting

nucleoplasmin-coated gold particles into the cytoplasm of the oocytes

(Feldherr et al., 1984). It was found, by electron microscopy, that

transport of the colloidal particles occurred through channels, at least

200 A in diameter, located in the centers of the nuclear pores. Thus,

the regions of the pores available for transport are considerably larger

than those available for diffusion.









40

The exit of mRNA from isolated nuclei has been studied in a number

of laboratories (reviewed by Clawson et al., 1985). These

investigations have shown that efflux is a temperature-dependent,

energy-requiring process, indicating that some form of transport is

involved (Agutter, 1985a; Clawson et al., 1978). In support of this

view, isolated nuclear envelope preparations were found to contain

nucleoside triphosphatase activity which is affected by the same factors

(ionic composition, pH, etc.) that regulate RNA efflux (Agutter, 1985a;

Clawson et al., 1980; 1984). It has also been suggested, on the basis

of results obtained in several laboratories, that the poly(A) tail of

mRNA is involved in transport across the envelope (Agutter, 1985b; Bernd

et al., 1982).

The efflux of tRNA from the nucleus has been investigated by Zasloff

(1983) using in vivo procedures. He obtained evidence for a saturable,

carrier-mediated transport mechanism for tRNAmet in the amphibian

oocyte. De Robertis et al. (1982) have demonstrated that microinjected

5S RNA can migrate either into or out of the nucleus in Xenopus oocytes;

however, the mechanism of exchange was not determined.

It is generally believed that RNA transport occurs through the

nuclear pores. Indirect evidence supporting this view has been reviewed

by Franke and Scheer (1974); direct evidence was obtained by Stevens and

Swift (1966). These latter investigators observed that 400 A RNP

granules synthesized at the Balbiani rings in Chironomus salivary gland

cells exit through the pores. As the particles enter the pores they are

transformed into rod-like structures with diameters of about 200-250 A.









41

There is evidence that RNA is not freely diffusible within the

nucleus and cytoplasm but is associated with structural elements of the

nuclear matrix and cytoskeleton (Agutter, 1985a; Feldherr, 1980; Fey et

al., 1986; van Eekelen and van Venrooij, 1981). Based partly on these

findings, Agutter (1985a) postulated that the efflux of RNA occurs in

three stages by a solid-phase mechanism; first, transport to the nuclear

envelope along the nuclear matrix, second, translocation through the

pores, and third, transport along the cytoskeletal matrix.

The focus of the present investigation is on the second step of the

efflux process, i.e., translocation across the envelope. The main

objectives are; first, to localize and characterize the specific regions

of the nuclear pores involved in the translocation of different classes

of RNA-coated gold to the cytoplasm and, second, to determine whether

individual pores can function in both RNA efflux and protein uptake, or

whether separate classes of nuclear pores exist. In order to obtain the

resolution necessary to examine individual exchange sites, coated

colloidal gold particles are used as tracers. The particles are

microinjected into oocytes, and subsequently localized by electron

microscopy.

The results are summarized as follows: first, gold particles

coated with tRNA (met or phe), 5S RNA, or poly(A) are all translocated

into the cytoplasm through central channels, at least 230 A in diameter,

located within the nuclear pores. Particles stabilized with

nonphysiological polynucleotides are also translocated; however, the

number and distribution of the particles associated with the pores

varied for different coating agents. Second, approximately 97% of the









42

pores in Xenopus oocytes can function in the translocation of RNA-coated

gold. Third, the accumulation of particles in the pores is a saturable

process. Fourth, individual pores can function in both RNA efflux and

protein uptake. Control experiments ruled out the possibilities of

fixation artifacts and non-specific exchange processes.



Materials and Methods

Xenopus laevis were purchased from Xenopus 1 (Ann Arbor, Michigan)

and maintained as reported previously (Feldherr, 1975).



Nucleoplasmin isolation

Nucleoplasmin was isolated from a starting volume of 30 ml of

Xenopus ovaries. The isolation procedure was similar to that described

by Dingwall et al. (1982), with the exception that an anti-

nucleoplasmin IgG affinity column was substituted for the DEAE-

cellulose and phenyl sepharose columns. Polyclonal antibodies against

nucleoplasmin were generated in rabbits and affinity purified. A

cleared cell homogenate, obtained from the lysed oocytes (Feldherr et

al., 1984), was passed over an anti-nucleoplasmin affinity column, and

the column was washed free of nonbound proteins. Nucleoplasmin was

eluted in 1 ml fractions with 50 mM glycine-HCl buffer (pH 2.5) into

microcentrifuge tubes containing sufficient 1 M tris (pH 9.0) to

increase the pH to 7.5. The protein was monitored at 280 nm, and the

fractions containing nucleoplasmin were pooled and treated with

ammonium sulfate (55% saturated) overnight in the cold. The soluble

(NH4)2S04 fraction was then dialyzed against a solution containing









43

0.05 M Tris and 0.05 M NaCI (pH 7.2), after which the nucleoplasmin was

precipitated in 80% alcohol and lyophilized as described previously

(Feldherr et al., 1984). Gel analysis was performed, as described by

Laemmeli (1970), to determine the purity of the preparation.



Gold preparation and stabilization

All glassware and solutions used in experiments involving RNA were

treated with 0.01% (vol/vol) diethyl pyrocarbonate and then autoclaved.

Colloidal gold particles were prepared by reducing chloroauric acid with

either trisodium citrate (Frens, 1973) or a saturated solution of white

phosphorus in ether (Feldherr, 1965). In this study, the trisodium

citrate method gave a particle distribution of 120-220 A in diameter,

whereas the phosphorus ether preparations ranged from either 20-50 A or

20-160 A, depending on the initial concentration of gold chloride used.

The gold sols were stabilized with tRNA (met or phe), 5S RNA,

poly(A) (3500 bases), poly(I) (500 bases), poly(dA) (500 bases),

nucleoplasmin, polyvinylpyrrolidone (PVP; 40 kd), polyglutamic acid,

ovalbumin, or bovine serum albumin (BSA). The RNAs were obtained from

Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical

Company (St. Louis, MO). The purity of the RNAs was tested by running

the preparations on 10% polyacrylamide gels containing 8.3 M ultrapure

urea; ethidium bromide was used as a stain. Polyglutamic acid, BSA,

ovalbumin and PVP were purchased from Sigma Chemical Company.

In each instance, the minimum amount of coating agent required to

stabilize the particles, that is, prevent precipitation in 1% NaCl, was

determined as described earlier (Feldherr, 1984). Before stabilization,









44

tRNA, poly(A), poly(I), and poly(dA) were dissolved in 10 mM KC1, 7.2 mM

K2HPO4 and 4.8 mM KH2PO4 (pH 7.0). 5S RNA was rehydrated in 2 ml of

sterile ion-free water resulting in a salt concentration of 1 mM Tris-

HC1, 10 mM NaCl and 0.1 mM MgCl2 (pH 7.5). Nucleoplasmin, polyglutamic

acid, PVP, BSA, and ovalbumin were solubilized in a low ionic strength

buffer containing 7.2 mM K2HPO4 and 4.8 mM kH2PO4 (pH 7.0).

Concentrations of coating agents and volumes used to stabilize the gold

preparations are given in Table I.

After stabilization, the 20-160 A preparations were centrifuged at

6,000g (at the bottom of the tube) at 4C for 10 minutes to remove any

large aggregates of gold. This step was not necessary for the 20-50 A

and 120-220 A fractions. Five to 7 ml of stabilized colloid were then

concentrated to 70-100 pl in Minicon concentrators (Amicon Corp.,

Danvers, MA). Finally, the samples were dialyzed against intracellular

medium consisting of 102 mM KC1, 11.1 mM NaCl, 7.2 mM K2HP04, and 4.8 mM

KH2PO4 (pH 7.0) for 3 h at 4C.


Injection

Frogs were anesthetized on ice for one hour and the ovaries

removed. Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually

defolliculated with watchmakers forceps and maintained in Ringer's

solution (Diberardino et al., 1977) at 22C. The defolliculated cells

were centrifuged at approximately 650g for 8-10 minutes as described

previously (Feldherr, 1980; Kressmann and Bernstiel, 1980). During

centrifugation, the nucleus migrates to a position just underneath the

plasma membrane at the animal pole and its outline can be visualized due



















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46

to the displacement of pigment granules in the cortex. Nuclear

injections could then be accomplished. Calcium-free Ringer's was used

as an extracellular medium during injection to prevent possible

precipitations of the colloid as the micropipettes were introduced into

the cells. Immediately after injection the cells were returned to

complete Ringer's solution until subsequent fixation. The total

exposure to calcium-free Ringer's was less than 30 minutes. The tip

diameters of the micropipettes were 10-15 pm.



Electron microscopy and analysis

The cells were fixed using a procedure similar to that described by

Kalt and Tandler (1971). The oocytes were fixed initially overnight at

4C in 100 mM HEPES containing 3% glutaraldehyde (vol/vol), 2%

paraformaldehyde (wt/vol), 2.5% DMSO (vol/vol) and 1 mM CaCl2 (pH 7.2).

The nuclei were then dissected out with their surrounding cytoplasm,

post-fixed in 2% Os04 (wt/vol) for one hour, and stained with 0.5%

p-phenylenediamine (wt/vol) in 70% acetone for 30 minutes. Finally, the

samples were dehydrated in a graded series of acetone and embedded in

Spurr's medium. Thin sections were cut on a Reichert microtome and

analyzed with a JEOL 100S electron microscope (JEOL USA, Cranford, NJ).

In the RNA efflux studies it was presumed that gold particles

located within the pores were in the process of translocation, and that

particles present in the cytoplasm just adjacent to the pores had

completed the translocation process. These areas will be referred to as

regions 1 and 2, respectively (see Fig. 1). Pores that contained one or

more particles in either or both of these regions were considered to be
























Nucleoplasm


Cytoplasm


Figure 1. A schematic representation of the regions used for
electron microscopic analysis of the particle distribution
within the pores. It is assumed that the particles in region 1
are undergoing translocation and that particles in region 2 have
completed the process. The arrow which defines the outer limits
of region 2 represents a distance of approximately 500 A.









48

actively involved in efflux. To determine the functional diameter of

the transport channels, the size distribution of the particles in

regions 1 and 2 were obtained and compared with the size of the injected

particles (i.e., particles within the nucleus). To standardize the

results, pores were analyzed in regions where the gold concentration in

the adjacent nucleoplasm was about 100 particles/0.36pm2.

Negative staining procedures were used to estimate the overall

diameters of the particles, that is, the gold plus the adsorbed coat

material.



Results

Purity of stabilizing agents

Approximately 600 pg of nucleoplasmin was isolated from 30 ml of

ovary using affinity chromatography. SDS-polyacrylamide gels of the

isolated protein showed major bands with apparent molecular weights of

165 kd and 145 kd, and a minor band with a molecular weight of

approximately 33 kd. The gel pattern (not shown) was identical to that

obtained for nucleoplasmin isolated using DEAE and phenyl sepharose

columns (Feldherr et al., 1984).

On a 10% polyacrylamide gel containing urea, tRNA ran as a single

distinct band corresponding to approximately 70 nucleotides, whereas the

pattern obtained for 5S RNA contained one major band corresponding to

approximately 130 nucleotides and minor bands of smaller sizes, probably

representing breakdown products. Gel scans of the tRNA and 5S RNA

showed the major band in each sample to be >80% of the total RNA.









49

Nuclear injection of tRNA-, 5S RNA- and poly(A)-coated gold

It was determined initially that varying the amount of the

injectate from 8-20 nl (the RNA content ranged from 30-100 ng) had no

effect on the results. Routinely, 8 nl of colloid were injected. This

volume contained a sufficient number of particles for electron

microscopic analysis and minimized possible damage to the nucleus. The

oocytes were fixed 15 min, 1 h, or 6 h after injection. Eighteen to 20

cells were examined for each type of RNA at each time interval.

The results were essentially the same for 20-160 A particles

coated with tRNA (met or phe), 5S RNA, or poly(A). At all time

intervals, particles were distributed randomly throughout the

nucleoplasm, except for aggregates of gold that were occasionally

observed at the surface of the nucleoli. After 15 min and 1 h,

particles were associated with almost all of the nuclear pores.

Representative results for tRNA and 5S RNA are shown in Figs. 2 and 3,

respectively. Based on the assumption that the presence of gold

particles within the pores or in the adjacent cytoplasm (regions 1 and

2 in Fig. 1) is indicative of nucleocytoplasmic exchange, it was

concluded that over 97% of the pores were involved in translocation 15

min and 1 h after injection (see Table II). There was an obvious

decrease in the percentage of pores that contained particles in the 6 h

experiments, but these results were not quantitated. In all instances,

particles were observed in the cytoplasm beyond the immediate vicinity

of the pores (i.e., beyond region 2). However, even after 6 h the

cytoplasmic to nuclear concentration ratio was only 1:14.
























Figure 2. tRNA-gold, nuclear injection 15 min experiment.
Gold particles (20-160 A) are observed evenly distributed throughout
the nucleus (N). Particles are seen within the centers of the majority
of the pores and also in the adjacent cytoplasm (C).
Bar, 0.2 pm.














Figure 3. 5S RNA-gold, nuclear injection 60 min experiment.
The tracer particles (20-160 A) show a similar distribution as
described in Figure 2. Particles are present within the pores
and the adjacent cytoplasm (C). N, nucleus. Bar, 0.2 pm.

































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The size distributions of tRNA- and 5S RNA-coated particles present

within the nucleoplasm and also associated with the nuclear pores

(regions 1 and 2) are given in Table III. Since the thickness of the

RNA coat, in both cases, was estimated to be 15-20 A, the results

indicate that particles with an overall diameter of at least 170 A can

pass through the centers of the pores. Since larger particles could not

be stabilized with tRNA or 5S RNA, it was not possible to determine an

upper size limit for translocation using these coating agents. In

contrast, 120-220 A particles could be stabilized with poly(A). The

results obtained wtih poly(A)-gold are illustrated in Fig. 4. Despite

the fact that relatively few particles were injected into the nuclei,

gold was found in over 70% of the pores after 15 min and 1 h. The size

distribution of poly(A)-coated particles associated with the pores is

shown in Table III. It is apparent from the data, which is based on the

examination of 12 cells, that particles at least 230 A in diameter

(including the coat) are translocated through the pores.



Nuclear injection of poly(I)- and poly(dA)-coated gold

In addition to the studies performed with tRNA, 5S RNA and poly(A),

nuclei were also injected with poly(I)- or poly(dA)-coated particles.

The volumes injected, and the procedure used for analysis of these

nonphysiological tracers, were the same as described above. In these

experiments, the oocytes were fixed 1 h after injection, and 5 cells

were examined in each group. The results are shown in Table II.

Particles coated with either poly(I) or poly(dA) were translocated

through the centers of the pores; however, differences were observed in













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'-4 *i 0e QO SL


-le -o
a) ~ ~ ~ ~ / lc) E)( JU Jf
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lo:
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E

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u

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r
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Figure 4. Poly(A)-gold, nuclear injection 60 mmin experiment.
Large gold particles (120-220 A) are observed extending through
the nulcear pores and are present in the adjacent cytoplasm (C).
N, nucleus. Bar, 0.2pm.


Figure 5. PVP-gold, nuclear injection 60 min experiment.
Gold particles are retained within the nucleoplasm and are rarely
seen associated with the envelope or within the nuclear pores.
C, cytoplasm. N, nucleus. Bar, 0.2 pm.























* 06
v *.*


0


* 8..
4$t
* .r.


.-s.. >.*'



IA^ ^


S

S


- -- .-,.-.


*






C


* 0
*
..S


4!


N


S


--a..-. -~









57

the numbers and distribution of the particles associated with these

structures. Compared with poly(dA)-coated gold, almost twice as many

poly(I)-coated particles were present in the pore areas and, in

addition, a higher proportion of these particles were located in

region 2. These results suggest that some polynucleotides are

translocated more efficiently than others.



Nuclear injection of BSA-, ovalbumin-, polyglutamic acid-, and PVP-

coated gold

To determine if the translocation of RNA-gold is a selective

process, nuclei were injected with gold fractions that had been

stabilized with the exogenous molecules PVP, BSA, or ovalbumin. The

volumes injected, and the experimental times (15 min, 1 h and 6 h), were

the same as those used in the RNA studies. The data are based on the

examination of 6-9 cells per time interval for each coating agent that

was used. The result of a 1 h nuclear PVP-gold injection is

illustrated in Fig. 5. Similar distributions were observed when

particles stabilized with BSA or ovalbumin were injected. It was

found, at all time intervals, that essentially all of the particles

coated with exogenous substances were retained within the nuclei and

less than 6% of the pores contained gold particles (Table II). It can

also be seen in Table II that the total number of control particles

translocated per 200 pores was only about 1% of the total number of

translocated RNA-coated particles. These results demonstrate that the

translocation of RNA-gold was due to the specific properties of the

adsorbed coat material.









58

To determine if the translocation of polynucleotide-coated

particles is due simply to a high negative charge density, gold

particles were coated with polyglutamic acid, which, like RNA, is a

polyanion. Since polyglutamic acid is not a highly effective

stabilizing agent, the colloid preparations that were injected were

relatively dilute. To compensate for this factor, tRNA-gold

preparations having an equivalent particle concentration were injected

in parallel experiments. The results, which are based on an analysis of

5 cells, are shown in Table IV. Tracer particles coated with

polyglutamic acid were observed in only 8% of the pores, compared with a

value of 83% obtained for tRNA-gold. These findings demonstrate that

the translocation of the RNA-coated particles is not simply a charge

effect.



Controls

Control experiments were performed on 6 oocytes to establish if the

results obtained for RNA-gold could be due to a redistribution of the

particles during fixation. In this study, the nuclei were injected with

tRNA-coated gold and the oocytes fixed within 10 seconds. The

particles near the site of injection were rarely observed within the

pores or in the adjacent cytoplasm. These data indicate that the

presence of RNA-gold within the pores at 15 min and 1 h intervals was

not a fixation artifact, but reflected an in vivo exchange process.

In order to ascertain whether the presence of excess RNA itself

could alter the properties of the pores, nuclei of 6 oocytes were

simultaneously injected with PVP-coated gold and 100 ng of soluble tRNA.























) rC
> 0








UIf
*t U









o u
0
) (-
9- r_
0 to
I-
4-
0 C






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5-

a.


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o
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00
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0 C.






U
C 0n
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*-,-
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60

Fewer than 4% of the 200 pores analyzed were involved in translocation.

Thus, the injection of RNA in amounts greater than those normally used

in this investigation, have no apparent effect on the physical

properties of the pores.



Concentration dependence of RNA translocation

To determine the concentration dependence of the translocation

process, the gold distribution in the pores was analyzed in areas in

which different concentrations of colloid were present in the adjacent

nucleoplasm. These experiments were performed with tRNA-coated gold

particles, and the cells were fixed after 1 h. To ensure that an

appropriate concentration range was obtained, different dilutions of

colloid were injected. The amount of tRNA in the injectate varied from

6-30 ng. The results are shown in Table V, and are based on the

examination of 6 oocytes. It can be seen that a maximum number of

particles per pore is obtained at a concentration of 90

particles/0.36pm2. Above this concentration, no differences were

observed either in the numbers or distribution of the particles,

suggesting that the translocation process is saturable.



Double label experiment

The high percentage of pores involved in RNA translocation

suggested that each pore may be a bidirectional channel, capable of both

protein and RNA transport. To address this possibility directly, large

gold fractions (120-220 A) coated with nucleoplasmin and small gold

fractions (20-50 A) stabilized with tRNA were used. The tRNA-coated























C
0)0


u U
.t 0
WO c
CD c
I- mU
0 f-
0- I-
4- C


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0)
5-
0


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4 -
EU



0)

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t-U
E
=L






in
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u 0
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IB0







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61














ee a
4- CM4 00 00 00
to co m 0 cr














C tD to to
10 CO Cr> CT> Cr















00 --4 (D CT> CMi
,-i r- Lo Un Zo








oo 1-4 m On Ct


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mo 4oo
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w
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00




N4.)
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4--
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0-
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0) U.


'--









62

particles were injected into the nucleus and nucleoplasmin-coated gold

was injected into the cytoplasm of the same cell. The sequence of the

injections varied in different experiments; however, in all instances

the interval between the first and second injection was 15 min, and the

oocytes were fixed after a total of 45 min. In all, 12 cells were

examined. The results, which were the same regardless of the injection

sequence, are illustrated in Fig. 6, a-d. Small RNA particles can be

observed immediately adjacent to the cytoplasmic surface of the pores

and large nucleoplasmin particles are present on the nuclear side of the

same pores. These distributions clearly demonstrate that individual

pores can function in both protein uptake and RNA efflux.



Cytoplasmic injections

The RNA-gold preparations [tRNA, 5S RNA and poly(A)] were injected

into the cytoplasm to determine if transport is reversible.

Approximately 40 nl of colloid (containing approximately 200 ng of RNA)

were introduced adjacent to the nuclear envelope in a total of 20

centrifuged cells. Similar distributions were obtained for all RNAs

used. In both 1 h and 6 h experiments, gold was seen within the

poresnear the site of injection, but relatively few particles were found

in the nucleoplasm. The nuclear to cytoplasmic concentration ratios

were approximately 0.01 after 1 h. The 5S RNA-gold results are

illustrated in Fig. 7. It should be pointed out that the number of

pores that contained gold varied considerably from cell to cell,

therefore, no effort was made to quantitate these results.























Figure 6. Double injection experiment 45 min.
tRNA-gold (20-50 A) was injected into the nucleus (N), and the adjacent
cytoplasm (C) was injected with nucleoplasmin-gold (120-220 A). The
large nucleoplasmin-coated particles can be seen just on the nuclear
sides of the pores and small RNA particles are present on the
cytoplasmic side. Bar, 0.1 pm.















Figure 7. 5S RNA-gold, cytoplasm injection 15 min experiment.
Gold particles are present in the cytoplasm (C) and can be seen
extending through the nuclear pores. Only a small percentage of
particles enter the nucleus (N). Bar, 0.1 pm.



























* N



* *


, 4


~* S


0


* .*


'- *-*.*--.
4


C ."


*


'p


"


* LDq 11 *


.*-.-.









65

Discussion

Colloidal gold procedures have been used previously to identify and

characterize the regions of the pores that are involved in the transport

of karyophilic proteins across the nuclear envelope (Feldherr et al.,

1984). It was found that the transport channels are located in the

centers of the pores and have functional diameters of at least 200 A.

In this study, the same experimental approach was used to characterize

the pathways for RNA efflux and, in addition, to determine whether

individual pores have the capability of transporting both RNA and

protein.

The presence of RNA-coated gold particles within and along the

cytoplasmic surface of the nuclear pores provides direct evidence that

these tracers are translocated across the envelope at these sites.

Furthermore, almost all of the pores (97% or more) can function in the

translocation process. The biological significance of these findings;

however, is dependent on demonstrating that the pathways visualized for

the colloidal gold tracers are the same as those normally used for the

exchange of endogenous RNA. In this regard, we have attempted to

demonstrate that the translocation of RNA-gold through the nuclear

pores is both a selective and active process, and that the observed gold

distributions were not due to technical artifacts.

The fact that gold was not present in the pores of cells fixed 10

seconds after injection shows that accumulation in these structures is

not simply a result of the injection procedures per se or a

redistribution of the particles during fixation and processing for

electron microscopy. Control studies also demonstrated that the









66

injection of excess RNA does not alter the overall properties of the

pores.

Gold particles coated with synthetic polymers (PVP or polyglutamic

acid), exogenous proteins (BSA or ovalbumin) and even endogenous

karyophilic proteins (nucleoplasmin, Feldherr et al., 1984) are

essentially excluded from the pores after nuclear injection,

demonstrating that the translocation of RNA-gold is a selective process.

The data obtained with poly(I) and poly(dA) indicate that the capacity

for translocation is not necessarily restricted to physiologically

active RNAs but may be a general property of polynucleotides. The

chemical and/or physical characteristics of RNA that facilitate

translocation are not known, although a comparison of the results

obtained with poly(A) and poly(dA) suggests that the sugar moieties

could be involved. The high negative charge density of RNA does not

appear to be a major contributing factor since particles coated with

polyglutamic acid are largely excluded from the pores.

In view of the results obtained by Zasloff (1983), which showed

that the transport of labelled tRNA is markedly reduced by a single

substitution of G-to-U at position 57, one might expect a greater

degree of specificity for the translocation of polynucleotides. It

should be kept in mind, however, that Zasloff measured the overall

efflux of RNA whereas our results relate specifically to the

translocation step across the envelope. Thus, the decreased rate of

transport of the variant is not necessarily due to an effect on

translocation through the pores, but could be due to increased binding









67

within the nucleoplasm or changes in the rate of migration in the

cytoplasm.

As discussed in the introduction, there is evidence that

macromolecules larger than 90 A in diameter are unable to diffuse across

the nuclear envelope, whereas particles as large as 200 A in diameter,

which contain nuclear targeting signals, can be transported through

central channels in the pores. It was found in this study that RNA-

coated gold particles as large as 230 A in diameter (including the coat)

readily penetrate the pores. Since this far exceeds the upper limit for

diffusion, these results suggest that a transport process is involved.

Consistent with this interpretation is the finding that the accumulation

of RNA-gold in the pores is a saturable process. Saturation indicates

the presence of a carrier-mediated transport process. However,

saturation would also occur if the particles simply occupied all of the

available space within and adjacent to the pore channels. The latter

explanation, which would have little bearing on the mechanism of

exchange, is unlikely for two reasons. First, direct electron

microscopic examination of region 1 shows that these areas are not fully

occupied at saturation levels. Second, particles coated with

nucleoplasmin can reach more than twice the concentration in region 2,

than tRNA-coated gold (data not shown), demonstrating that the nature of

the coat material rather than the total number of tracer particles is

the determining factor.

Colloidal tracers, although well suited for localizing and

characterizing exchange sites, are not appropriate for detailed kinetic

studies, which require numerous time points and involve the analysis of









68

large samples. For this reason a comprehensive examination of the

temperature dependence of translocation was not attempted. However, a

cursory study was performed at 4C and at 21C, comparing the

accumulation and distribution of tRNA-coated gold particles in the

pores. After 15 minutes, the average number of particles per pore

(based on the analysis of 100 pores) at 21C and 4C was 3.8 and 1.5,

respectively. Furthermore, at 4C only 25% of the particles were

located in region 2, compared to 50% in cells kept at 21C. The

temperature effects are very likely influenced by two separate

processes, binding to receptors and movement through the pores; thus,

the results are difficult to interpret. Despite this problem, however,

the overall effect of temperature is greater than would be expected for

a physical process, such as diffusion, and is consistent with the view

that an energy requiring step is involved.

Since the translocation of gold particles through the pores is i)

dependent on the properties of the adsorbed RNA-coat, ii) likely

involved in a transport process and iii) not an artifact of the

technique, it is concluded that the pathways visualized for the

translocation of RNA-coated colloidal tracers are the same as those

normally used for endogenous RNA. These pathways are located in the

centers of the nuclear pores and have apparent functional diameters of

approximately 230 A. These results are consistent with those obtained

previously by Stevens and Swift (1966) for mRNA efflux in Chironomus

salivary gland cells.

It is not known whether tRNA and 5S RNA complex with specific

proteins prior to exiting the nucleus. There is evidence that the









69

poly(A) tails of mRNA bind different polypeptides in the nucleus and

cytoplasm (Baer and Kornberg, 1983; Sachs and Kornberg, 1985; Setyono

and Greenberg, 1981); however, it has not been determined if these

proteins are involved in transport.

The fact that over 97% of the pores are involved in RNA efflux,

combined with the earlier observation that the majority of the pores

contained nucleoplasmin-coated particles following cytoplasmic

injections (Feldherr et al., 1984), suggests that these pathways are

bifunctional. The double injection experiments provided direct evidence

supporting this view. Thus, it appears that the central channels within

the pores can function in the translocation of both RNA and protein.

However, this does not necessarily mean that the same molecular

mechanisms are employed.

Six hours after injection, the nuclear to cytoplasmic concentration

ratio of RNA-coated gold was found to be 14:1. Correcting for the

difference in the volumes of the two compartments, it is estimated that

no more than 36% of the particles entered the cytoplasm. Based on data

presented by Zasloff (1983, Table I) an equivalent amount of

radiolabelled tRNA would be expected to leave the nucleus in about 3 h.

There are several factors that could account for this difference.

First, the conformation of tRNA could be modified after adsorption to

the gold particle. In this regard, Tobian et al. (1985) found that

point mutations which alter the conformation of tRNA also decrease its

rate of transport to the cytoplasm. Second, since several tRNA

molecules are adsorbed to the gold particles (the exact number has not

been determined) the binding avidity to components within the nucleus,









70

pores or even the adjacent cytoplasm could be affected. Third, the size

of the tracers could influence the transport rates. Although RNA-coated

particles leave the nucleus at a reduced rate, it should be emphasized

that the properties of RNA required for translocation through the pores

are retained.

Zasloff (1983) and De Robertis et al. (1982) reported that

radiolabelled tRNA is unable to enter the nucleus following injection

into the cytoplasm; however, the exchange of 5S RNA across the envelope

does appear to be bidirectional (De Robertis et al., 1982). Gold

particles coated with tRNA, 5S RNA, or poly(A) were able to pass from

the cytoplasm into the nucleus, but exchange across the envelope was

greatly restricted. Even after 1 h the nuclear to cytoplasmic

concentration ratio was only about 0.01. Considering these low rates of

uptake, it was surprising to find that RNA-coated particles were

observed extending through the centers of the pores. These results

could be interpreted to mean that the translocation of RNA through the

pores is a reversible process, but that release into the nucleoplasm,

which could require a separate mechanism, might be a limiting factor.

However, before any definitive conclusion can be drawn it will be

necessary to obtain reliable quantitative data concerning the percentage

of pores that contain gold and additional information relating to the

nuclear uptake rates. Furthermore, it is not known whether

translocation is initiated by the adsorbed RNA itself or whether the

particles fortuitously bind karyophilic proteins which, in turn, induce

transport (Mattaj, 1986). Hopefully it will be possible to resolve some

of these questions using isolated nuclei.














CHAPTER III
THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE
OF TARGETING SIGNALS ON NUCLEAR UPTAKE

Introduction


The nuclear pore complex has been identified as the major, if not

the exclusive site for macromolecular diffusion and transport between

the nucleus and cytoplasm of eukaryotic cells (Feldherr et al., 1962;

Feldherr et al. 1984). Evidence for mediated protein transport was

determined initially for RN1 (Feldherr et al., 1983) and nucleoplasmin

(Dingwall et al., 1982), both of which are major karyophilic proteins

found in amphibian oocytes. From electron microscopic analysis of the

intracellular distribution of nucleoplasmin-coated gold particles, it

was established that the transport channels, located in the centers of

the pores, are at least 200 A in diameter (Feldherr, et al., 1984). In

contrast, the channel has a functional diameter of approximately 90 A

with respect to diffusion (Paine et al., 1975).

Several laboratories have utilized recombinant DNA methodology and

single amino acid substitutions to obtain probes useful for the

localization and characterization of transport signals that target

specific proteins to the nucleus. These approaches have been used to

study nuclear uptake of the following proteins; nucleoplasmin (Burglin

and De Robertis, 1987; Dingwall et al., 1987), simian virus (SV 40)

large T-antigen (Kalderon et al., 1984a,b), the yeast regulatory

proteins MATa2 (Hall et al., 1984) and GAL4 (Silver et al., 1984), yeast

71









72

histones 2A and 2B (Moreland et al., 1987), the yeast ribosomal protein

L3 (Moreland et al., 1985), polyoma large T-antigen (Richardson et al.,

1986), and the adenovirus Ela protein (Lyons et al., 1987). Although

there is no consensus signal, it appears that nuclear targeting is

dependent on short, basic amino acid sequences.

Of the proteins listed above, SV 40 large T-antigen has been

studied most extensively. Kalderon et al. (1984b) constructed hybrid

proteins by linking various amino acid sequences found in SV 40 large

T-antigen to the amino terminus of pyruvate kinase and found that the

shortest sequence capable of targeting the enzyme to the nucleus was

Pro-Lys-Lys128-Lys-Arg-Lys-Val. Transport is especially sensitive to a

point mutation at the Lys128 position (Kalderon et al., 1984a; Lanford

et al., 1986). Amino acid mutations in the vicinity of the Lys128

position reduces, but does not necessarily abolish transport of SV 40

large T-antigen into the nucleus. Roberts et al. (1987) have shown that

multiple copies of a partially defective signal can cooperate to enhance

nuclear localization.

Lanford et al. (1986) synthesized peptides that contained the SV 40

large T transport signal and cross-linked them to several carrier

proteins (ovalbumin, BSA, IgG, sIgA, ferritin and IgM). When the

conjugates were injected into the cytoplasm of cultured cells, all but

IgM (m.w. 970 kd) entered the nucleus. Qualitative evidence, utilizing

indirect immunofluorescence, suggested that the number of signals per

carrier protein can affect the rate of uptake. Goldfarb et al. (1986)

found that BSA conjugated with SV 40 large T-antigen signals accumulates









73

in the nuclei of Xenopus oocytes with saturable uptake kinetics,

suggesting the involvement of a receptor-mediated process.

Nucleoplasmin, a 110 kd pentameric karyophilic protein (as

determined by sequence analysis), also has been used extensively to

study nuclear transport in oocytes (Dingwall et al., 1982; Feldherr et

al., 1984) and cultured cells (Schulz and Peters, 1986; Sugawa et al.,

1985). It has recently been determined that each monomeric subunit

contains 1 targeting signal. Furthermore, removal of one or more signal

domains by proteolytic cleavage markedly reduces the rate of nuclear

uptake (Dingwall et al., 1982). The region containing the signal has

been sequenced and although it is similar to the SV 40 targeting signal,

it is not identical (Dingwall, personal communication).

The data summarized above suggest that variations in the nuclear

targeting signal could significantly influence nucleocytoplasmic

exchange of proteins. To better understand the nature of these effects,

experiments were performed to determine how macromolecules containing

different signal sequences and different numbers of signals interact

with, and modulate the properties of the nuclear pores. Various size

colloidal gold particles were coated with 1) BSA conjugated with

different numbers of synthetic peptides containing the SV 40 targeting

signal, 2) BSA conjugated with inactive SV 40 signals, 3) large

T-antigen, or 4) nucleoplasmin. These tracers were microinjected into

oocytes and their distribution within the oocytes was later determined

by electron microscopy.

The data indicate that as the number of signals per molecule

increases, both the relative uptake of the tracer particles into the









74

nucleus and the functional size of the transport channels increase. The

SV 40 and nucleoplasmin targeting sequences varied in their ability to

facilitate transport. This could be related to differences in their

binding affinity for nuclear envelope (pore) receptors. Double

labelling experiments demonstrated that different targeting signals can

be recognized by and transported through the same pore. Control

experiments ruled out the possibilities of nonspecific exchange

processes.



Materials and Methods

Xenopus laevis were purchased from Xenopus I (Ann Arbor, Michigan)

and maintained as reported previously (Feldherr, 1975).



Colloidal gold coating agents

The synthesis of peptides containing the SV 40 large T-antigen

targeting signals and their conjugation to BSA was described in detail

by Lanford et al. (1986). In brief, the thirteen amino acid sequence

was synthesized on a glycyl-Merrifield resin (Merrifield, 1963) using a

Biosearch Sam Two (Biosearch, San Rafael, CA) automated peptide

synthesizer. The synthetic peptides were conjugated to BSA using the

heterobifunctional cross-linking agent m-maleimido benzoyl-N-

hydroxysuccimide ester (MBS; Pierce Chemical Co., Rockford, IL).

Dialysis in phosphate-buffered saline followed by repeated concentration

and dilution of the conjugates separated the unconjugated peptides from

the carrier proteins. By varying the molar ratios of the reactants,

different protein-peptide coupling ratios were obtained. Amino acid









75

analyses were performed with a Beckman Model 7300 analyzer (Beckman

Instruments Inc., Palo Alto, CA) to obtain a number-average ratio of

signal peptides to carrier protein BSA. The peptide to carrier protein

ratios obtained by SDS-PAGE were similar to those calculated by amino

acid analysis.

Large T-antigen was expressed in insect cells using the baculovirus

expression vector system (Luckow and Summers, 1988) and was purified by

immunoaffinity chromatography as described previously (Siamins and Lane,

1985). Nucleoplasmin was isolated using an anti-nucleoplasmin IgG

affinity column as described previously (see Chapter II, Materials and

Methods). BSA used in the control experiments was purchased from Sigma

Chemical Co. (St. Louis, MO).

The BSA conjugates used as coating agents are listed in Table VI.

The table shows the synthetic 13 amino acid sequence conjugated to BSA

and the average number of peptides per BSA molecule. The synthetic

peptide contains the seven amino acid sequence required for nuclear

localization of the SV 40 large-T antigen (Lanford et al., 1986). The

conjugates denoted BSA-WT represent preparations that contain active

nuclear targeting signals. In the BSA-cT conjugates, neutral asparagine

was substituted for the second lysine (equivalent to Lys128 in SV 40

large T-antigen) in the synthetic peptide (see Table VI). The resulting

signal is similar to the mutation present in the SV 40(cT)-3 mutant

(Lanford and Butel, 1984), and is defective in transport. To obtain a

coating agent containing an average of 3 signals per carrier molecule,

the BSA-WT8 conjugate was diluted 3-fold with the BSA-cT7 preparation

prior to stabilizing the gold particles. The use of BSA-cT7 to dilute































4:
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76





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s- f.

0CL 0.












-4 c


CL
0)
01 C
4-' E
c U)
10 10
I ,--

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10 3


co
C/i
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77

the active signal assured that the overall properties (shape and size)

of the particles were consistent with those of other tracers used in

this investigation. Purified SV 40 large T-antigen, which has 1 signal

per monomer, and nucleoplasmin, which has a different signal sequence,

also were used as coating agents. BSA alone was employed as an

additional control.



Preparation and stabilization of colloidal gold

The colloidal gold fractions that contained particles 20-50 A and

20-160 A in diameter were both prepared by reducing chloroauric acid

with a solution of white phosphorus in ether (Feldherr, 1965). 50-280 A

gold particles were obtained by adding 2.5 ml of 0.6% gold chloride to a

freshly prepared 20-160 A fraction, 1 ml of additional reducing agent

was then added, and the preparation was boiled for 2-3 minutes.

Fractions containing 120-280 A particles were prepared by reducing

chloroauric acid with trisodium citrate, as described previously (Frens,

1973). The 50-280 A and 120-280 A gold sols, were brought to pH 7.0

with 0.72 N K2CO3.

Prior to stabilization, all of the coating agents were either

dialyzed against, or dissolved in, a low ionic strength buffer (7.2 mM

K2HPO4 and 4.8 mM KH2PO4, pH 7.0). The volumes of the different agents

required to stabilize 1 ml of the gold sols are listed in Table VII.

The procedure used to determine these volumes is outlined in Feldherr et

al. (1984).

After stabilization, the 20-160 A and 50-280 A preparations were

centrifuged at 2000g at 4C for 10 minutes to remove any aggregates of





























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79

gold. Centrifugation of the 20-50 A and 120-280 A fractions was not

necessary. In each instance, 5-7 ml of stabilized colloid were

concentrated to 70-100 pl in Minicon concentrators (Amicon Corp.,

Danvers, MA) and dialyzed against intracellular injection medium (102 mM

KC1, 11.1 mM NaCl, 7.2 mM K2HPO4 and 4.8 mM KH2PO4, pH 7.0) at 4C prior

to injection.



Injection

Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually

defolliculated in amphibian Ringer's solution and centrifuged at

approximately 650g for 8-10 minutes (Kressman and Birnstiel, 1980). The

cells were then microinjected with approximately 40 nl of gold sol at a

site adjacent to the nucleus and fixed at intervals of 15 min, 1 h, 6 or

20 h. The tip diameters of the micropipettes were 10-15 pm.



Electron microscopy and analysis

The cells were fixed for electron microscopy and prepared for

sectioning as described previously (see Chapter 2, Materials and

Methods). Relative nuclear uptake of gold particles stabilized with the

different coating agents was determined by counting particles in equal

and adjacent areas of the nucleus and cytoplasm close to the site of

injection. Yolk granules and mitochondria were excluded from the

analyses. The counts are reported as nuclear to cytoplasmic ratios

(N/C). The size distributions of particles present within the nucleus

and cytoplasm were determined by direct measurement from electron

micrograph negatives. The envelope to cytoplasm ratios were obtained by









80

comparing the number of particles associated with the cytoplasmic

surface of the envelope (i.e., at or within 650 A of the nuclear

surface) to the number of particles in an equal, randomly selected area

of cytoplasm.

Negative staining with 1% phosphotungstic acid was used to estimate

the thickness of the adsorbed protein coats.

By extrapolating from data published by De Roe et al. (1984) and

correcting for additional mass contributed by the peptides, estimates

were made of the number of BSA molecules adsorbed onto the surfaces of

different size gold particles. For example, particles with diameters of

35 A, 80 A, 140 A, and 180 A (the mean size of the 4 different gold

preparations injected) would have 2, 8, 24, and 39 molecules of BSA

conjugates, respectively. Knowing the number of BSA molecules adsorbed

and the synthetic peptide to carrier protein ratios, it was possible to

estimate the total number of SV 40 large T-antigen targeting signals on

the different size gold particles; however, the proportion of signal

actually available for transport (i.e., exposed signals) is not known.



Results

Cytoplasmic injections of the tracer particles

It was determined initially that microinjection of approximately

40 nl of stabilized gold adjacent to the nucleus delivered a sufficient

number of particles for electron microscopic analysis. The protein

content of the injectate ranged from 50-300 ng depending on the size of

the gold fraction and the specific coating agent used.









81

All preparations containing active transport signals (BSA-WT

conjugates, large T-antigen or nucleoplasmin) were translocated into the

nucleus through central channels located within the nuclear pores. In

the region of injection, the particles were uniformly distributed in the

cytoplasm; however, at longer time intervals, 6 and 20 h, the BSA-WT

conjugates occasionally formed aggregates. The reason for this is not

known, but it appeared to be dependent on the presence of active signals

since similar aggregates were not observed with BSA-cT conjugates. At

all time intervals, particles containing active transport signals were

present both within the pores and the nucleoplasm. With increasing time

there was a concomitant increase in the number of particles present in

the nucleus (data not shown). Gold coated with the BSA-cT conjugates or

BSA alone were essentially excluded from the pores and nucleoplasm.

These general distributions are illustrated in Figs. 8 and 9, which show

the results obtained 1 h after injecting particles coated with BSA-WT11

and BSA-cT7, respectively.



Relative uptake

Quantitative data, dependent on the specific properties of the

individual coating agents, were obtained 1 h after injection. This time

interval allowed for sufficient particle distribution within the

cytoplasm, and minimized possible loss or redistribution of soluble cell

components caused by the injection procedures (Miller et al, 1984). The

N/C ratios obtained at 1 h do not represent equilibrium values since

nuclear uptake was observed to increase with time, but reflect relative
























Figure 8. BSA-WT11-gold. 1 h experiment.
Gold particles (20-160 A), near the site of injection, are observed
evenly distributed throughout the cytoplasm (C). Particles are seen
passing through the centers of the pores and also within the nucleus
(N). Bar, 0.1 Pm.













Figure 9. BSA-cT7-gold 1 h experiment.
Tracer particles (20-50 A), near the site of injection, are distributed
evenly throughout the cytoplasm (C), but are rarely seen within the
nucleus (N) or along the envelope. Bar, 0.1 pm.













83
















N





3 . "* ... -. .
V. ". A



J...
c" *



* ' 'I t. *' ** __^ ' -v -

;' ~ ~~~~~~ ~ '.' .: I;
8 .-
8Q








N












S'
St.. *

9









84

uptake of the tracer particles. Four to 6 cells were examined for each

coating agent within each size fraction.

The nuclear to cytoplasmic (N/C) ratios for different coating

agents are given in Table VIII. After 1 h, N/C ratios obtained with the

20-50 A fraction stabilized with BSA-WT5 and BSA-WT8 were 0.58. and 0.76,

respectively. The difference in these ratios, as determined by the

Student's t-test, is not statistically significant (p>0.25). When the

particle size was increased to 20-160 A or 50-280 A in diameter, the

differences in N/C ratios between the BSA-WT5 and BSA-WT8 conjugates

were highly significant (p<0.002). In addition, BSA-WT5 was more

effective in facilitating transport than large T-antigen which contains

1 signal per monomer (p<0.002). However, increasing the number of

signals from 8 (BSA-WT8) to 11 (BSA-WT11) did not significantly increase

the N/C ratio (p values obtained for the 20-160 A and 120-280 A

fractions were p>0.8 and 0.1>p>0.05, respectively).

The N/C ratio obtained for the BSA-WT8-cT7 dilution (approximately

3 signals per BSA molecule) was significantly lower than that obtained

for the BSA-WT5 conjugate (p<0.002); however, it was also significantly

lower than that observed for large T-antigen-gold (0.01
could be due to variation in the availability and/or spatial

distribution of the signal at the surface of the gold particles.

The N/C values for BSA-cT7-, BSA-cT13-, and BSA-coated gold were

significantly lower than all gold preparations containing active nuclear

targeting signals (p
Overall, it is concluded from these results that there is a direct

correlation between the number of transport signals and the relative












85











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86

uptake of particles into the nucleus. Furthermore, the data suggest

that as the size of the particles increases, more signals are required

for their transport across the envelope.

To compare the effectiveness of a different targeting signal,

parallel studies were performed with nucleoplasmin-gold. The relative

uptake of different size nucleoplasmin-coated particles by the nucleus

is shown in Table VIII. The N/C ratios determined for nucleoplasmin-

coated particles were significantly greater than those obtained for BSA-

WT5-, BSA-WT8-, or BSA-WT11-coated gold. In all instances, the

probability values were p<0.002. Since there are only 5 targeting

signals per nucleoplasmin molecule, these differences in uptake suggest

that the nucleoplasmin transport signal is more effective than the SV 40

large T-antigen targeting sequence, at least in oocytes.



Size distributions

In view of the above results, the size distributions of particles

in different regions of the oocytes were analyzed in more detail. The

nuclear and cytoplasmic size distributions determined for BSA-WT5 and

BSA-WT8 1 h after injection are given in Table IX and Fig. 10.

Seventeen percent of the BSA-WT8-coated particles that entered the

nucleus were larger than 157 A, compared to 5.6% for particles coated

with BSA-WT5. When particles larger than 185 A are compared, the

difference in uptake is almost 10-fold. The difference in the size of

the particles able to penetrate the pores is statistically significant

as determined by Chi-square analysis.

















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Full Text
REFERENCES
Aaronsen, R.P., and G. Blobel. 1975. Isolation of nuclear pore
complexes in association with a lamina. Proc. Natl. Acad. Sci. USA
72:1007-1011.
Abelson, P.H., and W.R. Duryee. 1949. Radioactive sodium permeability
and exchange in frog eggs. Biol. Bui. 96:205-217.
Agutter, P.S. 1985a. RNA processing, RNA transport and nuclear
structure. Ijn Nuclear Envelope Structure and RNA Maturation.
UCLA Symposia on Molecular and Cellular Biology. E.A. Smuckler
and G.A. Clawson, editors. Vol. 26. Alan R. Liss, Inc., New
York. 539-559.
Agutter, P.S. 1985b. Nuclear Envelope NTPase and RNA efflux. Iji
Nuclear Envelope Structure and RNA Maturation. UCLA Symposia on
Molecular and Cellular Biology. E.A. Smuckler and G.A. Clawson,
editors. Vol. 26. Alan R. Liss, Inc., New York. 561-578.
Agutter, P.S., J.R. Harris, and I. Stevenson. 1977. RNA stimulation
of mammalian liver nuclear envelope nucleoside triphosphatase. A
possible marker for the nuclear envelope. Biochem. J. 162:671-
679.
Agutter, P., H. McArdle, and B. McCaldin. 1976. Evidence for the
involvement of nuclear envelope NTPase in nucleocytoplasmic
translocation of ribonucleoprotein. Nature (Lond.) 263:165-167.
Agutter, P.S., B. McCaldin, and H.J. McArdle. 1979. Importance of
mammalian nuclear-envelope nucleoside triphosphatase in
nucleocytoplasmic transport of ribonucleoproteins. Biochem. J.
182:811-819.
Austerberry, C.F., and P.L. Paine. 1982. In vivo distribution of
proteins within a single cell. Clin. Chem. 28:1011-1014.
Baer, B., and R.D. Kornberg. 1983. The protein responsible for the
repeating structure of cytoplasmic poly(A)-ribonucleoprotein. vh
Cell Biol. 96:717-721.
117


113
permeability properties of the nuclear pores and/or ii) altering the
number of pores per envelope. It has been shown in amoebae that the
nuclear pores have some variability with respect to diffusion that
occurs during different stages of the cell cycle (Feldherr, 1966). In
addition, it was demonstrated that there are changes in the functional
properties of the pores during different metabolic states of the amoebae
and the differences in uptake were not due to a change in pore number or
size (Feldherr, 1971).
Maul et al. (1972) have demonstrated that pore formation in HeLa
cells during the cell cycle is biphasic. They found two significant
increases in pore formation during mitosis, the first being within an
hour after division and the second, shortly after the beginning of
S phase. Furthermore, they found that the number of pores per nucleus
can vary with a change in cellular activity. Thus, the cell, in
principle, can regulate the rate of accumulation by changing the number
of pores available for nucleocytoplasmic exchange.
In contrast to diffusion (see above), a question that remains to be
investigated is whether the transport properties of the pores change
significantly during the cell cycle and/or during changes in cellular
activity. Dreyer et al. (1985) have suggested that the temporal
accumulation of different proteins can be due to the nature and number
of the transport signals. However, as mentioned above, pore variability
can be dependent on the physiological state of the cell. Since the
proteins do not undergo posttranslational modification and they
distribute differently at various times throughout development, they
might be responding to changes in the envelope. At present, it is not


27
length of 77 A and an average thickness of 20 A (Kim et al., 1973). A
Stokes's radius of 34 A was derived for tRNAP*16 from its translational
diffusion coefficient in water (Peters, 1986). A molecule having these
dimensions will not readily diffuse through the nuclear pores.
Injection of the cloned tRNAtyr yeast gene into the nuclei of oocytes
produces a precursor transcript with a length of 108 nucleotides (Melton
et al., 1980). After processing, mature tRNA (78 nucleotides long) is
then exported into the cytoplasm. De Robertis et al. (1982) injected
labelled tRNA into the cytoplasm of oocytes and showed it to be excluded
from the nucleus, suggesting that tRNA efflux is vectorial
(unidirectional).
Zasloff (1983) studied tRNA transport in amphibian oocytes. This
is a convenient system since oocytes can transcribe genes that have been
injected into the nucleus. In addition, analysis is facilitated by the
fact that nucleus and cytoplasm can be readily separated. Two genes
were used in this study; the first was normal human tRNAmet and the
second was a variant human tRNAmet with a G-to-U substitution at
position 57 in the highly conserved loop IV region. Nuclei were
injected with either the genes encoding for the two tRNAs or prelabelled
tRNAs, and the cells were fractionated at various times thereafter. It
was found by electrophoretic analysis of both the nucleus and cytoplasm
that 80% of the normal tRNA entered the cytoplasm within 5 min; in
contrast, 90% of the variant tRNA remained intranuclear after 15 min.
Precise kinetic analysis, performed by liquid scintillometry, showed the
rate of transport of the variant tRNA to be 20-fold slower than normal
tRNA. In addition, normal tRNA transport is saturable and displays


129
van Eekelen, C.A.G, and W.J. van Venrooij. 1981. hnRNA and its
attachment to a nuclear protein matrix. J. Cel 1 Biol. 88:554-563.
Warren, P., and B. Dobberstein. 1978. Protein transfer across
microsomal membranes reassembled from separated membrane
components. Nature (Lond.) 273:569-571.
Watson, M.L. 1955. The nuclear envelope: Its structure and relation
to cytoplasmic membranes. J. Biophysic. Biochem. Cytol. 1:257-279.
Watson, M.L. 1959. Further observations on the nuclear envelope of
the animal cell. J. Biophysic. Biochem. Cytol. 6:147-171.
Wischnitzer, S. 1958. An electron microscope study of the nuclear
envelope of amphibian oocytes. J. Ultrastr. Res. 1:201-222.
Wunderlich, F., G. Giese, and H. Falk. 1983. In vitro nuclear
transport of ribosomal ribonucleoprotein: Temperature affects
quantity but not quality of exported particles. Mol. and Cel 1.
Biol. 3:693-698.
Wychowski, C., D. Benichou, and M. Girard. 1986. A domain of SV40
capsid polypeptide VPl that specifies migration into the cell
nucleus. EMBO J. 5:2569-2576.
Yoneda, Y., T. Ariuka, N. Imamoto-Sonobe, H. Sugawa, Y. Shimonishi, and
T. Uchida. 1987a. Synthetic peptides containing a region of SV40
large T-antigen involved in nuclear localization direct the
transport of proteins into the nucleus. Exp. Cell Res. 170:439-
452.
Yoneda, Y., N. Imamoto-Sonobe, M. Yamaizumi, and T. Uchida. 1987b.
Reversible inhibition of protein import into the nucleus by wheat
germ agglutinin injected into cultured cells. Exp. Cell Res.
173:586-595.
Zasloff, M. 1983. tRNA transport from the nucleus in a eukaryotic
cell: Carrier-mediated translocation process. Proc. Natl. Acad.
Sci. USA 80:6436-6440.
Zeller, R., T. Nyffenegger, and E.M. De Robertis. 1983.
Nuceocytoplasmic distribution of snRNPs and stockpiled snRNA-
binding proteins during oogenesis and early development in Xenopus
laevis. Cell 32:425-434.


% of Total Nuclear Particles
60
50 -
30 -
BSA-WT-8
BSA-WT-11
Nucleoplasmin
20 -
10 ^
120-145
145-170
170-195
195-220
1
220-245 >245
40 -
Particle Size (A)
Figure 11. Nuclear particle distributions: BSA-WTg, BSA-WT^, and nucleoplasmin
o


109
within the synthetic peptide signal on protein transport. They found
the rate of transport decreased when different basic amino acids were
substituted in the lysine 128 position, and that transport activity was
abolished when neutral asparagine was used. Based on these findings, I
would propose that a change of amino acids within the signal sequence
can alter the affinity of the signal for the receptor, resulting in
slower uptake.
Proposed Model
The transport channel is not a fixed structure since it can
fluctuate between 90 A (patent diffusion channel) and 260 A (maximum
size of the transport channel) in diameter, the functional diameter
being dependent on the number of binding events between receptors and
transport signals. There appear to be at least two factors involved in
the variability of the transport channel: the signal number and
affinity. From this data, I would propose that the mechanism for
protein transport through the nuclear pores of oocytes occurs by a gated
process. Normally, in an unactivated state, the channel within the pore
would be 90 A in diameter and available for diffusion into and out of
the nucleus. However, activation by transport signals would result in
the dilation of the transport channel to an extent which is directly
related to the number of active signals bound to the receptors at or
within the pore complex. Furthermore, a small number of high affinity
binding signals might be as effective in regulating the size of the
transport channel as a larger number of low affinity signals.
Alternatively, the dilation of the pore might be an all or none process


75
analyses were performed with a Beckman Model 7300 analyzer (Beckman
Instruments Inc., Palo Alto, CA) to obtain a number-average ratio of
signal peptides to carrier protein BSA. The peptide to carrier protein
ratios obtained by SDS-PAGE were similar to those calculated by amino
acid analysis.
Large T-antigen was expressed in insect cells using the baculovirus
expression vector system (Luckow and Summers, 1988) and was purified by
immunoaffinity chromatography as described previously (Siamins and Lane,
1985). Nucleoplasmin was isolated using an anti-nucleoplasmin IgG
affinity column as described previously (see Chapter II, Materials and
Methods). BSA used in the control experiments was purchased from Sigma
Chemical Co. (St. Louis, MO).
The BSA conjugates used as coating agents are listed in Table VI.
The table shows the synthetic 13 amino acid sequence conjugated to BSA
and the average number of peptides per BSA molecule. The synthetic
peptide contains the seven amino acid sequence required for nuclear
localization of the SV 40 large-T antigen (Lanford et al., 1986). The
conjugates denoted BSA-WT represent preparations that contain active
nuclear targeting signals. In the BSA-cT conjugates, neutral asparagine
was substituted for the second lysine (equivalent to Lys12 in SV 40
large T-antigen) in the synthetic peptide (see Table VI). The resulting
signal is similar to the mutation present in the SV 40(cT)-3 mutant
(Lanford and Bute!, 1984), and is defective in transport. To obtain a
coating agent containing an average of 3 signals per carrier molecule,
the BSA-WT3 conjugate was diluted 3-fold with the BSA-cTy preparation
prior to stabilizing the gold particles. The use of BSA-cTy to dilute


NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES
By
STEVEN IRA DWORETZKY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988


5
Discrete subunits, 100-250 A in diameter, are distributed evenly
around both the cytoplasmic and nuclear margins of the pore (Watson,
1955; Wischnitzer, 1958). Franke (1966) using a multiple rotational
exposure technique on negative-stained preparations of isolated
envelopes, describes the arrangement of annular globules (granules) to
be in eight-point radial symmetry. Furthermore, the subunits lying on
the pore margin within the nucleus overlap the same sites as subunits on
the cytoplasmic pore margin.
Within the centers of some pores, an electron-dense granule with
diameters ranging from 40-350 A can be observed. This central granule
has been visualized with different preparative techniques; however, the
observed frequency of its presence is highly variable (Franke and
Scheer, 1970). It has been suggested that the granule is actually
material in transit, e.g. ribonucleoprotein (RNP), and that a
correlation exists between the frequency of the central granule and the
activity of RNA synthesis (Scheer, 1970). Cytochemical studies
performed by Franke and Falk (1970) support this view. Even with this
data it still remains controversial whether the granule is a permanent
structural component of the pore complex or material passing through the
pore.
Thin filaments, 25-50 A in diameter, have also been observed in the
interior of the pore. These filaments radiate laterally from the pore
margins and appear to connect with the central granule (Franke, 1974).
The filaments have a spokelike radial appearance and exhibit eightfold
symmetry similar to that of the annular globules.


60
Fewer than 4% of the 200 pores analyzed were involved in translocation.
Thus, the injection of RNA in amounts greater than those normally used
in this investigation, have no apparent effect on the physical
properties of the pores.
Concentration dependence of RNA translocation
To determine the concentration dependence of the translocation
process, the gold distribution in the pores was analyzed in areas in
which different concentrations of colloid were present in the adjacent
nucleoplasm. These experiments were performed with tRNA-coated gold
particles, and the cells were fixed after 1 h. To ensure that an
appropriate concentration range was obtained, different dilutions of
colloid were injected. The amount of tRNA in the injectate varied from
6-30 ng. The results are shown in Table V, and are based on the
examination of 6 oocytes. It can be seen that a maximum number of
particles per pore is obtained at a concentration of 90
particles/0.36pm^. Above this concentration, no differences were
observed either in the numbers or distribution of the particles,
suggesting that the translocation process is saturable.
Double label experiment
The high percentage of pores involved in RNA translocation
suggested that each pore may be a bidirectional channel, capable of both
protein and RNA transport. To address this possibility directly, large
gold fractions (120-220 A) coated with nucleoplasmin and small gold
fractions (20-50 A) stabilized with tRNA were used. The tRNA-coated


Figure 13. Coinjection of gold particles coated with BSA-conjugates
and nucleoplasmin 1 h experiments.
13a and 13b, large nucleoplasmin-coated particles (120-280 A) and small
BSA-WT8-coated particles (20-50 A) can both be seen within the pore
region or just at the nuclear surface of the pores. 13c, large
nucleoplasmin-coated particles are seen entering the nucleus (N) while
small BSA-cTycoated particles are retained in the cytoplasm (C).
Bar, 0.1 pm.


124
Lanford, R.E., P.. Kanda, and R.C. Kennedy. 1986. Induction of
nuclear transport with a synthetic peptide homologous to the SV40 T
antigen transport signal. Cel 1 46:575-582.
Lanford, R.E., R.G. White, R.G. Dunham, and P. Kanda. Effects of basic
and nonbasic amino acid substitutions on transport induced by SV40
T antigen synthetic peptide nuclear transport signals. (Submitted
for publication).
Luckow, V.A., and M.D. Summers. 1988. Trends in the development of
bacolovirus expression vectors. Bio/Technology 6:47-55.
Lyons, R.H., B.Q. Ferguson, and M. Rosenberg. 1987. Pentapeptide
nuclear localization signal in adenovirus Ela. Mol. Cell. Biol.
7:2451-2456.
Mattaj, I.W. 1986. The role of RNA-Protein interactions in
intracellular targeting. In Nucleocytoplasmic Transport. R.
Peters and M. Trendelenburg, editors. Springer-Verlag, Berlin.
275-285.
Mattaj, I.W., and E.M. De Robertis. 1985. Nuclear segregation of U2
snRNA requires binding of specific snRNP proteins. Cel 1
40:111-118.
Mattaj, I.W., S. Lienhard, R. Zeller, and E.M. De Robertis. 1983.
Nuclear exclusion of transcription factor 111A and the 42S particle
transfer RNA-binding protein in Xenopus oocytes: A possible
mechanism for gene control? J. Cell Biol. 97:1261-1265.
Maul, G.G. 1977. The nuclear and the cytoplasmic pore complex:
Structure, dynamics, distribution, and evolution. Int. Rev.
Cytol. Suppl. 6:75-186.
Maul, G.G., and F.A. Baglia. 1983. Localization of a major nuclear
envelope protein by differential solubilization. Exp. Cell Res.
145:285-292.
Maul, G.G., H.M. Maul, J.E. Scogna, M.W. Lieberman, G.S. Stein, B.Y.
Hsu, and T.W. Borun. Time sequence of nuclear pore formation in
phytohemagglutinin-stimulated lymphocytes and in HeLa cells during
the cell cycle. J. Cell Biol. 55:433-447.
McDonald, J.R., and P.S. Agutter. 1980. The relationship between
polyribonucleotide binding and the phosphorylation and
dephosphorylation of nuclear envelope protein. FEBS Lett. 116:145-
148.


APPENDIX
Top View
Side View
A schematic representation of the nuclear pore complex based on
data from Franke's (1974) and Unwin and Milligan's (1982) model. G,
annular globules; S, spokes; C, central granule.
116


121
Feldherr, C.M., and J.A. Ogburn. 1980. Mechanism for the selection of
nuclear polypeptides in Xenopus oocytes. II. Two-dimensional gel
analysis. J. Cell Biol. 87:589-593.
Feldherr, C.M., and J. Pomerantz. 1978. Mechanism for the selection
of nuclear polypeptides in Xenopus oocytes. J. Cell Biol. 78:168-
175.
Fey, E.G., D.A. Ornelles, and S. Penman. 1986. Association of RNA
with the cytoskeleton and the nuclear matrix. J. Cell Sci.
5(Suppl.):99-119.
Finlay, D.R., D.D. Newmeyer, T.M. Price, and D.J. Forbes. 1987.
Inhibition of in vitro nuclear transport by a lectin that binds to
nuclear pores. J. Cell Biol. 104:189-200.
Forbes, D.J., M.W. Kirschner, and J.W. Newport. 1983. Spontaneous
formation of nucleus-like structures around bacteriophage DNA
microinjected into eggs. Cel 1 34:13-23.
Ford, P.J. 1971. Non-coordinated accumulation and synthesis of 5S
ribonucleic acid by ovaries of Xenopus laevis. Nature (Lond.)
233:561-564.
Frank, M., and S.B. Horowitz. 1975. Nucleocytoplasmic transport and
distribution of an amino acid, i_n situ. J. Cell Sci. 19:127-139.
Franke, W.W. 1966. Isolated nuclear membranes. J. Cell Biol. 31:
619-623.
Franke, W.W. 1974. Structure, biochemistry, and functions of the
nuclear envelope. Int. Rev. Cytol. Suppl. 4:71-236.
Franke, W.W., and H. Falk. 1970. Appearance of nuclear pore complexes
after Bernhard's staining procedure. Histochemie 24:266-278.
Franke, W.W., and U. Scheer. 1974. Pathways of nucleocytoplasmic
translocation of ribonucleoproteins. Symp. Soc. Exp. Biol.
XXVIII:249-282.
Frens, G. 1973. Controlled nucleation for the regulation of the
particle size in monodisperse gold suspensions. Nature Physical
Science 241:20-22.
Gerace, L., and G. Blobel. 1982. Nuclear lamina and the structural
organization of the nuclear envelope. Cold Spring Harbor Symp.
Quant. Biol. 46:967-978.


4
are different, although they have not been analyzed by biochemical
methods.
As visualized by electron microscopy, the nuclear lamina is a
discrete layer of material, varying in thickness from 30-100 nm, lining
the entire inner surface of the nuclear envelope, excluding the pore
complexes. A lamina enriched fraction is obtained by non-ionic
detergent and high salt extraction of isolated envelopes (Aaronsen and
Blobel, 1975; Dwyer and Blobel, 1976). Protein analysis of the residual
fraction shows the presence of three predominant proteins, referred to
as lamins A, B, and C. They comprise approximately 40% of this
fraction. During interphase, these polypeptides have been localized, by
immunocytochemistry, exclusively to the lamina at the nuclear periphery
(Gerace et al., 1978). Gerace and Blobel (1982) have shown that the
lamina forms a fibrous network that is a component of the nuclear
skeleton. The lamina has been implicated to function as a structural
framework for the nuclear envelope and an anchoring site at the nuclear
periphery for chromatin. In addition to its structural role, the lamina
is thought to be involved in reversible nuclear envelope assembly and
disassembly during mitosis (Gerace et al., 1984).
The pores are filled with diffuse electron-opaque material that
extends a short distance into both the nucleus and cytoplasm. In
tangential sections, this electron-opaque material has a ring-like
appearance and has been referred to as the annular material (Watson,
1955; Wischnitzer, 1958). The pore with its associated annulus is
called the pore complex.


110
rather than a channel opening incremently. Although the protein uptake
data favors the latter, the two mechanisms cannot be distinguished at
this time.
Consistent with the gating model are the results presented by
Newmeyer and Forbes (1988) and Richardson et al. (1988). They show that
the transport of proteins across the envelope i_n vitro can be
experimentally separated into at least two steps. The first step is
the binding of the proteins to the cytoplasmic pore surface which
involves the specific recognition of the transport signal by the nuclear
pore complex. Binding to this region is signal sequence dependent, thus
again demonstrating selectivity by the pore complex, and ATP
independent. The second step is the ATP dependent translocation through
the pores.
According to the model, multiple interactions need occur prior to
distention of the transport channel. If so, then multiple copies of
receptors should reside in the pore complex. Components speculated to
be part of the translocation machinery are the group of glycoproteins
localized to the pore complex by Snow et al. (1987), Finlay et al.
(1967), and Davis and Blobel (1986). Although these glycoproteins have
not been demonstrated to function as receptors, they are present in
multiple copies within each pore complex, consistent with the gating
model. Furthermore, in an attempt to define the role of the
glycoproteins in protein transport it was shown that WGA binds to the
pore region and inhibits the uptake of nucleoplasmin in synthetic nuclei
(Finlay et al., 1987) and cultured cells (Yoneda et al., 1987), but does
not block the diffusion channel.


Figure 8. BSA-WTij-gold. 1 h experiment.
Gold particles (20-160 A), near the site of injection, are observed
evenly distributed throughout the cytoplasm (C). Particles are seen
passing through the centers of the pores and also within the nucleus
(N). Bar, 0.1 pm.
Figure 9. BSA-cTygold 1 h experiment.
Tracer particles (20-50 A), near the site of injection, are distributed
evenly throughout the cytoplasm (C), but are rarely seen within the
nucleus (N) or along the envelope. Bar, 0.1 pm.


Table III Size Distribution of Gold Particles Present in the Nuclei and Pores
Total No. % of Particles in Each Size Class*
Experiment of Particles 20-40 40-60 60-80 80-100 100-120 120-140 140-160 160-180 180-200 >200
(1 h) Measured A A A A A A A A A A
tRNAmet
Nuclei**
486
0.3
27.9
51.5
12.8
4.2
1.5
1.5
--
--
--
Pores
462
0.4
22.1
52.6
15.4
6.1
1.3
2.1


5S RNA
Nuclei
1110
3.2
35.8
48.4
10.0
1.8
0.45
0.36
--
--
--
Pores
773
2.6
37.0
48.4
10.3
1.3
0.25
0.15



Poly (A)
Nuclei
906





6.6
35.5
43.8
9.8
4.2
Pores
593
--




5.2
37.3
42.0
13.6
1.9
* Particle dimensions do not include the thickness of the coat. Negative staining indicated that the coat
thickness adds 30-40 A to the overall diameters of the particles.
** Particles present within the nuclei.
*** Total number of particles present within the pores and in the cytoplasm immediately adjacent to the
pores.


Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES
By
STEVEN IRA DWORETZKY
August 1988
Chairman: Carl M. Feldherr
Major Department: Anatomy and Cell Biology
To localize and characterize the pathways for RNA translocation to
the cytoplasm, gold particles coated with tRNA, 5S RNA, or poly(A) were
microinjected into the nuclei of Xenopus oocytes. At various times
after injection, cells were fixed and the particle distributions
analyzed by electron microscopy. Similar results were obtained with all
RNAs used. Particles ranging from 20-230 A in diameter were observed
within central channels of the nuclear pores and in the adjacent
cytoplasm. Particles larger than 90 A would not be expected to diffuse
readily through the pores, suggesting that mediated transport occurred.
Furthermore, approximately 97% of the pores analyzed were involved in
the translocation of gold particles. After nuclear injection, particles
coated with exogenous macromolecules were essentially excluded from the


Table VI Protein Preparations Used as Coating Agents
Coating Agent
BSA
BSA-WT5
BSA-WT8
BSA-WTn
Average No.
of Signals Sequence Conjugated to BSA
0
5
8
11
Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arq-Lys-Val-Gly-Gly
BSA-cTy
BSA-cT13
BSA-WTg + cT7*
Large T-antigen
Nucleoplasmin
7
13
3
1 per monomer
1 per monomer
Cys-Gly-Tyr-Gly-Pro-Lys-Asn-Lys-Arg-Lys-Val-Gly-Gly
The underlined sequence represents the active (WT preparations) and inactive (cT preparations)
SV 40 large T nuclear transport signal.
*The average number of signals obtained for this coating agent was performed by diluting
BSA-WT8 with BSA-cTy.


101
obtained evidence that transport involves two separate events; the first
is binding to the pores, which is signal sequence dependent, and the
second is translocation into the nucleus, which is ATP dependent.
Overall, the data obtained in this study are consistent with the view
that transport occurs through the pores by a gated process. I would
suggest that a 90 A channel is normally present within the pores
allowing for the diffusion of smaller macromolecules into and out of the
nucleus (Paine, 1975). However, in response to an appropriate transport
signals, the dimensions of the channel can increase in size to
accommodate the uptake of transportable (nondiffusive) macromolecules.
The results obtained with nucleoplasmin and the BSA conjugates
indicate that the extent of channel dilation might be variable and
dependent on the number of simultaneous interactions between signals and
receptors. Thus, the degree to which the channels are dilated is likely
to be modulated by a combination of two factors, 1) the number of
transport signals available and 2) the binding affinity of the signals
for the receptors. According to this model, a small number of high
affinity signals might be as effective in regulating the size of the
transport channel as a larger number of low affinity signals.
In evaluating the effect of signal number on the translocation
process, it should be kept in mind that endogenous karyophilic proteins
would also contribute to the total pool of transport signals. The
degree to which endogenous proteins might influence the uptake of the
tracer particles cannot be determined at this time.
Coinjection of different size gold particles, coated with proteins
containing different nuclear targeting signals (BSA-WT and


TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF FIGURES vii
LIST OF TABLES viii
ABBREVIATIONS ix
ABSTRACT xii
CHAPTERS
I A REVIEW OF NUCLEAR PERMEABILITY: DIFFUSION AND
TRANSPORT 1
Introduction 1
Morphology 2
Nuclear Permeability Experiments 7
Nuclear Efflux 25
Statement of Research 37
II TRANSLOCATION OF RNA-COATED GOLD PARTICLES THROUGH THE
NUCLEAR PORES OF OOCYTES 39
Introduction 39
Materials and Methods 42
Results 48
Discussion 65
III THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE OF
TARGETING SIGNALS ON NUCLEAR UPTAKE 71
Introduction 71
Materials and Methods 74
Results 80
Discussion 97
v


64
**> ~
N


Figure 4. Poly(A)-gold, nuclear injection 60 min experiment.
Large gold particles (120-220 A) are observed extending through
the nulcear pores and are present in the adjacent cytoplasm (C)
N, nucleus. Bar, 0.2pm.
Figure 5. PVP-gold, nuclear injection 60 min experiment.
Gold particles are retained within the nucleoplasm and are rarely
seen associated with the envelope or within the nuclear pores.
C, cytoplasm. N, nucleus. Bar, 0.2 pm.


21
Kalderon et al. (1984a) used site-directed mutagenesis to identify
a seven amino acid sequence within large T-antigen that is required for
nuclear localization, with the lysine at position 128 being the critical
residue. A change in this lysine residue to asparagine, threonine,
isoleucine, leucine, methionine, or glutamine caused the large T-antigen
to become localized in the cytoplasm of the cells. Mutations within
the signal sequence on either side of the lysine influenced the rate of
uptake and qualitative distribution of large T-antigen, but did not
abolish transport activity. The minimum sequence required for nuclear
localization is Pro-Lys-Lys^-Lys-Arg-Lys-Val (Kalderon et al., 1984b).
One important aspect of this signal sequence is its ability to function
when removed from its normal environment and placed in a different
region of a protein. For example, if the signal is linked to the amino
terminus of either a mutated large T-antigen or chicken muscle pyruvate
kinase (a nonnuclear protein), it can still confer nuclear localization.
In addition, a synthetically synthesized signal peptide cross-linked to
a carrier protein has the ability to target the conjugate into the
nucleus of cultured cells (Lanford et al., 1986; Yoneda et al., 1987) as
well as oocytes (Goldfarb et al., 1986). Thus, the functional autonomy
of the targeting signal suggests that it does not require a fixed
secondary structure.
Recently, nucleoplasmin has been cloned and sequenced (Burglin and
De Robertis, 1987; Dingwall et al., 1987). The fifty amino acids from
the carboxy terminal tail domain have two regions similar in homology
with the nuclear localization signal from the SV 40 large T-antigen
(Dingwall et al., 1987). In addition, two other regions have been


86
uptake of particles into the nucleus. Furthermore, the data suggest
that as the size of the particles increases, more signals are required
for their transport across the envelope.
To compare the effectiveness of a different targeting signal,
parallel studies were performed with nucleoplasmin-gold. The relative
uptake of different size nucleoplasmin-coated particles by the nucleus
is shown in Table VIII. The N/C ratios determined for nucleoplasmin-
coated particles were significantly greater than those obtained for BSA-
WT5-, BSA-WTg-, or BSA-WT^-coated gold. In all instances, the
probability values were p<0.002. Since there are only 5 targeting
signals per nucleoplasmin molecule, these differences in uptake suggest
that the nucleoplasmin transport signal is more effective than the SV 40
large T-antigen targeting sequence, at least in oocytes.
Size distributions
In view of the above results, the size distributions of particles
in different regions of the oocytes were analyzed in more detail. The
nuclear and cytoplasmic size distributions determined for BSA-WT5 and
BSA-WTg 1 h after injection are given in Table IX and Fig. 10.
Seventeen percent of the BSA-WTg-coated particles that entered the
nucleus were larger than 157 A, compared to 5.6% for particles coated
with BSA-WT5. When particles larger than 185 A are compared, the
difference in uptake is almost 10-fold. The difference in the size of
the particles able to penetrate the pores is statistically significant
as determined by Chi-square analysis.


58
To determine if the translocation of polynucleotide-coated
particles is due simply to a high negative charge density, gold
particles were coated with polyglutamic acid, which, like RNA, is a
polyanion. Since polyglutamic acid is not a highly effective
stabilizing agent, the colloid preparations that were injected were
relatively dilute. To compensate for this factor, tRNA-gold
preparations having an equivalent particle concentration were injected
in parallel experiments. The results, which are based on an analysis of
5 cells, are shown in Table IV. Tracer particles coated with
polyglutamic acid were observed in only 8% of the pores, compared with a
value of 83% obtained for tRNA-gold. These findings demonstrate that
the translocation of the RNA-coated particles is not simply a charge
effect.
Controls
Control experiments were performed on 6 oocytes to establish if the
results obtained for RNA-gold could be due to a redistribution of the
particles during fixation. In this study, the nuclei were injected with
tRNA-coated gold and the oocytes fixed within 10 seconds. The
particles near the site of injection were rarely observed within the
pores or in the adjacent cytoplasm. These data indicate that the
presence of RNA-gold within the pores at 15 min and 1 h intervals was
not a fixation artifact, but reflected an i_n vivo exchange process.
In order to ascertain whether the presence of excess RNA itself
could alter the properties of the pores, nuclei of 6 oocytes were
simultaneously injected with PVP-coated gold and 100 ng of soluble tRNA.


15
amoebae (120-140 A, Feldherr, 1965), and hepatocytes (100-110 A, Peters,
1984). These differences in size can markedly affect the exclusion
limits and flux rates of macromolecules. For example, it would take
approximately 1 h for a molecule, 50 A in diameter, to diffuse through a
pore with a diameter of 110 A. In contrast, it would take the same
molecule 8 h to pass through a pore with a diameter of 90 A (estimates
from Fig. 6; Paine et al., 1975). This can explain the 2-5 orders of
magnitude difference in dextran flux rates between oocytes and
hepatocytes.
Endogenous macromolecules
Subsequent nuclear permeability studies have focused on the
selective uptake of endogenous proteins. Labelled endogenous proteins
can serve as probes to investigate the normal molecular interactions and
behavior found within cells, in contrast to exogenous molecules, which
are unlikely to have intracellular interactions and solely used to
characterize the passive permeability properties of the nuclear
envelope. It soon became evident that the rates of nuclear accumulation
of endogenous molecules were much greater than exogenous molecules of
comparable sizes. The data demonstrated that exchanges of endogenous
proteins between the nucleus and cytoplasm were controlled by selective
processes, explaining in part, the observation that endogenous proteins
distribute differently than exogenous tracers.
There are two possible mechanisms that can account for the rapid
rate of endogenous protein accumulation within the nucleoplasm. First,
proteins can diffuse freely through the nuclear pores and then can be


74
nucleus and the functional size of the transport channels increase. The
SV 40 and nucleoplasmin targeting sequences varied in their ability to
facilitate transport. This could be related to differences in their
binding affinity for nuclear envelope (pore) receptors. Double
labelling experiments demonstrated that different targeting signals can
be recognized by and transported through the same pore. Control
experiments ruled out the possibilities of nonspecific exchange
processes.
Materials and Methods
Xenopus laevis were purchased from Xenopus I (Ann Arbor, Michigan)
and maintained as reported previously (Feldherr, 1975).
Colloidal gold coating agents
The synthesis of peptides containing the SV 40 large T-antigen
targeting signals and their conjugation to BSA was described in detail
by Lanford et al. (1986). In brief, the thirteen amino acid sequence
was synthesized on a glycyl-Merrifield resin (Merrifield, 1963) using a
Biosearch Sam Two (Biosearch, San Rafael, CA) automated peptide
synthesizer. The synthetic peptides were conjugated to BSA using the
heterobifunctional cross-linking agent m-maleimido benzoyl-N-
hydroxysuccimide ester (MBS; Pierce Chemical Co., Rockford, IL).
Dialysis in phosphate-buffered saline followed by repeated concentration
and dilution of the conjugates separated the unconjugated peptides from
the carrier proteins. By varying the molar ratios of the reactants,
different protein-peptide coupling ratios were obtained. Amino acid


43
0.05 M Tris and 0.05 M NaCl (pH 7.2), after which the nucleoplasmin was
precipitated in 80% alcohol and lyophilized as described previously
(Feldherr et al., 1984). Gel analysis was performed, as described by
Laemmeli (1970), to determine the purity of the preparation.
Gold preparation and stabilization
All glassware and solutions used in experiments involving RNA were
treated with 0.01% (vol/vol) diethyl pyrocarbonate and then autoclaved.
Colloidal gold particles were prepared by reducing chloroauric acid with
either trisodium citrate (Frens, 1973) or a saturated solution of white
phosphorus in ether (Feldherr, 1965). In this study, the trisodium
citrate method gave a particle distribution of 120-220 A in diameter,
whereas the phosphorus ether preparations ranged from either 20-50 A or
20-160 A, depending on the initial concentration of gold chloride used.
The gold sols were stabilized with tRNA (met or phe), 5S RNA,
poly(A) (3500 bases), poly(I) (500 bases), poly(dA) (500 bases),
nucleoplasmin, polyvinylpyrrolidone (PVP; 40 kd), polyglutamic acid,
ovalbumin, or bovine serum albumin (BSA). The RNAs were obtained from
Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical
Company (St. Louis, M0). The purity of the RNAs was tested by running
the preparations on 10% polyacrylamide gels containing 8.3 M ultrapure
urea; ethidium bromide was used as a stain. Polyglutamic acid, BSA,
ovalbumin and PVP were purchased from Sigma Chemical Company.
In each instance, the minimum amount of coating agent required to
stabilize the particles, that is, prevent precipitation in 1% NaCl, was
determined as described earlier (Feldherr, 1984). Before stabilization,


Table VII Volumes of Coating Agent (ul) Required to Stabilize 1 ml of Gold Sol*
Fraction Size
Coating Agents
(cone.)
20-50 A
20-160 A
50-280
A 120-280 A
BSA
(1.0mg/ml)
60



BSA-WT5
(1.3mg/ml)
10
60
40

BSA-WTg
(0.6mg/ml)
30
150
150
20
BSA-WTn
(0.5mg/ml)

200

15
BSA-cTy
(1.4mg/ml)
20
80


BSA-cT13
(0.6mg/ml)


140

Large T-Ag
(0.4mg/ml)

400


Nucleoplasmin
(0.6mg/ml)

40
150
25
*These are average values,
coating agents required for
intended to serve as a guide. The
stabilization should be determined
exact amounts
for each
individual gold preparation.


7
of eight glycoproteins exist in multiple copies per pore complex and
their function is unknown at this time.
Compiling the data available on the components of the pore complex,
many investigators have suggested models or schematic representations of
the nuclear envelope architecture. These models, in conjunction with
the biochemical data, provide a framework for investigating how the
nuclear pores might be involved in regulating nucleocytoplasmic
exchanges. The appendix presents a schematic that is based on Franke's
(1974) and Unwin and Milligan's (1982) data.
Nuclear Permeability Experiments
It is clear from morphological evidence that the nuclear envelope
with its associated pore complexes is different from the plasma membrane
and other intracellular membranes. Since cellular processes both within
the nucleus and cytoplasm are dependent on a mutual exchange of
material, an important question arises as to the permeability properties
of the nuclear envelope and to what extent the envelope might regulate
nucleocytoplasmic exchange.
Three general approaches are used to study intracellular exchanges
across the nuclear envelope. First, microinjection of labelled tracers
(fluorescent, radioactive or electron-dense) is employed extensively to
study the permeability of the nuclear envelope to molecules within
intact cells. Second, permeability experiments can be performed on
isolated nuclei; however, these results must be carefully interpreted as
the permeability characteristics can change as a result of the isolation
(e.g., Paine et al., 1983; Peters, 1986). Third, recombinant DNA


19
rapidly accumulates in the nucleus. Nucleoplasmin is a thermostable,
pentameric molecule present within the nucleus in a high concentration
(Mills et al., 1980). It appears in the electron microscope as a disc
with a molecular dimension of 74 A in diameter (Earnshaw et al., 1980).
Pepsin digestion selectively cleaves nucleoplasmin into two structural
domains, a protease-resistant core and five protease-sensitive tails
(Dingwall et al., 1982). Protease removal of the five tail domains
leaves a pentameric core that is excluded from the nucleus after
microinjection into the cytoplasm. In contrast, the tail domains, after
cytoplasmic injection, are capable of nuclear accumulation. When the
core is injected directly into the nucleus, it migrates throughout the
nucleoplasm but is still excluded from the cytoplasm, indicating that
the core has the ability to be retained by the nucleus but not the
ability to migrate into the nucleus. These results demonstrate that the
tail domain contains the necessary information to specify selective
entry. By reducing the time of digestion, a mixture of pentameric cores
that contain different numbers of tail regions can be prepared. A core
molecule with only one tail is capable of entering the nucleus, but at
one-tenth the rate of intact nucleoplasmin (Dingwall et al., 1982).
Amino acid analysis of the tail domain (C-terminus) indicates an
enrichment in lysine residues and a depletion in hydrophobic amino
acids.
The second approach, utilizing genetic engineering, entails the
construction of plasmids encoding proteins normally found in the
cytoplasm linked to putative nuclear signal sequences. The end product
is a hybrid protein, converting an otherwise cytoplasmic protein into a


73
in the nuclei of Xenopus oocytes with saturable uptake kinetics,
suggesting the involvement of a receptor-mediated process.
Nucleoplasmin, a 110 kd pentameric karyophilic protein (as
determined by sequence analysis), also has been used extensively to
study nuclear transport in oocytes (Dingwall et al., 1982; Feldherr et
al., 1984) and cultured cells (Schulz and Peters, 1986; Sugawa et al.,
1985). It has recently been determined that each monomeric subunit
contains 1 targeting signal. Furthermore, removal of one or more signal
domains by proteolytic cleavage markedly reduces the rate of nuclear
uptake (Dingwall et al., 1982). The region containing the signal has
been sequenced and although it is similar to the SV 40 targeting signal,
it is not identical (Dingwall, personal communication).
The data summarized above suggest that variations in the nuclear
targeting signal could significantly influence nucleocytoplasmic
exchange of proteins. To better understand the nature of these effects,
experiments were performed to determine how macromolecules containing
different signal sequences and different numbers of signals interact
with, and modulate the properties of the nuclear pores. Various size
colloidal gold particles were coated with 1) BSA conjugated with
different numbers of synthetic peptides containing the SV 40 targeting
signal, 2) BSA conjugated with inactive SV 40 signals, 3) large
T-antigen, or 4) nucleoplasmin. These tracers were microinjected into
oocytes and their distribution within the oocytes was later determined
by electron microscopy.
The data indicate that as the number of signals per molecule
increases, both the relative uptake of the tracer particles into the


Figure 6. Double injection experiment 45 min.
tRNA-gold (20-50 A) was injected into the nucleus (N), and the adjacent
cytoplasm (C) was injected with nucleoplasmin-gold (120-220 A). The
large nucleoplasmin-coated particles can be seen just on the nuclear
sides of the pores and small RNA particles are present on the
cytoplasmic side. Bar, 0.1 pm.
Figure 7. 5S RNA-gold, cytoplasm injection 15 min experiment.
Gold particles are present in the cytoplasm (C) and can be seen
extending through the nuclear pores. Only a small percentage of
particles enter the nucleus (N). Bar, 0.1 pm.


14
Stacey and Allfrey (1984) also used well characterized proteins to
study the permeability of the nuclear envelope of cultured cells.
Thirteen proteins ranging from 13 kd to 669 kd with pi's from 4.4 to 11
were microinjected into the nucleus or cytoplasm of HeLa cells.
Proteins less than 40 kd entered almost immediately, in contrast to
proteins between 40 kd and 60 kd which entered more slowly. Many of the
proteins larger than 60 kd were excluded from the nucleus. The rates of
diffusion into and out of the nucleus were indistinguishable.
To estimate the patent pore diameter in cultured liver cells,
Peters (1984) used fluorescent microphotolysis to study the permeability
properties of the nuclear envelope. Fluorescein-labelled dextrans
ranging from 3 to 150 kd were injected into hepatocytes and allowed to
reach equilibrium. A pulse from an argon laser depleted the fluorescent
signal within a region of the nucleus and then microfluorimetry was used
to measure the influx of the tracer back into the depleted region. His
results showed the influx of the dextrans, from the cytoplasm to the
nucleus, to have an exclusion limit between 17 and 41 kd. Using similar
equations as Paine et al. (1975), Peters derived the functional pore
diameter to be 100-110 A with respect to diffusion.
All of the experiments involving the microinjection of exogenous
tracers support the view that the nuclear envelope has properties of a
molecular sieve. The entry of exogenous tracers into the nucleus occurs
by non-selective mechanisms, that is, diffusion through the pores. The
rate of influx is inversely related to size which reflects some of the
limitations imposed by the pore complex. The pore diameter has been
estimated for amphibian oocytes (90 A; Paine et al., 1975),


28
temperature dependence. Furthermore, tRNAP^e can act as an effective
competitor of tRNAmet transport. These results strongly suggest that
tRNA transport into the cytoplasm involves a carrier-mediated process
rather than simple diffusion.
In an attempt to define the critical region of the tRNA molecule
required for transport, Tobian et al. (1985) used site-directed
mutagenesis to obtain 30 different mutants with single nucleotide
substitutions. Many of the mutations perturbed the rate of transport.
The most deleterious effects were in the highly conserved T stem-loop
regions; however, many of these tRNAs had impaired processing. It is
suggested from this study that transport, in addition to processing, is
dependent on maintenance of the secondary structure.
The nuclear pores can serve as diffusion pathways for exogenous
tracers and as a transport pathway for some karyophilic proteins. As
mentioned above, the diffusion of tRNA out of the nucleus seems unlikely
due to i) its size and ii) the involvement of a carrier-mediated
process. Zasloff (1983) has suggested that the pores serve as the
pathway for exchange and that peripheral ribosome-like particles on the
cytoplasmic surface of the nuclear envelope provide the motor mechanism
required for transport.
Small nuclear RNAs
The snRNAs comprise 0.1-1.0% of the total cellular RNA. They are
small (100-250 nucleotides), stable, uridylic-acid rich RNAs that are
localized in the nucleus and are transiently found in the cytoplasm. At
least eight snRNAs, U1-U8, have been identified, and all are associated


83


25
dependent. In addition, the lectin wheat germ agglutinin (WGA)
effectively inhibits the transport of nucleoplasmin and the inhibitory
effects could be blocked with the addition of N-acetylglucosamine
(Finlay et al., 1987). Ferritin-labelled WGA was shown to be localized
to the cytoplasmic face of the nuclear pores. Furthermore, iodinated
WGA stains a glycoprotein, 63-65 kd, on nitrocellulose blots of rat
liver nuclei (Finlay et al., 1987). This glycoprotein is similar to the
one detected by Davis and Blobel (1986) and Snow et al. (1987). It has
been suggested that this glycoprotein is part of the pore complex and
might be involved in the nuclear transport of proteins.
Nuclear Efflux
Introduction
The nucleus synthesizes the RNAs required for protein synthesis;
thus, the efflux of messenger RNA (mRNA), ribosomal RNA (rRNA), and
transfer RNA (tNRA) is an obligatory step in the process of gene
expression. Heterogeneous nuclear RNA (hnRNA), small nuclear RNAs
(snRNA) and tRNA precursors are found normally within the nucleus.
Prior to mature mRNA export to the cytoplasm, hnRNA must undergo i)
processing, which involves removal of the introns and splicing together
of the exons, and ii) posttranscriptional modifications, which can
include polyadenylation and methylation. tRNA precursors must also be
processed before efflux and precursor rRNA is processed into 28S, 18S,
and 5.8S RNAs and are present in 40S and 60S RNP particles prior to
transport into the cytoplasm.


IV SUMMARY AND PROSPECTUS
103
Summary of Results 103
Nuclear Envelope Selectivity 104
Dynamic Aspects of the Transport Channels 107
Proposed Model 109
Future Trends Ill
APPENDIX 116
REFERENCES 117
BIOGRAPHICAL SKETCH 130
vi


3
procedures listed above to accumulate a vast amount of ultrastructural
information concerning both the nuclear envelope and pore structure from
a range of cell types, resulting in many interpretations of the pore's
three-dimensional architecture. Comprehensive reviews of the morphology
of the nuclear envelope and pore complexes have been published by Franke
(1974), Harris (1978), and Maul (1977). The following discussion will
be limited to a general morphological description of the nuclear
envelope and its associated pore complex, without describing some of the
finer substructures, since their existence remains controversial.
The nuclear envelope is considered to have four structural
components: the inner and outer membranes, the pore complexes, and the
nuclear lamina. The envelope, seen in thin sections perpendicular to
the nucleus, is composed of a double membrane system with the inner and
outer membrane parallel to one another until they approach the pore
complexes. In general, each membrane is approximately 75 A in diameter.
A homogenous region, which varies in width from 100-600 A depending on
the cell type, lies between the two membranes and is referred to as the
perinuclear cisterna (Wischnitzer, 1958). Often, the luminal space of
the perinuclear cisterna is observed to be in direct continuity with the
luminal cavity of the endoplasmic reticulum (Watson, 1955). The outer
and inner membranes are associated with different structures.
Polyribosomes are seen to be attached to the outer membrane, whereas,
the inner surface of the inner membrane appears to have an intimate
association with a distinct morphological layer, referred to as the
lamina. These observations suggest that the inner and outer membranes


pores and cytoplasm. These results indicate that translocation of RNA-
coated gold was due to the presence of RNA associated with the
particles.
To determine if the number of targeting signals affects the
transport of proteins into the nucleus, oocytes were injected with gold
particles coated with BSA cross-linked with various numbers of short
synthetic peptides containing the SV 40 large T-antigen nuclear
transport signal. Large T-antigen also was used as a coating agent.
Particles carrying active targeting signals entered the nucleus through
central channels within the nuclear pores. Analysis of the
intracellular distribution and size of the tracers entering the nucleus
indicated that the number of signals per molecule affects both the
uptake of particles and the functional size of the channels available
for translocation. In control experiments, particles coated with BSA or
BSA conjugated with inactive SV 40 transport signals were virtually
excluded from the nucleus. Nucleoplasmin-coated gold was transported
through the nuclear pores more effectively than the BSA conjugates. The
accumulation of tracers along the nuclear envelope suggests that
differences in the activity of the two targeting signals might be
related to their binding affinity for envelope receptors.
Double injection experiments demonstrated that individual pores are
capable of transporting both protein and RNA and they can also recognize
and transport proteins containing different nuclear targeting signals.
xi 11


Figure 2. tRNA-gold, nuclear injection 15 min experiment.
Gold particles (20-160 A) are observed evenly distributed throughout
the nucleus (N). Particles are seen within the centers of the majority
of the pores and also in the adjacent cytoplasm (C).
Bar, 0.2 pm.
Figure 3. 5S RNA-gold, nuclear injection 60 min experiment.
The tracer particles (20-160 A) show a similar distribution as
described in Figure 2. Particles are present within the pores
and the adjacent cytoplasm (C). N, nucleus. Bar, 0.2 pm.


TABLE X Size Distribution of BSA-WTr-, WTii~ and Nucleoplasmin-coated Particles in Injected Cells*
Experiment
(1 h)
Total No.
of Particles
Measured
Percentage of Particles in Each Size Class#
120-145 145-170 170-195 195-220 220-245 >245
A A A A A A
BSA-WT8
Nucleus
371
3.2
21.6
55.5
18.6
1.1
--
Cytoplasm
556
2.9
16.5
51.3
21.9
5.9
1.4
BSA-WTn
Nucleus
358
4.2
21.5
53.6
19.6
1.1

Cytoplasm
557
3.2
14.7
50.6
22.8
7.0
1.6
Nucleoplasmin
Nucleus
514
3.5
18.9
55.6
19.0
2.4

Cytoplasm
525
3.2
16.4
49.3
20.4
7.2
3.4
*These experiments were performed with 120-260 A gold particles and the mean size of the fraction
was 180 A.
#The size of the gold particles does not include the thickness of the coating agent.


12
nucleus accumulated ^5¡_|1ist-one to at least twice the concentration of
the cytoplasm. In contrast, the ^I-BSA was more concentrated in the
cytoplasm, even after 12 hours. It was clear from these results that
the nuclear envelope shows some selectivity for inhibiting passage of
large exogenous proteins.
The nucleocytoplasmic exchange of a range of exogenous
macromolecules was studied quantitatively by Paine and Feldherr (1972).
Well characterized macromolecules with respect to size, shape, and
electrical charge were used: ferritin (mol wt 465,000; pi 4.4), BSA
(mol wt 67,000; pi 4.71), ovalbumin (mol wt 45,000; pi 4.7), myoglobin
(mol wt 17,500; pi 6.9), lysozyme (mol wt 14,500; pi 11.8); and
cytochrome c (mol wt 13,000; pi 10.7). All the proteins were
f1uorescently labelled, with the exception of ferritin which is
electron-opaque, and injected into the cytoplasm of oocytes from
Periplaneta americana. Their results showed that ferritin and BSA had
extremely low N/C ratios even after 5 h. Ovalbumin slowly entered the
nucleus; however, a higher concentration was present in the cytoplasm as
compared to the nucleus after 5 h. On the other hand, cytochrome c
entered the nucleus within 5 min, whereas lysozyme and myoglobin had N/C
ratios greater than 1.0 within 3 min. These results demonstrated that
neutrally or positively charged proteins, less than 20 A in diameter,
can readily enter the nucleus. Furthermore, the nuclear uptake of the
proteins, 20-95 A in diameter, was inversely related to size and not
simply based on charge. Similar conclusions were reached by Bonner
(1975a) who studied the nuclear uptake of the small basic proteins;


44
tRNA, poly(A), poly(I), and poly(dA) were dissolved in 10 mM KC1, 7.2 mM
K2HPO4 and 4.8 mM KH2PO4 (pH 7.0). 5S RNA was rehydrated in 2 ml of
sterile ion-free water resulting in a salt concentration of 1 mM Tris-
HC1, 10 mM NaCl and 0.1 mM MgCl2 (pH 7.5). Nucleoplasmin, polyglutamic
acid, PVP, BSA, and ovalbumin were solubilized in a low ionic strength
buffer containing 7.2 mM K2HPO4 and 4.8 mM kH2P04 (pH 7.0).
Concentrations of coating agents and volumes used to stabilize the gold
preparations are given in Table I.
After stabilization, the 20-160 A preparations were centrifuged at
6,000g (at the bottom of the tube) at 4C for 10 minutes to remove any
large aggregates of gold. This step was not necessary for the 20-50 A
and 120-220 A fractions. Five to 7 ml of stabilized colloid were then
concentrated to 70-100 pi in Minicon concentrators (Amicon Corp.,
Danvers, MA). Finally, the samples were dialyzed against intracellular
medium consisting of 102 mM KC1, 11.1 mM NaCl, 7.2 mM K2HPO4, and 4.8 mM
KH2P04 (pH 7.0) for 3 h at 4C.
Injection
Frogs were anesthetized on ice for one hour and the ovaries
removed. Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually
defolliculated with watchmakers forceps and maintained in Ringer's
solution (Diberardino et al., 1977) at 22C. The defolliculated cells
were centrifuged at approximately 650g for 8-10 minutes as described
previously (Feldherr, 1980; Kressmann and Bernstiel, 1980). During
centrifugation, the nucleus migrates to a position just underneath the
plasma membrane at the animal pole and its outline can be visualized due


CHAPTER II
TRANSLOCATION OF RNA-COATED GOLD PARTICLES
THROUGH THE NUCLEAR PORES OF OOCYTES
Introduction
The nuclear envelope in eukaryotic cells is the site of continual
macromolecular exchange between the nucleoplasm and the cytoplasm. Such
exchanges can occur either by passive diffusion or mediated transport
through the nuclear pores (Dingwall and Laskey, 1986). Microinjection
of various sized exogenous molecules has been used in previous studies
to determine the dimensions of the diffusion channels within the pores.
The channels were estimated to be 90 A in diameter in amphibian oocytes
(Paine et al., 1975), and are available for diffusion both into and out
of the nucleus (Paine, 1975). Evidence for protein transport across the
envelope was initially obtained for RN1 (Feldherr et al., 1983) and
nucleoplasmin (Dingwall et al., 1982), both of which are major
karyophilic proteins found in amphibian oocytes. The specific sites of
transport for nucleoplasmin have been identified by microinjecting
nucleoplasmin-coated gold particles into the cytoplasm of the oocytes
(Feldherr et al., 1984). It was found, by electron microscopy, that
transport of the colloidal particles occurred through channels, at least
200 A in diameter, located in the centers of the nuclear pores. Thus,
the regions of the pores available for transport are considerably larger
than those available for diffusion.
39


40
The exit of mRNA from isolated nuclei has been studied in a number
of laboratories (reviewed by Clawson et al., 1985). These
investigations have shown that efflux is a temperature-dependent,
energy-requiring process, indicating that some form of transport is
involved (Agutter, 1985a; Clawson et al., 1978). In support of this
view, isolated nuclear envelope preparations were found to contain
nucleoside triphosphatase activity which is affected by the same factors
(ionic composition, pH, etc.) that regulate RNA efflux (Agutter, 1985a;
Clawson et al., 1980; 1984). It has also been suggested, on the basis
of results obtained in several laboratories, that the poly(A) tail of
mRNA is involved in transport across the envelope (Agutter, 1985b; Bernd
et al., 1982).
The efflux of tRNA from the nucleus has been investigated by Zasloff
(1983) using i_n vivo procedures. He obtained evidence for a saturable,
carrier-mediated transport mechanism for tRNAmet in the amphibian
oocyte. De Robertis et al. (1982) have demonstrated that microinjected
5S RNA can migrate either into or out of the nucleus in Xenopus oocytes;
however, the mechanism of exchange was not determined.
It is generally believed that RNA transport occurs through the
nuclear pores. Indirect evidence supporting this view has been reviewed
by Franke and Scheer (1974); direct evidence was obtained by Stevens and
Swift (1966). These latter investigators observed that 400 A RNP
granules synthesized at the Balbiani rings in Chironomus salivary gland
cells exit through the pores. As the particles enter the pores they are
transformed into rod-like structures with diameters of about 200-250 A.


30
then allows for the accumulation within the nucleus. Whether the signal
is in the protein, the RNA, or possibly a combination of both, has yet
to be determined.
Ribosomal RNA
The ribosomal DNA transcript, located in the nucleoli, contains the
18S, 5.8S, and 28S genes. After transcription, the rRNA is complexed
with proteins to form a 80S ribosomal ribonucleoprotein particle, which
is processed to the mature 40S and 60S particles prior to cytoplasmic
transport. The 5S RNA is transcribed, processed, and transported
separately. These rRNPs are the major components involved in ribosome
biogenesis.
Previtellogenic oocytes (stages I and II; Dumont, 1972) synthesize
and store 5S RNA which comprises 30-40% of the total cellular RNA.
TFIIIA is a transcription factor with a molecular weight of 39 kd that
binds to 5S genes. In addition, TFIIIA also binds to cytoplasmic 5S RNA
to form a 7S ribonucleoprotein particle. By employing immunocyto-
chemical procedures on sections of Xenopus ovaries, Mattaj et al. (1983)
demonstrated that TFIIIA is located predominantly in the cytoplasm. In
previtellogenic oocytes, TFIIIA is associated with the 5S RNA (Picard
and Wegnez, 1979); thus, the immunocytochemical staining patterns are
actually localizing 7S RNPs. Pelham and Brown (1980) studied the
i_n vitro transcription of the 5S RNA gene and showed that transcription
could be inhibited when 5S RNA, in excess of the 5S gene, competed for
TFIIIA binding. They suggested from these results that TFIIIA might be
involved in a feed-back mechanism to control 5S gene expression. It is


TABLE XI ~ Size Distribution of BSA-WTfl-cT7- and Large T-Ag-coated Particles in Injected Cells*
Total No.
Percentage of Particles
in Each Size
Class#
Experiment
(1 h)
of Particles
Measured
10-30 30-50 50-70
AAA
70-90
A
90-110
A
>110
A
Large T-Ag
Nucleus 386
Cytoplasm 600
BSA-WTg-cTy (1:3 dilution)
Nucleus 574
Cytoplasm 621
22.8 49.0 26.2
16.2 30.3 39.3
26.0 40.1 27.4
11.6 23.7 37.6
2.0
8.8 3.7 1.7
5.2 0.7 0.2
16.7 7.1 3.6
*These experiments were performed with 20-120 A gold particles and the mean size of the
fraction was 60 A.
#The size of the gold particles does not include the thickness of the coating agent.


Table II Translocation of Gold Particles as a Function of the Coating Agent
No. of Particles
Experiment (1 h)Translocated*
% of Pores Active
Particles/Pore in Translocation*
Region
1 Region 2
Total
tRNA-gold
437
414
851
4.3
97%
5S RNA-gold
458
425
883
4.4
98%
Poly(A)-gold
309
479
788
3.9
98%
Poly(dA)-gold
334
144
478
2.4
98%
Poly(I)-gold
397
543
940
4.7
96%
PVP-gold**
3
5
8
0.04
4%
BSA-gold**
5
3
8
0.04
4%
Ovalbumin-gold
0
t-
9
11
0.05
6%
* Data based on
the analysis
of 200 pores
all within
equivalent
concentrations of
gold particles.
**The size of the gold particles in these experiments ranged from 20-50 A. In all
other experiments the particle sizes ranged from 20-160 A.


CHAPTER IV
SUMMARY AND PROSPECTUS
Summary of Results
In the present study, microinjection of colloidal gold particles
coated with different RNAs or proteins was used to establish
morphological criteria concerning the nucleocytoplasmic exchange of
macromolecules j_n vivo. The results from the injection of RNA-coated
particles (Chapter II) can be summarized as follows: first, central
channels located within the nuclear pores are visualized as the major,
if not exclusive, site for RNA translocation into the cytoplasm.
Second, poly(A)-coated particles, at least 230 A in diameter, can
penetrate the envelope. Third, most, if not all, of the nuclear pores
have the ability to transport the RNA tracer particles. Finally,
individual pores can be bifunctional, i.e., they can recognize and
transport both RNA and protein. The results presented in chapter III
have established how variations in the number and amino acid sequence of
protein transport signals can affect protein uptake. First, it was
found that particles coated with BSA conjugated with synthetic peptides
that contained the SV 40 large T-antigen transport signal translocate
into the nucleus through the centers of the nuclear pores. It was then
demonstrated that variations in the number and sequence of protein
transport signals can affect both the functional size of the transport
channel and the relative uptake of particles into the nucleus. The
103


47
Nucleoplasm
Cytoplasm
Figure 1. A schematic representation of the regions used for
electron microscopic analysis of the particle distribution
within the pores. It is assumed that the particles in region 1
are undergoing translocation and that particles in region 2 have
completed the process. The arrow which defines the outer limits
of region 2 represents a distance of approximately 500 A.


126
Paine, P.L. 1975. Nucleocytoplasmic movement of fluorescent tracers
microinjected into living salivary gland cells. J. Cell Biol.
66:652-657.
Paine, P.L., C.F. Austerberry, L.J. Desjarlais, and S.B. Horowitz.
1983. Protein loss during nuclear isolation. J. Cell Biol.
97:1240-1242.
Paine, P.L., and C.M. Feldherr. 1972. Nucleocytoplasmic exchange of
macromolecules. Exp. Cell Res. 74:81-98.
Paine, P.L., and S.B. Horowitz. 1980. The movement of material
between nucleus and cytoplasm. I_n Cell Biology. D.M. Prescott
and L. Goldstein, editors. Vol. 4. Academic Press, New York.
299-338.
Paine, P.L., L.C. Moore, and S.B. Horowitz. 1975. Nuclear envelope
permeability. Nature (Lond.) 254:109-114.
Pelham, H.R.B., and D.D. Brown. 1980. A specific transcription factor
that can bind either the 5S RNA gene or 5S RNA. Proc. Natl. Acad.
Sci. USA 77:4170-4174.
Peters, R. 1983. Nuclear envelope permeability measured by
fluorescence microphotolysis of single liver cell nuclei.
J. Cell Biol. 258:11427-11429.
Peters, R. 1984. Nucleo-cytoplasmic flux and intracellular mobility
in single hepatocytes measured by fluorescence microphotolysis.
EMBO J. 3:1831-1836.
Peters, R. 1986. Fluorescence microphotolysis to measure
nucleocytoplasmic transport and intracellular mobility. Biochim.
Biophys. Acta. 864:305-359.
Picard, B., and M. Wegnez. 1979. Isolation of a 7S particle from
Xenopus laevis oocytes: A 5S RNA-protein complex. Proc. Natl.
Acad. Sci. USA 76:241-245.
Purrello, F., R. Vigneri, G.A. Clawson, and I.D. Goldfine. 1982.
Insulin stimulation of nucleoside triphosphatase activity in
isolated nuclear envelopes. Science 216:1005-1007.
Richardson, W.D., A.D. Mills, S.M. Dilworth, R.A. Laskey, and C.
Dingwall. 1988. Nuclear protein migration involves two steps:
Rapid binding at the nuclear envelope followed by slower
translocation through nuclear pores. Cel 1 52:655-664.
Richardson, W.D., B.L. Roberts, and A.E. Smith. 1986. Nuclear
location signals in polyoma virus large-T. Cel 1 44:77-85.


128
Siamins, V., and D.P. Lane. 1985. An immunoaffinity purification
procedure for SV 40 large T-antigen. Virology 144:88-100.
Silver, P.A., L.P. Keegan, and M. Ptashne. 1984. Amino terminus of
the yeast GAL4 gene product is sufficient for nuclear localization.
Proc. Natl. Acad. Sci. USA 81:5951-5955.
Skoglund, U., K. Andersson, B. Bjorkroth, M.M. Lamb, and B. Daneholt.
1983. Visualization of the formation and transport of a specific
hnRNP particle. Cell 34:847-855.
Smith, C.D., and W.W. Wells. 1983. Phosphorylation of rat liver
nuclear envelopes. I. Characterization of i_n vitro
phosphorylation. J. Biol. Chem. 258:9360-9367.
Snow, C.M., A. Senior, and L. Gerace. 1987. Monoclonal antibodies
identify a group of nuclear pore complex glycoproteins. J. Cell
Biol. 104:1143-1156.
Stacey, D.W., and V.G. Allfrey. 1984. Microinjection studies of
protein transit across the nuclear envelope of human cells.
Exp. Cell Res. 154:283-292.
Steer, R.C., M.J. Wilson, and K. Ahmed. 1979a. Protein phosphokinase
activity of rat liver nuclear membrane. Exp. Cell Res. 119:403-
406.
Steer, R.C., M.J. Wilson, and K. Ahmed. 1979b. Phosphoprotein
phosphatase activity of rat liver nuclear membrane. Biochem.
Biophys. Res. Commun. 89:1082-1087.
Stevens, B.J., and H. Swift. 1966. RNA transport from nucleus to
cytoplasm in Chironomus salivary glands. J. Cell Biol. 31:55-77.
Stuart, S.E., G.A. Clawson, F.M. Rottmann, and R.J. Patterson. 1977.
RNA transport in isolated myeloma nuclei: Transport from
membrane-denuded nuclei. J. Cell Biol. 72:57-66.
Sugawa, H., N. Imamoto, M. Wataya-Kaneda, and T. Uchida. 1985.
Foreign protein can be carried into the nucleus of mammalian cell
by conjugation with nucleoplasmin. Exp. Cell Res. 159:419-429.
Tobian, J.A., L. Drinkard, and M. Zasloff. 1985. tRNA nuclear
transport: Defining the critical regions of human tRNA^met by
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Unwin, P.N.T., and R.A. Milligan. 1982. A large particle associated
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93:63-75.


68
large samples. For this reason a comprehensive examination of the
temperature dependence of translocation was not attempted. However, a
cursory study was performed at 4C and at 21C, comparing the
accumulation and distribution of tRNA-coated gold particles in the
pores. After 15 minutes, the average number of particles per pore
(based on the analysis of 100 pores) at 21C and 4C was 3.8 and 1.5,
respectively. Furthermore, at 4C only 25% of the particles were
located in region 2, compared to 50% in cells kept at 21C. The
temperature effects are very likely influenced by two separate
processes, binding to receptors and movement through the pores; thus,
the results are difficult to interpret. Despite this problem, however,
the overall effect of temperature is greater than would be expected for
a physical process, such as diffusion, and is consistent with the view
that an energy requiring step is involved.
Since the translocation of gold particles through the pores is i)
dependent on the properties of the adsorbed RNA-coat, ii) likely
involved in a transport process and iii) not an artifact of the
technique, it is concluded that the pathways visualized for the
translocation of RNA-coated colloidal tracers are the same as those
normally used for endogenous RNA. These pathways are located in the
centers of the nuclear pores and have apparent functional diameters of
approximately 230 A. These results are consistent with those obtained
previously by Stevens and Swift (1966) for mRNA efflux in Chironomus
salivary gland cells.
It is not known whether tRNA and 5S RNA complex with specific
proteins prior to exiting the nucleus. There is evidence that the


6
Unwin and Milligan (1982) have used thin section electron
microscopy coupled with Fourier averaging methods to describe the pore's
three-dimensional organization to a resolution of 90 A. They found the
pores, in Xenopus oocytes, to occupy 20-30 percent of the envelope
surface and are composed of several discrete components: plugs, spokes,
particles, and rings. The rings have internal and external diameters of
800 A and 1200 A, respectively. Spokes extend radially from the margins
of the channel toward a plug (30-350 A in diameter) located in the
center of the pore. Particles are seen on the cytoplasmic surface of
the pores but do not appear to be integral components, as they can be
released with high salt treatments. This model, with its high
resolution of analysis, confirms many of the components put forth in the
model described by Franke (1974).
Until recently, little was known about the molecular composition of
the pore complex. Gerace et al. (1982) have identified a 190 kd
intrinsic membrane glycoprotein that is specifically localized to the
nuclear pores in liver nuclei. After detergent treatment, which
solubilizes most nuclear envelope intrinsic proteins, the 190 kd
glycoprotein remains associated with the pore complex-nuclear lamina
fraction. Based on this and its biochemical properties, it is suggested
that the 190 kd glycoprotein is involved in anchoring the pore complex
to the envelope. Davis and Blobel (1986), using monoclonal antibodies
and immunoelectron microscopy, have identified a 62 kd protein that
localizes within the annuli of rat liver nuclei. Snow et al. (1987)
have described a group of eight glycoproteins, with common epitopes,
that are localized exclusively to the nuclear pore complex. The group


31
important to mention that TFIIIA alone can enter the nucleus to bind to
the 5S gene; however, when bound to 5S RNA, it remains in the cytoplasm.
Free 5S RNA also is capable of entering the nucleus and it accumulates
in the nucleoli (De Robertis et al., 1982). The 7S RNP is able to
diffuse throughout the cytoplasm of oocytes (Ford, 1971) but is excluded
from the nucleus. The mechanism of exclusion is not yet known; however,
it is possible that the formation of the 7S RNP in the cytoplasm masks a
transport signal, either on the RNA or the protein that is required for
nuclear entry.
Wunderlich and co-workers have studied 18S and 28S rRNA transport
from isolated Tetrahymena macronuclei. They reported that rRNP efflux
is ATP dependent and the amount of RNP transport is sensitive to a shift
in temperature (Giese and Wunderlich, 1983). It was also found that
rRNA is integrated into the nuclear matrix which might control the
transport process since removal of the nuclear envelope had no effect on
the temperature dependence of rRNP export (Wunderlich et al., 1983).
Although 28S and 18S RNA leave the nucleus complexed with ribosomal
proteins, it is not clear whether the RNA or protein contains the efflux
signals.
The role of the nuclear envelope in regulating rRNA translocation
was investigated jn vivo (Feldherr, 1980) by disrupting the nuclear
envelope with glass needles, and i_n vitro (Stuart et al., 1977) by using
membrane-denuded nuclei. The efflux of rRNA from the nucleus to the
cytoplasm was not affected by either experimental procedure suggesting
that rRNA is most likely bound to nondiffusible nuclear elements.


66
injection of excess RNA does not alter the overall properties of the
pores.
Gold particles coated with synthetic polymers (PVP or polyglutamic
acid), exogenous proteins (BSA or ovalbumin) and even endogenous
karyophilic proteins (nucleoplasmin, Feldherr et al., 1984) are
essentially excluded from the pores after nuclear injection,
demonstrating that the translocation of RNA-gold is a selective process.
The data obtained with poly(I) and poly(dA) indicate that the capacity
for translocation is not necessarily restricted to physiologically
active RNAs but may be a general property of polynucleotides. The
chemical and/or physical characteristics of RNA that facilitate
translocation are not known, although a comparison of the results
obtained with poly(A) and poly(dA) suggests that the sugar moieties
could be involved. The high negative charge density of RNA does not
appear to be a major contributing factor since particles coated with
polyglutamic acid are largely excluded from the pores.
In view of the results obtained by Zasloff (1983), which showed
that the transport of labelled tRNA is markedly reduced by a single
substitution of G-to-U at position 57, one might expect a greater
degree of specificity for the translocation of polynucleotides. It
should be kept in mind, however, that Zasloff measured the overall
efflux of RNA whereas our results relate specifically to the
translocation step across the envelope. Thus, the decreased rate of
transport of the variant is not necessarily due to an effect on
translocation through the pores, but could be due to increased binding


100
number per gold particle increases. Furthermore, the maximum size of
the transport channel is estimated to be 260 A in diameter. Control
experiments, utilizing BSA conjugated with inactive signals,
demonstrated that differences in the uptake of gold particles coated
with different BSA-WT conjugates were due to variations in the number of
active signals and not to nonspecific factors such as alterations in
particle size and charge.
Nucleoplasmin-coated gold was used as a stabilizing agent to
compare the effectiveness of a different nuclear targeting signal. It
was found that nucleoplasmin, BSA-WTg, and BSA-WT^ all had similar
effects on the functional size of the transport channel, even though
nucleoplasmin has fewer signals than either of the conjugates. However,
the relative uptake of nucleoplasmin-coated gold was significantly
greater than that observed for particles coated with the BSA-WT
conjugates. The fact that nucleoplasmin-gold accumulated along the
nuclear surface to a greater degree than other tracers suggests that the
relative effectiveness of different targeting sequences might be related
to their binding affinity for transport receptors.
The possibility that binding might be an important step in the
transport process was originally suggested by Feldherr et al. (1984),
and was based on the observation that nucleoplasmin-gold particles
accumulate at the surface of the pores during translocation. Other data
in support of this view are the kinetic studies by Goldfarb et al.
(1986) which demonstrate that the nuclear uptake of BSA conjugated with
large T targeting signals is saturable and, therefore, likely to be a
receptor-mediated process. Furthermore, Newmeyer and Forbes (1988)


72
histones 2A and 2B (Moreland et al., 1987), the yeast ribosomal protein
L3 (Moreland et al., 1985), polyoma large T-antigen (Richardson et al.,
1986), and the adenovirus Ela protein (Lyons et al., 1987). Although
there is no concensus signal, it appears that nuclear targeting is
dependent on short, basic amino acid sequences.
Of the proteins listed above, SV 40 large T-antigen has been
studied most extensively. Kalderon et al. (1984b) constructed hybrid
proteins by linking various amino acid sequences found in SV 40 large
T-antigen to the amino terminus of pyruvate kinase and found that the
shortest sequence capable of targeting the enzyme to the nucleus was
Pro-Lys-Lys^-Lys-Arg-Lys-Val. Transport is especially sensitive to a
point mutation at the Lys1^ position (Kalderon et al., 1984a; Lanford
et al., 1986). Amino acid mutations in the vicinity of the Lys1^
position reduces, but does not necessarily abolish transport of SV 40
large T-antigen into the nucleus. Roberts et al. (1987) have shown that
multiple copies of a partially defective signal can cooperate to enhance
nuclear localization.
Lanford et al. (1986) synthesized peptides that contained the SV 40
large T transport signal and cross-linked them to several carrier
proteins (ovalbumin, BSA, IgG, slgA, ferritin and IgM). When the
conjugates were injected into the cytoplasm of cultured cells, all but
IgM (m.w. 970 kd) entered the nucleus. Qualitative evidence, utilizing
indirect immunofluorescence, suggested that the number of signals per
carrier protein can affect the rate of uptake. Goldfarb et al. (1986)
found that BSA conjugated with SV 40 large T-antigen signals accumulates


106
particles and they can also recognize and transport proteins containing
different nuclear targeting signals. Based upon these results it is
suggested that nuclear pores have a broad range of specificity and
distinct functional classes of pores might be nonexistent.
Since the signals from large T-antigen cross-linked to BSA and
different RNAs are capable of translocating through the same nuclear
pores as nucleoplasmin, it is of interest whether they all use the same
mechanism for translocation. It is not known if the RNAs complex with
proteins which subsequently initiate their transport or whether RNA
itself has a putative transport signal. Since BSA-WT- and
nucleoplasmin-coated particles can translocate through the same pore it
remains to be determined if there are different receptors for different
transport signals within individual pores. Although a concensus
sequence for protein transport signals does not exist, signal sequences
usually contain a short stretch of basic amino acids. In this respect,
nucleoplasmin and large T-antigen share partial homology.
It is clear that specificity observed for the nucleocytoplasmic
exchange of endogeneous macromolecules cannot be accounted for entirely
by the properties of the pores. Thus, it is likely that selectivity of
proteins and RNA designated for transport can also occur within either
the nuclear or cytoplasmic compartments. For example, mRNA must be
released from the intranuclear matrix with the hydrolysis of ATP prior
to efflux (Schroder et al., 1987). The presence of poly(A) tails on
mRNA might be part of the selection process; however, poly(A)mRNA also
must somehow be selected since histone mRNA is not polyadenylated. In
addition, many of the proteins can be bound within the cytoplasm or


Table VIII N/C Ratios* 1 h
Coating Agents
20-50 A
20-160 A
50-280 A
120-260
BSA
0.009



BSA-cTy
0.01
0.009


BSA-cT13


0.006

bsa-wt8 +
BSA-CT7 (1:3 dilution)

0.035


BSA-WT5
0.58
0.18
0.06

bsa-wt8
0.76
0.79
0.38
0.14
BSA-WTn

0.80

0.24
Large T-Antigen

0.077


Nucleoplasmin

2.43
0.71
0.51
*N/C ratios were calculated from 500-3000 particle counts per data point.


33
to the envelope is likely to involve the processing of the precursor,
posttranscriptional modification, and release from the nuclear matrix.
Once at the inner surface of the nuclear envelope, the second step is
the translocation across the envelope, presumably through the pores.
Translocation of mRNA across the nuclear envelope appears to
involve a series of reactions requiring nucleoside triphosphatase
(NTPase), protein kinase, protein phosphohydrolase, and poly(A) binding
sites. Using isolated nuclei, Agutter et al. (1976) first reported a
correlation between RNA efflux and the presence of NTPase activity which
was localized in nuclear envelope fractions. Further studies on the
energy requirement of poly(A)+mRNA translocation were performed by
Clawson et al. (1978). These investigations determined that both ATP
and GTP could serve as energy sources.
Clawson et al. (1984) used photoaffinity labelling on rat liver
nuclear envelopes and localized the NTPase activity to a 46 kd protein.
Schroder et al. (1986a) subsequently purified and characterized a 40 kd
NTPase from rat liver nuclear envelopes which is thought to be the same
polypeptide identified by Clawson. In addition to the nuclear envelope,
NTPase activity was also found to be present in the matrix (Clawson et
al., 1984; Maul and Baglia, 1983). There are several experiments that
correlate the energy-dependent transport of poly(A)+mRNA with the NTPase
activity. First, an increase in the rate of RNA efflux by thioacetamide
treatment (Clawson et al., 1980), insulin administration (Purrello et
al., 1982), or tryptophan feeding (Murty et al., 1980), causes a
parallel increase in NTPase activity. Second, in a transport assay
using isolated nuclei, NTPase activity and mRNA efflux have similar


NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES
By
STEVEN IRA DWORETZKY
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1988

DEDICATED TO MY GRANDPARENTS, LEON AND FRANCES DWORETZKY

ACKNOWLEDGEMENTS
The studies presented in this dissertation could not have been
performed without the guidance and assistance of Dr. Carl Feldherr. He
served as an excellent role model for conducting scientific research of
the highest caliber. I would like to express my gratitude to him for
allowing me to work in his laboratory throughout the past three years.
Special thanks are extended to Dr. Robert Cohen for serving as a
committee member and providing insight to the biophysical aspects
associated with my research. I would like to thank Drs. C. West and G.
Stein for serving as members of my committee and providing critical
review of the manuscripts submitted for publication.
I am grateful to Dr. Robert Lanford at the Southwest Foundation
for Biomedical Research in San Antonio, Texas, for providing all of the
BSA-conjugates that were used to study the effects of signal number on
protein uptake.
The work presented in Chapter II is reproduced from the Journal of
Cell Biology, 1988, volume 106, number 3, pages 575-584 by copyright
permission of The Rockefeller University Press.
I would like to thank Denifield Player for advice concerning
techniques in electron microscopy and Linda Mobley for typing of
manuscripts and other pertinent material throughout my graduate career.

Finally, and most importantly, I wish to express my utmost
gratitude to Barbara Ludwig. She was a constant source of moral
support and encouragement throughout the trials and tribulations of my
graduate education. In addition, I would like to thank my parents for
their words of encouragement and steadfast support.
IV

TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS iii
LIST OF FIGURES vii
LIST OF TABLES viii
ABBREVIATIONS ix
ABSTRACT xii
CHAPTERS
I A REVIEW OF NUCLEAR PERMEABILITY: DIFFUSION AND
TRANSPORT 1
Introduction 1
Morphology 2
Nuclear Permeability Experiments 7
Nuclear Efflux 25
Statement of Research 37
II TRANSLOCATION OF RNA-COATED GOLD PARTICLES THROUGH THE
NUCLEAR PORES OF OOCYTES 39
Introduction 39
Materials and Methods 42
Results 48
Discussion 65
III THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE OF
TARGETING SIGNALS ON NUCLEAR UPTAKE 71
Introduction 71
Materials and Methods 74
Results 80
Discussion 97
v

IV SUMMARY AND PROSPECTUS
103
Summary of Results 103
Nuclear Envelope Selectivity 104
Dynamic Aspects of the Transport Channels 107
Proposed Model 109
Future Trends Ill
APPENDIX 116
REFERENCES 117
BIOGRAPHICAL SKETCH 130
vi

LIST OF FIGURES
Figure Page
1. A schematic representation of regions 1 and 2 47
2. tRNA-gold, nuclear injection 51
3. 5S RNA-gold, nuclear injection 51
4. Poly(A)-gold, nuclear injection 56
5. PVP-gold, nuclear injection 56
6. Double injection experiment 64
7. 5S RNA-gold, cytoplasmic injection 64
8. BSA-WTn-gold 83
9. BSA-cT7-gold 83
10. Nuclear particle distributions: BSA-WTg and BSA-WT5 ... 88
11. Nuclear particle distributions: BSA-WTg, BSA-WT^ and
nucleoplasmin 90
12. Accumulation of tracers along the nuclear envelope .... 95
13. Coinjection of gold particles coated with BSA-conjugates
and nucleoplasmin 99

LIST OF TABLES
Table Page
I Amounts of Coating Agent Required to Stabilize Gold Sols. 45
II Translocation of Gold Particles as a Function of the
Coating Agent 52
III Size Distribution of Gold Particles Present in the Nuclei
and Pores 54
IV Translocation of Gold Particles Coated with Polyglutamic
Acid 59
V Concentration Dependence of tRNA-gold Translocation .... 61
VI Protein Preparations Used as Coating Agents 76
VII Volumes of Coating Agent (pi) Required to Stabilize 1 ml
of Gold Sol 78
VIII N/C ratios 1 h 85
IX Size Distribution of BSA-WTg- and WTg-coated Particles
in Injected Cells 87
X Size Distribution of BSA-WTg-, WT^i- and Nucleoplasmin-
coated Particles in Injected Cells 91
XI Size Distribution of BSA-WTg-cTy- and Large T-Ag-coated
Particles in Injected Cells 92
XII Envelope-Associated Particles 96
vi i i

LIST OF ABBREVIATIONS
a
A
ATP
Arg
3
BSA
cT
DMSO
DNA
G
3H
h
HEPES
125!
IgG
kd
M
min
ml
mM
A1 pha
Angstrom
Adenosine-51-triphosphate
Arginine
Beta
Bovine serum albumin
Mutant large T-antigen
Dimethyl sulfoxide
Deoxyribonucleic acid
Guanosine
Tritiated
Hour(s)
N-2-Hydroxyethylpiperazine-N1-2-
Ethanesulfuric acid
Iodinated
Immunoglobulin
Kilodaltons
Molar concentration
Minute(s)
Mi 11 i 1iter(s)
Mi 11imolar
ix

mol wt
Molecular weight
N
Normal concentration
N/C
Nuclear to Cytoplasmic
ng
Nanogram
ni
Nanoliter(s)
NTPase
Nucleotide triphosphatase
OSO4
Osmium tetroxide
P
Probability
PAGE
Polyacrylamide gel electrophoresis
Poly(A)
Polyadenylic acid
Poly d(A)
Poly-deoxy-adenylic acid
Poly(I)
Polyinosinic acid
Poly(U)
Polyuridylic acid
Pro
Proline
PVP
Polyvinylpyrrolidone
RNA
Ribonucleic acid
hnRNA
Heteronuclear ribonucleic acid
mRNA
Messenger ribonucleic acid
rRNA
Ribosomal ribonucleic acid
tRNA
Transfer ribonucleic acid
RNP
Ribonucleoprotein
s
Second(s)
SDS
Sodium dodecyl sulfate
SV 40
Simian virus 40
T-antigen
Tumor antigen
U
Uridine
X

Ml
nm
Val
vol/vol
WGA
WT
wt/vol
Microliter(s)
Micron(s)
Valine
Volume/Volume
Wheat germ agglutinin
Wild type large T-antigen
Weight/Volume
XI

Abstract of Dissertation Presented to the Graduate School of the
University of Florida in Partial Fulfillment of the Requirements
for the Degree of Doctor of Philosophy
NUCLEOCYTOPLASMIC TRANSPORT OF MACROMOLECULES
THROUGH THE NUCLEAR PORES OF XENOPUS OOCYTES
By
STEVEN IRA DWORETZKY
August 1988
Chairman: Carl M. Feldherr
Major Department: Anatomy and Cell Biology
To localize and characterize the pathways for RNA translocation to
the cytoplasm, gold particles coated with tRNA, 5S RNA, or poly(A) were
microinjected into the nuclei of Xenopus oocytes. At various times
after injection, cells were fixed and the particle distributions
analyzed by electron microscopy. Similar results were obtained with all
RNAs used. Particles ranging from 20-230 A in diameter were observed
within central channels of the nuclear pores and in the adjacent
cytoplasm. Particles larger than 90 A would not be expected to diffuse
readily through the pores, suggesting that mediated transport occurred.
Furthermore, approximately 97% of the pores analyzed were involved in
the translocation of gold particles. After nuclear injection, particles
coated with exogenous macromolecules were essentially excluded from the

pores and cytoplasm. These results indicate that translocation of RNA-
coated gold was due to the presence of RNA associated with the
particles.
To determine if the number of targeting signals affects the
transport of proteins into the nucleus, oocytes were injected with gold
particles coated with BSA cross-linked with various numbers of short
synthetic peptides containing the SV 40 large T-antigen nuclear
transport signal. Large T-antigen also was used as a coating agent.
Particles carrying active targeting signals entered the nucleus through
central channels within the nuclear pores. Analysis of the
intracellular distribution and size of the tracers entering the nucleus
indicated that the number of signals per molecule affects both the
uptake of particles and the functional size of the channels available
for translocation. In control experiments, particles coated with BSA or
BSA conjugated with inactive SV 40 transport signals were virtually
excluded from the nucleus. Nucleoplasmin-coated gold was transported
through the nuclear pores more effectively than the BSA conjugates. The
accumulation of tracers along the nuclear envelope suggests that
differences in the activity of the two targeting signals might be
related to their binding affinity for envelope receptors.
Double injection experiments demonstrated that individual pores are
capable of transporting both protein and RNA and they can also recognize
and transport proteins containing different nuclear targeting signals.
xi 11

CHAPTER I
A REVIEW OF NUCLEAR ENVELOPE PERMEABILITY:
DIFFUSION AND TRANSPORT
Introduction
The formation of the nuclear envelope around the cell's genetic
material is one of the major evolutionary differences between
prokaryotes and eukaryotes. The nucleus, site of both RNA and DNA
synthesis, contains the genetic information needed for phenotypic
expression. The cytoplasm, on the other hand, is the site of protein
synthesis, which requires certain RNAs that are exported from the
nucleus. Furthermore, the nucleus is dependent on proteins synthesized
in the cytoplasm. Therefore, cell growth, division, differentiation,
and maintenance require a continual exchange of information between both
the cytoplasm and nucleus. To better understand cellular function, it
is important to determine the factors that control the rate of movement
between the two compartments and the types of molecules that are
exchanged. For this reason, knowledge of the nuclear envelope's
structure and permeability properties is essential in understanding the
interrelationships that exist between the nucleus and cytoplasm of the
eukaryotic cel 1.
In the first part of this chapter an ultrastructural description of
the nuclear envelope is provided, followed by a review of permeability
experiments which have helped to elucidate the role of the envelope in
regulating nucleocytoplasmic exchanges.
1

2
Morphology
The discovery of the nucleus is documented as early as 1833. Fifty
years later, light microscopists began speculating that a membrane
surrounded the nucleus. It was Chambers and Fell (1931) who
microdissected cells and demonstrated the existence of a nuclear
envelope. With the use of the electron microscope, Callan and Tomlin
(1950) performed ultrastructural studies on manually isolated oocyte
nuclei and confirmed the existence of an envelope surrounding the
nucleus. They described it as a double membrane structure, with a
porous outer membrane adjacent to the cytoplasm and a continuous inner
membrane adjacent to the nucleoplasm.
With advances in the resolution of the electron microscope and
improvements in fixation procedures, the nuclear envelope and pore
structure became the subject of intense morphological studies. The
envelope and associated structures can be visualized with the electron
microscope by a variety of techniques including i) i_n situ fixation
followed by ultrathin sectioning, ii) negative staining of isolated
envelopes, iii) freeze-etching, and iv) high-resolution scanning
microscopy. One of the first detailed studies utilizing transmission
electron microscopy was performed by Watson (1955, 1959). He analyzed
nuclei from a wide variety of cell types and found that the inner and
outer membranes fuse at regular intervals along the envelope resulting
in the formation of nonmembranous regions ("holes"), ranging from 800-
1200 A in diameter, referred to as the nuclear pores. These pores form
channels that represent potential sites of interaction between the
nucleoplasm and cytoplasm. Since then, laboratories have used the

3
procedures listed above to accumulate a vast amount of ultrastructural
information concerning both the nuclear envelope and pore structure from
a range of cell types, resulting in many interpretations of the pore's
three-dimensional architecture. Comprehensive reviews of the morphology
of the nuclear envelope and pore complexes have been published by Franke
(1974), Harris (1978), and Maul (1977). The following discussion will
be limited to a general morphological description of the nuclear
envelope and its associated pore complex, without describing some of the
finer substructures, since their existence remains controversial.
The nuclear envelope is considered to have four structural
components: the inner and outer membranes, the pore complexes, and the
nuclear lamina. The envelope, seen in thin sections perpendicular to
the nucleus, is composed of a double membrane system with the inner and
outer membrane parallel to one another until they approach the pore
complexes. In general, each membrane is approximately 75 A in diameter.
A homogenous region, which varies in width from 100-600 A depending on
the cell type, lies between the two membranes and is referred to as the
perinuclear cisterna (Wischnitzer, 1958). Often, the luminal space of
the perinuclear cisterna is observed to be in direct continuity with the
luminal cavity of the endoplasmic reticulum (Watson, 1955). The outer
and inner membranes are associated with different structures.
Polyribosomes are seen to be attached to the outer membrane, whereas,
the inner surface of the inner membrane appears to have an intimate
association with a distinct morphological layer, referred to as the
lamina. These observations suggest that the inner and outer membranes

4
are different, although they have not been analyzed by biochemical
methods.
As visualized by electron microscopy, the nuclear lamina is a
discrete layer of material, varying in thickness from 30-100 nm, lining
the entire inner surface of the nuclear envelope, excluding the pore
complexes. A lamina enriched fraction is obtained by non-ionic
detergent and high salt extraction of isolated envelopes (Aaronsen and
Blobel, 1975; Dwyer and Blobel, 1976). Protein analysis of the residual
fraction shows the presence of three predominant proteins, referred to
as lamins A, B, and C. They comprise approximately 40% of this
fraction. During interphase, these polypeptides have been localized, by
immunocytochemistry, exclusively to the lamina at the nuclear periphery
(Gerace et al., 1978). Gerace and Blobel (1982) have shown that the
lamina forms a fibrous network that is a component of the nuclear
skeleton. The lamina has been implicated to function as a structural
framework for the nuclear envelope and an anchoring site at the nuclear
periphery for chromatin. In addition to its structural role, the lamina
is thought to be involved in reversible nuclear envelope assembly and
disassembly during mitosis (Gerace et al., 1984).
The pores are filled with diffuse electron-opaque material that
extends a short distance into both the nucleus and cytoplasm. In
tangential sections, this electron-opaque material has a ring-like
appearance and has been referred to as the annular material (Watson,
1955; Wischnitzer, 1958). The pore with its associated annulus is
called the pore complex.

5
Discrete subunits, 100-250 A in diameter, are distributed evenly
around both the cytoplasmic and nuclear margins of the pore (Watson,
1955; Wischnitzer, 1958). Franke (1966) using a multiple rotational
exposure technique on negative-stained preparations of isolated
envelopes, describes the arrangement of annular globules (granules) to
be in eight-point radial symmetry. Furthermore, the subunits lying on
the pore margin within the nucleus overlap the same sites as subunits on
the cytoplasmic pore margin.
Within the centers of some pores, an electron-dense granule with
diameters ranging from 40-350 A can be observed. This central granule
has been visualized with different preparative techniques; however, the
observed frequency of its presence is highly variable (Franke and
Scheer, 1970). It has been suggested that the granule is actually
material in transit, e.g. ribonucleoprotein (RNP), and that a
correlation exists between the frequency of the central granule and the
activity of RNA synthesis (Scheer, 1970). Cytochemical studies
performed by Franke and Falk (1970) support this view. Even with this
data it still remains controversial whether the granule is a permanent
structural component of the pore complex or material passing through the
pore.
Thin filaments, 25-50 A in diameter, have also been observed in the
interior of the pore. These filaments radiate laterally from the pore
margins and appear to connect with the central granule (Franke, 1974).
The filaments have a spokelike radial appearance and exhibit eightfold
symmetry similar to that of the annular globules.

6
Unwin and Milligan (1982) have used thin section electron
microscopy coupled with Fourier averaging methods to describe the pore's
three-dimensional organization to a resolution of 90 A. They found the
pores, in Xenopus oocytes, to occupy 20-30 percent of the envelope
surface and are composed of several discrete components: plugs, spokes,
particles, and rings. The rings have internal and external diameters of
800 A and 1200 A, respectively. Spokes extend radially from the margins
of the channel toward a plug (30-350 A in diameter) located in the
center of the pore. Particles are seen on the cytoplasmic surface of
the pores but do not appear to be integral components, as they can be
released with high salt treatments. This model, with its high
resolution of analysis, confirms many of the components put forth in the
model described by Franke (1974).
Until recently, little was known about the molecular composition of
the pore complex. Gerace et al. (1982) have identified a 190 kd
intrinsic membrane glycoprotein that is specifically localized to the
nuclear pores in liver nuclei. After detergent treatment, which
solubilizes most nuclear envelope intrinsic proteins, the 190 kd
glycoprotein remains associated with the pore complex-nuclear lamina
fraction. Based on this and its biochemical properties, it is suggested
that the 190 kd glycoprotein is involved in anchoring the pore complex
to the envelope. Davis and Blobel (1986), using monoclonal antibodies
and immunoelectron microscopy, have identified a 62 kd protein that
localizes within the annuli of rat liver nuclei. Snow et al. (1987)
have described a group of eight glycoproteins, with common epitopes,
that are localized exclusively to the nuclear pore complex. The group

7
of eight glycoproteins exist in multiple copies per pore complex and
their function is unknown at this time.
Compiling the data available on the components of the pore complex,
many investigators have suggested models or schematic representations of
the nuclear envelope architecture. These models, in conjunction with
the biochemical data, provide a framework for investigating how the
nuclear pores might be involved in regulating nucleocytoplasmic
exchanges. The appendix presents a schematic that is based on Franke's
(1974) and Unwin and Milligan's (1982) data.
Nuclear Permeability Experiments
It is clear from morphological evidence that the nuclear envelope
with its associated pore complexes is different from the plasma membrane
and other intracellular membranes. Since cellular processes both within
the nucleus and cytoplasm are dependent on a mutual exchange of
material, an important question arises as to the permeability properties
of the nuclear envelope and to what extent the envelope might regulate
nucleocytoplasmic exchange.
Three general approaches are used to study intracellular exchanges
across the nuclear envelope. First, microinjection of labelled tracers
(fluorescent, radioactive or electron-dense) is employed extensively to
study the permeability of the nuclear envelope to molecules within
intact cells. Second, permeability experiments can be performed on
isolated nuclei; however, these results must be carefully interpreted as
the permeability characteristics can change as a result of the isolation
(e.g., Paine et al., 1983; Peters, 1986). Third, recombinant DNA

8
methodology has been used to probe structural features of proteins that
are specifically targeted to the nucleus.
Ion permeability
Ions are known to interact with chromatin and activate specific
genes. During mitosis, meiosis, DNA synthesis, and hormonal
stimulation, ionic changes are occurring within the cell; thus, it is
important to understand intracellular ion distributions and how the
envelope might regulate their exchange between the nucleus and
cytoplasm.
Earlier permeability studies were qualitative in nature and based
on the structural or colloidal changes that occurred within isolated
nuclei following incubation in different salt solutions. It was
concluded from these studies that both cations and anions can rapidly
penetrate the nuclear envelope.
The use of radioactive isotopes as tracer molecules provided a new
method to give precise data on the permeability of the envelope.
Abelson and Duryee (1949) incubated individual oocytes in ^4Na+ and
later studied the intracellular ion distribution by quick freezing and
radioautography. The results demonstrated that the nuclear envelope in
amphibian oocytes was readily permeable to ^Na+. in addition to these
findings, the nucleus, at equilibrium, contained twice as much
radioactive tracer per unit volume as the cytoplasm. The measurement of
water content in both compartments indicated the nucleus to have a
higher percentage of water than the cytoplasm, which could account for
the difference in Na+ concentrations. Other molecules, ^K+, ^pg^

9
35S0=4> leucine-^C, and alanine-^C were shown by Naora et al. (1962)
to pass readily across the nuclear envelope.
To measure precisely the intracellular distribution of Na+, Horowitz
and Fenichel (1970) utilized ultra-low temperature autoradiographic
techniques. They described at least three intracellular Na+ fractions:
a cytoplasmic fast fraction, a cytoplasmic slow fraction, and a nuclear
fast fraction. The fast fractions were thought to represent freely
diffusible Na+, whereas the slow fraction is interpreted to be Na+ bound
to nondiffusible elements within the cytoplasm. This slow diffusion
fraction could account for the higher concentration of the Na+ ion found
within the cytoplasm. Therefore, the asymmetric ion concentrations can
be accounted for by two factors: first, the differences in water
content between the nucleus and cytoplasm, and second, possible binding
in the cytoplasm.
Another method for studying the permeability of the nuclear
membrane to ions is by measuring the envelope's electrical resistance.
Inserting intracellular microelectrodes into the nucleus and cytoplasm
of amphibian oocytes, Kanno and Loewenstein (1963) found no appreciable
resistance, indicating that ions are able to diffuse freely across the
envelope. Comparing the results from oocytes, the flux measurements and
electrical resistance data are in good agreement.
The results presented above suggest that the nuclear envelope is
not a barrier against ion diffusion. Since the nuclear envelope does
not impede ion passage and ion transport by the nuclear envelope is not
indicated (Century et al., 1970), it is likely that ions pass through
the aqueous channel formed by the nuclear pores.

10
Small molecules
Permeability of the nuclear envelope to small molecules was studied
by microinjecting sucrose (Horowitz, 1972), inulin (Horowitz and Moore,
1974), and the non-metabolizable amino acid a-aminoisobutyric acid (AIB)
(Frank and Horowitz, 1975) into oocytes. All of these small molecules
rapidly crossed the envelope such that standard flux measurements could
not accurately define the rate of movement, suggesting that the nuclear
envelope is of equivalent permeability to an equal thickness of
cytoplasm. To better define the uptake kinetics of small molecules,
Kohen et al. (1971) used high resolution microfluorimetry to quantitate
rapid movement across the envelope. They investigated the permeability
of glycolytic intermediates. Their results demonstrate that the
metabolites move across the nuclear envelope with a 35 millisecond
delay, indicating that for small molecules the nuclear envelope does not
constitute a permeability barrier. Thus, similar to ions, it is likely
that small molecules migrate into the nucleus through the nuclear pores.
Exogenous macromolecules
The nuclear uptake of radio-labelled or fluorescein-labelled
exogenous molecules has been investigated by microinjecting these
tracers into the cytoplasm of cells and following their intracellular
distribution with time. Colloidal gold particles and ferritin, both of
which can be visualized with the electron microscope, have also been
used to study nuclear permeability. Microinjection of these exogenous
tracers provides a method to better understand some of the physical
characteristics of the nuclear envelope with respect to diffusion.

11
The pores, from a morphological standpoint, would appear to be a
likely pathway for movement into and out of the nucleus. In support of
this view, Feldherr (1962) injected colloidal gold particles coated with
polyvinylpyrrolidone (PVP) into the cytoplasm of the amoebae Chaos chaos
and localized the distribution of the tracers by electron microscopy.
After 1-2 minutes, gold particles were observed in the centers of the
pores and 24 hours after injection, gold particles were found to
accumulate in the nucleus. Injection of different size gold fractions,
20-50 A and 20-160 A in diameter, showed that small gold particles
localized within the nucleus after 3 min, in contrast to the larger gold
fraction which had negligible uptake after 10 min. These results
indicate that the rate of movement across the nuclear envelope is
inversely proportional to the molecular size of the tracer. After
longer time intervals, it was determined for Amoeba proteus that PVP-
coated gold particles up to 120 A can penetrate the pores of the nuclear
envelope, whereas in Chaos chaos, particles as large as 140 A can enter
the nucleus (Feldherr, 1965). It is important to note that the maximum
size of the particles able to penetrate the envelope is smaller than the
800 A internal diameter of the pores. Overall, it was concluded from
these studies that pores can serve as pathways for nucleocytoplasmic
exchange but can restrict the movement of large molecules since the
entire area of the pore is not available for free exchange.
Gurdon (1970) studied permeability properties of the nuclear
envelope by injecting ^5¡_iabelled histone (10,000-20,000 kd) and
bovine serum albumin (67,500 kd), into amphibian oocytes. After 50
minutes, it was demonstrated by autoradiographic analysis that the

12
nucleus accumulated ^5¡_|1ist-one to at least twice the concentration of
the cytoplasm. In contrast, the ^I-BSA was more concentrated in the
cytoplasm, even after 12 hours. It was clear from these results that
the nuclear envelope shows some selectivity for inhibiting passage of
large exogenous proteins.
The nucleocytoplasmic exchange of a range of exogenous
macromolecules was studied quantitatively by Paine and Feldherr (1972).
Well characterized macromolecules with respect to size, shape, and
electrical charge were used: ferritin (mol wt 465,000; pi 4.4), BSA
(mol wt 67,000; pi 4.71), ovalbumin (mol wt 45,000; pi 4.7), myoglobin
(mol wt 17,500; pi 6.9), lysozyme (mol wt 14,500; pi 11.8); and
cytochrome c (mol wt 13,000; pi 10.7). All the proteins were
f1uorescently labelled, with the exception of ferritin which is
electron-opaque, and injected into the cytoplasm of oocytes from
Periplaneta americana. Their results showed that ferritin and BSA had
extremely low N/C ratios even after 5 h. Ovalbumin slowly entered the
nucleus; however, a higher concentration was present in the cytoplasm as
compared to the nucleus after 5 h. On the other hand, cytochrome c
entered the nucleus within 5 min, whereas lysozyme and myoglobin had N/C
ratios greater than 1.0 within 3 min. These results demonstrated that
neutrally or positively charged proteins, less than 20 A in diameter,
can readily enter the nucleus. Furthermore, the nuclear uptake of the
proteins, 20-95 A in diameter, was inversely related to size and not
simply based on charge. Similar conclusions were reached by Bonner
(1975a) who studied the nuclear uptake of the small basic proteins;

13
histones, lysozyme, and trypsin inhibitor, and the neutral protein,
myoglobin.
To determine if exogenous macromolecules exit the nucleus, Paine
(1975) injected ovalbumin, horseradish peroxidase, myoglobin, and
cytochrome c directly into the nucleoplasm of Chironomus salivary gland
cells. The results showed that the fluorescein-labelled molecules enter
and leave the nucleus with similar kinetics, indicating that
nucleocytoplasmic exchange is bidirectional and the rate of movement
across the envelope is dependent on 1) the restrictions imposed by the
diameter of the diffusion channel and 2) the size of the molecule.
To estimate the patent diameter of the nuclear pores in amphibian
oocytes, Paine et al. (1975) injected tritiated dextrans with known
hydrodynamic radii of 12.0, 23.3, and 35.5 A. Dextrans are uncharged
polymers of D-glycopyranose and available in a range of molecular sizes.
To determine the intracellular localization of the tracers, the cells
were quickly frozen at different time intervals, sectioned, and
autoradiographed. Comparison of the grain profiles for all three
tracers showed that the nuclear envelope is less permeable to larger
dextrans, supporting the view that the envelope can limit the rate of
entry of larger macromolecules. The relationship between the size of
the dextrans and their rates of entry suggested that the nuclear
envelope had a sieving effect due to its porous nature. Developing a
mathematical model from the rates of uptake, diffusional coefficients,
and the length of the channel, the authors estimated the patent pore
diameter to be 90 A.

14
Stacey and Allfrey (1984) also used well characterized proteins to
study the permeability of the nuclear envelope of cultured cells.
Thirteen proteins ranging from 13 kd to 669 kd with pi's from 4.4 to 11
were microinjected into the nucleus or cytoplasm of HeLa cells.
Proteins less than 40 kd entered almost immediately, in contrast to
proteins between 40 kd and 60 kd which entered more slowly. Many of the
proteins larger than 60 kd were excluded from the nucleus. The rates of
diffusion into and out of the nucleus were indistinguishable.
To estimate the patent pore diameter in cultured liver cells,
Peters (1984) used fluorescent microphotolysis to study the permeability
properties of the nuclear envelope. Fluorescein-labelled dextrans
ranging from 3 to 150 kd were injected into hepatocytes and allowed to
reach equilibrium. A pulse from an argon laser depleted the fluorescent
signal within a region of the nucleus and then microfluorimetry was used
to measure the influx of the tracer back into the depleted region. His
results showed the influx of the dextrans, from the cytoplasm to the
nucleus, to have an exclusion limit between 17 and 41 kd. Using similar
equations as Paine et al. (1975), Peters derived the functional pore
diameter to be 100-110 A with respect to diffusion.
All of the experiments involving the microinjection of exogenous
tracers support the view that the nuclear envelope has properties of a
molecular sieve. The entry of exogenous tracers into the nucleus occurs
by non-selective mechanisms, that is, diffusion through the pores. The
rate of influx is inversely related to size which reflects some of the
limitations imposed by the pore complex. The pore diameter has been
estimated for amphibian oocytes (90 A; Paine et al., 1975),

15
amoebae (120-140 A, Feldherr, 1965), and hepatocytes (100-110 A, Peters,
1984). These differences in size can markedly affect the exclusion
limits and flux rates of macromolecules. For example, it would take
approximately 1 h for a molecule, 50 A in diameter, to diffuse through a
pore with a diameter of 110 A. In contrast, it would take the same
molecule 8 h to pass through a pore with a diameter of 90 A (estimates
from Fig. 6; Paine et al., 1975). This can explain the 2-5 orders of
magnitude difference in dextran flux rates between oocytes and
hepatocytes.
Endogenous macromolecules
Subsequent nuclear permeability studies have focused on the
selective uptake of endogenous proteins. Labelled endogenous proteins
can serve as probes to investigate the normal molecular interactions and
behavior found within cells, in contrast to exogenous molecules, which
are unlikely to have intracellular interactions and solely used to
characterize the passive permeability properties of the nuclear
envelope. It soon became evident that the rates of nuclear accumulation
of endogenous molecules were much greater than exogenous molecules of
comparable sizes. The data demonstrated that exchanges of endogenous
proteins between the nucleus and cytoplasm were controlled by selective
processes, explaining in part, the observation that endogenous proteins
distribute differently than exogenous tracers.
There are two possible mechanisms that can account for the rapid
rate of endogenous protein accumulation within the nucleoplasm. First,
proteins can diffuse freely through the nuclear pores and then can be

16
selectively retained within the nucleus, by binding to a nondiffusible
substrate. Second, the exchange of some or all of the endogenous
proteins, through the pores, can occur by facilitated uptake. This mode
of exchange is governed by the intrinsic properties of the molecule.
Following translocation across the envelope, accumulation in the nucleus
can occur by selective binding or by another mechanism for irreversible
transport.
It has been reported that many proteins accumulate in the nucleus
against a concentration gradient--e.g., for example, histones (Gurdon,
1970) and N1/N2 (De Robertis et al., 1978)--attain nuclear to
cytoplasmic ratios of 115 and 120, respectively. Austerberry and Paine
(1982) used cryomicrodissection and two-dimensional electrophoresis to
measure quantitatively the intracellular distribution of 90 proteins in
the living cell. Their results demonstrated that at least 35 proteins
within the nucleus have nuclear to cytoplasmic ratios greater than 10,
which suggests some form of selective retention. Additional evidence to
support nuclear binding comes from the experiments performed by Feldherr
and Ogburn (1980). When the permeability properties of the envelope are
altered, as shown by its failure to exclude labelled BSA, most of the
endogenous nuclear proteins still accumulate within the nucleus (see
below).
Bonner (1975b) studied the migration of iji vivo labelled endogenous
proteins into nuclei of amphibian oocytes. After injection of labelled
nuclear proteins into the cytoplasm of unlabelled oocytes, the
radioactivity was found to concentrate in the nucleus. If labelled
cytoplasmic proteins were injected into the cytoplasm of unlabelled

17
oocytes, radioactivity was concentrated in the cytoplasm although some
labelled protein entered the nucleus. From these results, Bonner
suggested the existence of three classes of proteins: N proteins, found
predominantly in the nucleus; C proteins, found predominantly in the
cytoplasm; and B proteins, found in equivalent amounts in both the
nucleus and cytoplasm. Feldherr (1975) measured the rate of nuclear
uptake for endogenous proteins that were labelled with tritiated amino
acids i_n vivo. His results demonstrated that newly synthesized nuclear
proteins, ranging from 94,000 to 150,000 kd, were three times more
concentrated within the nucleus as compared to the cytoplasm after 6 h.
The rates of accumulation for endogenous proteins of this size were
greater than expected in comparison to the results obtained for
exogenous proteins of similar size.
To better understand the role of the nuclear envelope in regulating
nucleocytoplasmic exchange of endogenous proteins, Feldherr and
Pomerantz (1978) altered the diffusion barrier by using glass needles to
puncture holes in the envelope and then measured the uptake of labelled
nuclear proteins. The results, as determined by one-dimensional gel
electrophoresis, showed no qualitative differences in the accumulation
of nuclear proteins in punctured nuclei as compared to nuclei from
nonpunctured control oocytes. Feldherr and Ogburn (1980) further
studied the mechanism of selecting nuclear proteins by first disrupting
the nuclear envelope and then using two-dimensional gel analysis,
fluorography, and double-labelling techniques to determine intracellular
distributions. Analysis of over 300 nuclear polypeptides showed less
than five percent of the proteins varied between experimentally

18
punctured oocytes and nonpunctured controls. It was concluded from
these results that nuclear binding plays a major role in regulating the
nucleocytoplasmic distribution of endogenous proteins, and that the
envelope does not function as a rate-limiting barrier.
It is apparent from the above studies that many of the nuclear
proteins accumulate in the nucleus at significantly higher rates than
exogenous molecules of similar size. As discussed previously, exogenous
proteins larger than 45 kd (ovalbumin) do not readily enter the nucleus;
however, endogenous proteins larger than 90 kd accumulate quite rapidly.
Evidence for facilitated transport was obtained by Feldherr et al.
(1983) for RN1, a 148 kd nuclear protein, in the oocytes of Rana
pipiens. After microinjection or i_n vivo labelling of RN1, the kinetic
results demonstrated rapid accumulation within the nucleoplasm.
Theoretical estimates of the diffusion rate of a 148 kd protein through
the 90 A channel indicated that the rate of uptake could not be
accounted for by simple diffusion through the pores and subsequent
binding. Thus, in this instance some form of mediated transport is
apparently required for rapid accumulation.
Recent research has shown that signal sequences of some nuclear
proteins are required to target macromolecules to the nucleus. Two
approaches have been used to study the polypeptide domains that contain
these sequences. The first approach is the partial digestion of a
protein followed by analysis of the fragments capable of accumulating in
the nucleus and the second approach employs DNA methodology.
The first approach has been successfully employed to study
nucleoplasmin, a 110 kd karyophilic protein in Xenopus oocytes, which

19
rapidly accumulates in the nucleus. Nucleoplasmin is a thermostable,
pentameric molecule present within the nucleus in a high concentration
(Mills et al., 1980). It appears in the electron microscope as a disc
with a molecular dimension of 74 A in diameter (Earnshaw et al., 1980).
Pepsin digestion selectively cleaves nucleoplasmin into two structural
domains, a protease-resistant core and five protease-sensitive tails
(Dingwall et al., 1982). Protease removal of the five tail domains
leaves a pentameric core that is excluded from the nucleus after
microinjection into the cytoplasm. In contrast, the tail domains, after
cytoplasmic injection, are capable of nuclear accumulation. When the
core is injected directly into the nucleus, it migrates throughout the
nucleoplasm but is still excluded from the cytoplasm, indicating that
the core has the ability to be retained by the nucleus but not the
ability to migrate into the nucleus. These results demonstrate that the
tail domain contains the necessary information to specify selective
entry. By reducing the time of digestion, a mixture of pentameric cores
that contain different numbers of tail regions can be prepared. A core
molecule with only one tail is capable of entering the nucleus, but at
one-tenth the rate of intact nucleoplasmin (Dingwall et al., 1982).
Amino acid analysis of the tail domain (C-terminus) indicates an
enrichment in lysine residues and a depletion in hydrophobic amino
acids.
The second approach, utilizing genetic engineering, entails the
construction of plasmids encoding proteins normally found in the
cytoplasm linked to putative nuclear signal sequences. The end product
is a hybrid protein, converting an otherwise cytoplasmic protein into a

20
protein localizing in the nucleus. After narrowing down a region of the
protein necessary for nuclear localization, oligonucleotide-directed
mutagenesis can be used to map out the essential amino acid sequence
necessary for targeting. Overall, these approaches have been used to
study an array of proteins that are targeted to specific organelles:
endoplasmic reticulum, mitochondria, nuclei, and peroxisomes.
The simian virus (SV) 40 large T-antigen targeting signal has been
the most extensively studied sequence that confers nuclear localization.
Large T-antigen, during lytic infection, is responsible for the
regulation of viral transcription and replication. The protein has 708
amino acids and in the monomeric form (can be tetrameric) has a
molecular weight of 94 kd. The size of this protein suggests that it
cannot enter the nucleus through the pores by simple diffusion, although
large T-antigen is found predominantly within the nucleoplasm. Butel et
al. (1969) described a large T-antigen from a mutant SV 40 hybrid virus
that was localized in the cytoplasm of an infected cell. Comparison of
the sequence analysis of wild type large T (WT) to the mutant large T-
antigen (cT) revealed the amino acid at the 128 position was changed
from lysine to asparagine (Lanford and Butel, 1984). The mutant large
T-antigen was found to be soluble in the cytoplasm and capable of
binding DNA (Lanford and Butel, 1980). Thus, the failure to accumulate
in the nucleus was not caused by its inability to either diffuse
throughout the cytoplasm or bind DNA, but caused by a mutation in a
critical region of the protein sequence that is required for nuclear
localization.

21
Kalderon et al. (1984a) used site-directed mutagenesis to identify
a seven amino acid sequence within large T-antigen that is required for
nuclear localization, with the lysine at position 128 being the critical
residue. A change in this lysine residue to asparagine, threonine,
isoleucine, leucine, methionine, or glutamine caused the large T-antigen
to become localized in the cytoplasm of the cells. Mutations within
the signal sequence on either side of the lysine influenced the rate of
uptake and qualitative distribution of large T-antigen, but did not
abolish transport activity. The minimum sequence required for nuclear
localization is Pro-Lys-Lys^-Lys-Arg-Lys-Val (Kalderon et al., 1984b).
One important aspect of this signal sequence is its ability to function
when removed from its normal environment and placed in a different
region of a protein. For example, if the signal is linked to the amino
terminus of either a mutated large T-antigen or chicken muscle pyruvate
kinase (a nonnuclear protein), it can still confer nuclear localization.
In addition, a synthetically synthesized signal peptide cross-linked to
a carrier protein has the ability to target the conjugate into the
nucleus of cultured cells (Lanford et al., 1986; Yoneda et al., 1987) as
well as oocytes (Goldfarb et al., 1986). Thus, the functional autonomy
of the targeting signal suggests that it does not require a fixed
secondary structure.
Recently, nucleoplasmin has been cloned and sequenced (Burglin and
De Robertis, 1987; Dingwall et al., 1987). The fifty amino acids from
the carboxy terminal tail domain have two regions similar in homology
with the nuclear localization signal from the SV 40 large T-antigen
(Dingwall et al., 1987). In addition, two other regions have been

22
identified to have weak homology with targeting signals from the yeast
MATa2 protein. It was postulated from these results that each tail
domain might have four targeting signals, with a total of 20 signals per
pentamer. Further experimentation on the tail domain has now
demonstrated that each tail contains only 1 signal sequence consisting
of approximately 14 amino acids and though it is similar to the SV 40 T-
antigen targeting signal, it is not homologous (Dingwall, personal
communication 1988).
The list of proteins containing nuclear targeting signals continues
to be expanded. In a search to find a conserved sequence, several
targeting signals have been compared to SV 40 large T-antigen. Protein
signals that have similar amino acid sequences include nucleoplasmin
(Dingwall et al., 1987), polyoma virus large T-antigen (Richardson et
al., 1986), SV 40 VP1 (Wychowski et al., 1986), and Xenopus laevis N1/N2
protein (Kleinschmidt et al., 1986). Nuclear localization sequences
from other proteins showing little homology to SV 40 large T-antigen
include the adenovirus Ela protein (Lyons et al., 1987), the yeast
proteins L3 (Moreland et al., 1985) and MATa2 (Hall et al., 1984), and
GAL4 (Silver et al., 1984).
The translocation of proteins directly across intracellular
membranes has been well documented for endoplasmic reticulum (Warren and
Dobberstein, 1978) and mitochondria (Schatz and Butow, 1983). Proteins
destined for either organelle contain a signal sequence, which is
cleaved after entry. Protein import into the endoplasmic reticulum
occurs by a cotranslational mechanism; however, this mechanism is
unlikely for nuclear uptake since cytoplasmically injected proteins,

23
extracted from the nucleus, can accumulate back into the nucleus.
Mitochondrial proteins have signal sequences that target them into
specific regions of the mitochondria and cross the membrane(s) by a
posttranslational mechanism. However, no evidence exists that nuclear
transport occurs by a posttranslational process directly across the
membrane, although it still remains a possibility.
An elegant approach to study the exchange sites of a known
transportable nuclear protein was performed by coating colloidal gold
particles with nucleoplasmin (Feldherr et al., 1984). Their results
demonstrate that nucleoplasmin-coated gold enter the nucleus through
channels located in the centers of the pores. Particles, ranging in
size from 50-200 A, were able to penetrate the pore region, thus
indicating that the functional size of the transport channel is
different than the size of the diffusion channel. The transported
particles are nondeformable and spherical, which argues against
structural deformation of nucleoplasmin prior to transport.
Furthermore, the particles are markedly larger than the diffusion
channel, which suggests the involvement of a selective uptake process.
In vitro uptake
The use of an i_n vitro assay system to investigate protein
transport has both advantages and disadvantages. The drawbacks are,
first, changes in the permeability characteristics of isolated nuclei.
Peters (1983) measured the permeability of single isolated liver nuclei
to dextrans using fluorescent microphotolysis and estimated the pore
diameter to be 112-118 A, which is 2-8 A larger than pores from i_n vivo

24
nuclei (see exogenous molecules section). According to the model of
Paine et al. (1975), a small change in pore diameter can have a marked
effect on flux rates. Second, after isolation of oocyte nuclei under
aqueous conditions, 95% of the proteins is lost with a half-time of
250 s (Paine et al., 1983). Third, assuming some nuclear proteins are
transported along cytoskeletal elements to the nuclear surface, the
isolation procedure removes the nucleus from its cytoplasmic anchorage,
thus removing a possible step in transport. In light of these
disadvantages, the results from the i_n vitro experiments must be
carefully interpreted.
An i_n vitro system is advantageous to study protein uptake since
the steps in the process can be easily dissected and experimentally
manipulated. In vitro assays can be used: i) to characterize
components needed for translocation, ii) to determine inhibitors of
protein transport, and iii) to determine if protein transport is ATP-
dependent.
Forbes et al. (1983) have shown that bacteriophage DNA
microinjected into Xenopus eggs can form nucleus-like structures.
Addition of DNA from either bacteriophage lambda or Xenopus to an
extract from Xenopus eggs results in the formation of synthetic nuclei
that have morphological similarities to intact nuclei (Newmeyer et al.,
1986). These synthetic nuclei have a double membrane envelope, however,
not all possess nuclear pore complexes. The reconstituted nuclei, in
conjunction with labelled nucleoplasmin, have been used to investigate
aspects of the protein transport process. Newmeyer et al. (1986)
demonstrated that nucleoplasmin uptake is ATP- and temperature-

25
dependent. In addition, the lectin wheat germ agglutinin (WGA)
effectively inhibits the transport of nucleoplasmin and the inhibitory
effects could be blocked with the addition of N-acetylglucosamine
(Finlay et al., 1987). Ferritin-labelled WGA was shown to be localized
to the cytoplasmic face of the nuclear pores. Furthermore, iodinated
WGA stains a glycoprotein, 63-65 kd, on nitrocellulose blots of rat
liver nuclei (Finlay et al., 1987). This glycoprotein is similar to the
one detected by Davis and Blobel (1986) and Snow et al. (1987). It has
been suggested that this glycoprotein is part of the pore complex and
might be involved in the nuclear transport of proteins.
Nuclear Efflux
Introduction
The nucleus synthesizes the RNAs required for protein synthesis;
thus, the efflux of messenger RNA (mRNA), ribosomal RNA (rRNA), and
transfer RNA (tNRA) is an obligatory step in the process of gene
expression. Heterogeneous nuclear RNA (hnRNA), small nuclear RNAs
(snRNA) and tRNA precursors are found normally within the nucleus.
Prior to mature mRNA export to the cytoplasm, hnRNA must undergo i)
processing, which involves removal of the introns and splicing together
of the exons, and ii) posttranscriptional modifications, which can
include polyadenylation and methylation. tRNA precursors must also be
processed before efflux and precursor rRNA is processed into 28S, 18S,
and 5.8S RNAs and are present in 40S and 60S RNP particles prior to
transport into the cytoplasm.

26
Goldstein and Plaut (1955) obtained direct evidence in Amoeba
proteus for the transfer of RNA to the cytoplasm by first labelling the
RNA and then transplanting the labelled nucleus to an unlabelled
recipient cell. As shown by autoradiography, the number of grains
within the cytoplasm increased with time. In addition, no radioactivity
was observed in the unlabelled recipient cell nucleus. Ribonuclease
digestion was performed to show that the radioactivity was associated
with the RNA. Due to the size of the RNA, it was postulated that simple
diffusion was not the mechanism involved in RNA efflux.
The movement of RNA from nucleus to cytoplasm is likely to be a
three-step process, involving 1) migration from its site of
transcription to the inner surface of the nuclear envelope, 2)
translocation across the envelope, presumably through the nuclear pores,
and 3) movement from the outer surface of the nuclear envelope to its
final destination within the cytoplasm. The first two steps have been
the focus of intense investigation in an attempt to understand the
mechanism(s) involved in RNA transport. The final step of directing RNA
to its destination within the cytoplasm is not as well characterized as
steps 1 and 2. In addition, Agutter (1985a) has suggested that RNA
efflux occurs by a solid-state process; that is, movement occurs along
structural elements both within the nucleus and cytoplasm.
Transfer RNA
The structure of tRNA and its involvement in protein synthesis have
been the subject of many investigations. Transfer RNAs are small,
compact molecules. In the crystallized state, yeast tRNAP^e has a

27
length of 77 A and an average thickness of 20 A (Kim et al., 1973). A
Stokes's radius of 34 A was derived for tRNAP*16 from its translational
diffusion coefficient in water (Peters, 1986). A molecule having these
dimensions will not readily diffuse through the nuclear pores.
Injection of the cloned tRNAtyr yeast gene into the nuclei of oocytes
produces a precursor transcript with a length of 108 nucleotides (Melton
et al., 1980). After processing, mature tRNA (78 nucleotides long) is
then exported into the cytoplasm. De Robertis et al. (1982) injected
labelled tRNA into the cytoplasm of oocytes and showed it to be excluded
from the nucleus, suggesting that tRNA efflux is vectorial
(unidirectional).
Zasloff (1983) studied tRNA transport in amphibian oocytes. This
is a convenient system since oocytes can transcribe genes that have been
injected into the nucleus. In addition, analysis is facilitated by the
fact that nucleus and cytoplasm can be readily separated. Two genes
were used in this study; the first was normal human tRNAmet and the
second was a variant human tRNAmet with a G-to-U substitution at
position 57 in the highly conserved loop IV region. Nuclei were
injected with either the genes encoding for the two tRNAs or prelabelled
tRNAs, and the cells were fractionated at various times thereafter. It
was found by electrophoretic analysis of both the nucleus and cytoplasm
that 80% of the normal tRNA entered the cytoplasm within 5 min; in
contrast, 90% of the variant tRNA remained intranuclear after 15 min.
Precise kinetic analysis, performed by liquid scintillometry, showed the
rate of transport of the variant tRNA to be 20-fold slower than normal
tRNA. In addition, normal tRNA transport is saturable and displays

28
temperature dependence. Furthermore, tRNAP^e can act as an effective
competitor of tRNAmet transport. These results strongly suggest that
tRNA transport into the cytoplasm involves a carrier-mediated process
rather than simple diffusion.
In an attempt to define the critical region of the tRNA molecule
required for transport, Tobian et al. (1985) used site-directed
mutagenesis to obtain 30 different mutants with single nucleotide
substitutions. Many of the mutations perturbed the rate of transport.
The most deleterious effects were in the highly conserved T stem-loop
regions; however, many of these tRNAs had impaired processing. It is
suggested from this study that transport, in addition to processing, is
dependent on maintenance of the secondary structure.
The nuclear pores can serve as diffusion pathways for exogenous
tracers and as a transport pathway for some karyophilic proteins. As
mentioned above, the diffusion of tRNA out of the nucleus seems unlikely
due to i) its size and ii) the involvement of a carrier-mediated
process. Zasloff (1983) has suggested that the pores serve as the
pathway for exchange and that peripheral ribosome-like particles on the
cytoplasmic surface of the nuclear envelope provide the motor mechanism
required for transport.
Small nuclear RNAs
The snRNAs comprise 0.1-1.0% of the total cellular RNA. They are
small (100-250 nucleotides), stable, uridylic-acid rich RNAs that are
localized in the nucleus and are transiently found in the cytoplasm. At
least eight snRNAs, U1-U8, have been identified, and all are associated

29
with a core of five common proteins. Functional roles that have been
identified for a few of the U-snRNAs include polyadenylation,
intranuclear transport, and splicing of hnRNA.
To investigate intracellular RNA transport within intact cells,
De Robertis et al. (1982) injected radiolabelled RNA from HeLa cells
into the cytoplasm of Xenopus oocytes. The results demonstrate that
snRNAs can migrate from the cytoplasm into the nucleus and concentrate
there 30- to 60-fold. It was also established that HeLa snRNAs complex
with oocyte proteins in the cytoplasm before their migration into the
nucleus. Zeller et al. (1983) analyzed the distribution of snRNAs and
their associated binding proteins during oogenesis and early
development. Mature Xenopus oocytes and embryos prior to gastrulation
are known to contain an excess of snRNA binding proteins (De Robertis et
al., 1982) and less U1 and U2 snRNAs than previtellogenic oocytes. In
embryos, the midblastula transition (Newport and Kirschner, 1982) marks
the onset of a burst in snRNA synthesis and the migration of snRNA
binding proteins into the nucleus. Injection of labelled snRNA into the
cytoplasm of mature oocytes can prematurely displace the cytoplasmic
binding proteins into the nucleus, suggesting that movement of the
proteins requires the formation of the snRNPs. To determine whether
nuclear accumulation occurs only when snRNA and its binding proteins are
complexed together, Mattaj and De Robertis (1985) used site-directed
mutagenesis to delete the protein binding site on 112 snRNA. As a result
of the snRNA's inability to bind its cytoplasmic proteins, it becomes
unable to enter the nucleus. It is suggested that the formation of the
snRNP within the cytoplasm might unmask a cryptic targeting signal which

30
then allows for the accumulation within the nucleus. Whether the signal
is in the protein, the RNA, or possibly a combination of both, has yet
to be determined.
Ribosomal RNA
The ribosomal DNA transcript, located in the nucleoli, contains the
18S, 5.8S, and 28S genes. After transcription, the rRNA is complexed
with proteins to form a 80S ribosomal ribonucleoprotein particle, which
is processed to the mature 40S and 60S particles prior to cytoplasmic
transport. The 5S RNA is transcribed, processed, and transported
separately. These rRNPs are the major components involved in ribosome
biogenesis.
Previtellogenic oocytes (stages I and II; Dumont, 1972) synthesize
and store 5S RNA which comprises 30-40% of the total cellular RNA.
TFIIIA is a transcription factor with a molecular weight of 39 kd that
binds to 5S genes. In addition, TFIIIA also binds to cytoplasmic 5S RNA
to form a 7S ribonucleoprotein particle. By employing immunocyto-
chemical procedures on sections of Xenopus ovaries, Mattaj et al. (1983)
demonstrated that TFIIIA is located predominantly in the cytoplasm. In
previtellogenic oocytes, TFIIIA is associated with the 5S RNA (Picard
and Wegnez, 1979); thus, the immunocytochemical staining patterns are
actually localizing 7S RNPs. Pelham and Brown (1980) studied the
i_n vitro transcription of the 5S RNA gene and showed that transcription
could be inhibited when 5S RNA, in excess of the 5S gene, competed for
TFIIIA binding. They suggested from these results that TFIIIA might be
involved in a feed-back mechanism to control 5S gene expression. It is

31
important to mention that TFIIIA alone can enter the nucleus to bind to
the 5S gene; however, when bound to 5S RNA, it remains in the cytoplasm.
Free 5S RNA also is capable of entering the nucleus and it accumulates
in the nucleoli (De Robertis et al., 1982). The 7S RNP is able to
diffuse throughout the cytoplasm of oocytes (Ford, 1971) but is excluded
from the nucleus. The mechanism of exclusion is not yet known; however,
it is possible that the formation of the 7S RNP in the cytoplasm masks a
transport signal, either on the RNA or the protein that is required for
nuclear entry.
Wunderlich and co-workers have studied 18S and 28S rRNA transport
from isolated Tetrahymena macronuclei. They reported that rRNP efflux
is ATP dependent and the amount of RNP transport is sensitive to a shift
in temperature (Giese and Wunderlich, 1983). It was also found that
rRNA is integrated into the nuclear matrix which might control the
transport process since removal of the nuclear envelope had no effect on
the temperature dependence of rRNP export (Wunderlich et al., 1983).
Although 28S and 18S RNA leave the nucleus complexed with ribosomal
proteins, it is not clear whether the RNA or protein contains the efflux
signals.
The role of the nuclear envelope in regulating rRNA translocation
was investigated jn vivo (Feldherr, 1980) by disrupting the nuclear
envelope with glass needles, and i_n vitro (Stuart et al., 1977) by using
membrane-denuded nuclei. The efflux of rRNA from the nucleus to the
cytoplasm was not affected by either experimental procedure suggesting
that rRNA is most likely bound to nondiffusible nuclear elements.

32
Overall, tRNA, 5S RNA, and rRNA are generally believed to
translocate into the cytoplasm through the nuclear pores. However, much
of the evidence is circumstantial and there are no definitive data to
indicate that the pores serve as the major pathways for the efflux of
these different classes of RNA.
Messenger RNA
Heterogeneous nuclear RNA is processed and undergoes extensive
posttranscriptional modifications before mature mRNA can be transported
into the cytoplasm. Unspliced mRNA is retained in the nucleus and only
mature mRNA appears in the efflux supernatant from isolated nuclei iji
vitro or in the cytoplasm of intact cells, suggesting that processing is
one of the many requirements necessary for export. The movement of
mature mRNA, from its site(s) of transcription and processing, to the
nuclear envelope is the first step involved in export (see above).
Agutter (1985a) has suggested that mRNA might not be freely diffusible
throughout the cells. To determine whether RNA is preferentially
associated with the nuclear matrix, nuclei were treated with nonionic
detergents, high salt, EDTA, and urea. It was shown that greater than
90% of the nuclear RNA was tightly associated with the insoluble nuclear
matrix (Berezney, 1980; van Eekelen and van Venrooij, 1981). Release of
the mature ovalbumin mRNA but not the immature mRNA from the nuclear
matrix occurs in the presence of ATP and does not require hydrolysis of
the phosphodiester bond (Schroder et al., 1987). This selective
detachment from the matrix might be another level of control in
regulating the transport of mRNA. Overall, the migration of mature mRNA

33
to the envelope is likely to involve the processing of the precursor,
posttranscriptional modification, and release from the nuclear matrix.
Once at the inner surface of the nuclear envelope, the second step is
the translocation across the envelope, presumably through the pores.
Translocation of mRNA across the nuclear envelope appears to
involve a series of reactions requiring nucleoside triphosphatase
(NTPase), protein kinase, protein phosphohydrolase, and poly(A) binding
sites. Using isolated nuclei, Agutter et al. (1976) first reported a
correlation between RNA efflux and the presence of NTPase activity which
was localized in nuclear envelope fractions. Further studies on the
energy requirement of poly(A)+mRNA translocation were performed by
Clawson et al. (1978). These investigations determined that both ATP
and GTP could serve as energy sources.
Clawson et al. (1984) used photoaffinity labelling on rat liver
nuclear envelopes and localized the NTPase activity to a 46 kd protein.
Schroder et al. (1986a) subsequently purified and characterized a 40 kd
NTPase from rat liver nuclear envelopes which is thought to be the same
polypeptide identified by Clawson. In addition to the nuclear envelope,
NTPase activity was also found to be present in the matrix (Clawson et
al., 1984; Maul and Baglia, 1983). There are several experiments that
correlate the energy-dependent transport of poly(A)+mRNA with the NTPase
activity. First, an increase in the rate of RNA efflux by thioacetamide
treatment (Clawson et al., 1980), insulin administration (Purrello et
al., 1982), or tryptophan feeding (Murty et al., 1980), causes a
parallel increase in NTPase activity. Second, in a transport assay
using isolated nuclei, NTPase activity and mRNA efflux have similar

34
kinetic properties, substrate specificities, and sensitivities to
inhibitors (Agutter et al., 1976; Clawson et al., 1978, 1980). One
additional line of evidence that mRNA and NTPase are interrelated is the
inhibition of mRNA efflux by antibodies against pore complex-lamina
components that affect NTPase activity (Schroder et al.; unpublished
results).
The effects of poly(A) on NTPase activity were observed initially
by Agutter et al. (1977) on isolated, intact nuclear envelopes. They
showed that addition of poly(A) or poly(G) to the media could enhance
NTPase activity. Furthermore, Bernd et al. (1982) demonstrated the
NTPase activity to be markedly stimulated by either poly(A)+mRNA or a
homopolymer of poly(A) at least 15-20 bases long. Additional evidence
comes from experiments by Riedel and Fasold (1987). They prepared
resealed nuclear-envelope vesicles that retain NTPase activity and found
that both poly(A) and mRNA can stimulate the NTPase activity associated
with these vesicles. It is suggested from these data that poly(A) can
modulate NTPase activity and possibly regulate the translocation of
mRNA.
It has been demonstrated that protein kinases and phosphohydrolases
are present in the nuclear envelope (Lam and Kasper, 1979; Smith and
Wells, 1983; Steer et al., 1979a, 1979b). McDonald and Agutter (1980)
have studied the effects of polyribonucleotide binding on the
phosphorylation and dephosphorylation of nuclear envelope proteins and
found that the same polyribonucleotides that stimulate NTPase, inhibit
protein kinase activity and stimulate one of the phosphohydrolases.
They also found the nuclear envelope to contain a population of poly(A)

35
binding sites that increase in affinity by a kinase-dependent
phosphorylation reaction. The actual role these components play in the
in vivo translocation of mRNA across the envelope has not yet been
clearly defined; however, poly(A)+mRNA efflux models have been proposed
by Agutter (1985a) and Schroder et al. (1988).
The model proposed by Agutter (1985a) integrates the components
involved in RNA translocation: NTPase, poly(A) binding sites, protein
kinases and phosphohydrolase, to explain nucleocytoplasmic transport of
poly(A)+mRNA. The following scheme is based on Agutter's model (1985a).
The NTPase exists in a complex with the poly(A) binding site. A protein
kinase uses MgATP as a substrate to phosphorylate the poly(A) binding
site. Binding of nuclear poly(A)+mRNA to the [phosphorylated binding
site-NTPase] complex stimulates dephosphorylation by the
phosphohydrolase. MgATP is also a substrate for NTPase, the binding of
MgATP to the NTPase of the [poly(A)+mRNA-poly(A) binding site-NTPase]
complex causes the displacement of poly(A)+mRNA, thus facilitating the
translocation of mRNA. The NTPase then hydrolyses ATP and the [NTPase-
poly(A) binding site] complex is available again to start another cycle.
At least two cytosolic proteins, 34 kd and 58 kd, with an affinity
for poly(A) can stimulate mRNA efflux j_n vitro (Moffett and Webb, 1981,
1983; Schroder et al., 1986b). The 34 kd protein, in the presence of
poly(A), stimulates the NTPase activity to a greater extent than poly(A)
by itself. In the absence of poly(A), p34 has no effect on kinase or
phosphohydrolase activity. The 34 kd protein is thought to enhance
NTPase activity by increasing the affinity of poly(A) to the poly(A)
binding site in the phosphorylated nuclear envelope. Schroder et al.

36
(1988) suggest that the 58 kd protein inhibits kinase activity by
promoting poly(A) binding to unphosphorylated envelopes, which prevents
the down-regulation of NTPase activity by the kinase.
The third step in the overall RNA transport is the involvement of
the cytoplasm. Although it is established that much of the mRNA is
bound to the cytoskeleton (Jeffery, 1982), it is not clear how the RNA
finds its final destination. The proteins associated with poly(A)+mRNA
in the cytoplasm are different from those that are bound to poly(A)
within the nucleus (Baer and Kornberg, 1983; Sachs and Kornberg, 1985;
Setyono and Greenberg, 1981). It is not known if these proteins are
involved in the transport of RNAs within the cytoplasm or serve as
anchors to cytoskeletal elements. In this regard, it is necessary to
further understand the functional role of the cytoplasmic proteins
associated with RNAs.
Ribonucleoproteins (RNPs) have been visualized exiting the nucleus
through the nuclear pores by electron microscopic cytochemical analysis
(Stevens and Swift, 1966). In a more recent study, Skoglund et al.
(1983) studied the formation and transport of an hnRNP particle, a
transcription product from the Balbiani ring genes in Chironomus. Their
results, by using the Miller spread technique, describe the growth of a
100 A wide filament into a large 400-500 A granule. Prior to export,
these mature RNP particles undergo conformational changes that result in
the formation of rod-shaped structures with an average length of 1350 A
and diameters of 250-300 A that pass through the centers of the nuclear
pores. Even though these RNPs undergo a shape change, they are still

37
too large to pass through the pores by passive diffusion, suggesting
that some facilitated mechanism is a requirement for export.
Statement of Research Topic
It has been demonstrated that there are selective mechanisms which
facilitate the passage of certain endogenous macromolecules through the
nuclear pores. The mechanism and selectivity of the pore by which
transport occurs is not known; for example, does translocation proceed
through the pores by a gated mechanism, or does transport occur through
a fixed channel? How does the nuclear targeting signal interact with
the pore region to initiate translocation? Do different nuclear
transport signals use the same pore for uptake? What effects do
variations in the number and sequence of targeting signals have on
nuclear uptake? In relation to RNA transport, what is the precise
location for the translocation of different classes of RNA to the
cytoplasm? Are there specific functional classes of pores that are
involved in RNA transport? or do all pores have the capability to
translocate both RNAs and proteins?
To better understand the role of the nuclear envelope in regulating
nucleocytoplasmic exchange, experiments were designed to elucidate
characteristics of the macromolecular transport process. The data
obtained will focus on the translocation of RNAs. More specifically,
analysis will include the proportion of functional pores, the precise
location and size of the transport channels, and the multifunctional
capability of individual pores (i.e., both protein uptake and RNA
efflux). In addition, data will be obtained for the effect of different

38
numbers of synthetic nuclear targeting signals on protein uptake and the
functional size of the transport channel. It will also be determined
whether individual pores have the capability to transport proteins
containing different nuclear targeting sequences.
Many of the previous nucleocytoplasmic exchange experiments were
based on quantitative and qualitative studies performed with
fluorescein- or radio-labelled molecules; however, there are certain
characteristics of the exchange sites that cannot be studied using these
type of tracer molecules. In this study, colloidal gold particles will
be used to identify and characterize the sites for macromolecular
exchange.
Colloidal gold tracers have some unique properties that make them
ideal for this type of experimentation. First, gold particles with
different diameters can be easily prepared. Second, the surface
properties of the particles can be modified by the adsorption of
different molecules and the particles will then acquire the
characteristics of their coating agents. Third, tracer particles can be
localized precisely to specific cell structures by electron microscopy.
The following study will focus primarily on nucleocytoplasmic
transport of macromolecules, especially with regard to the
characteristics of the exchange sites located within the nuclear pores.
The experiments are performed on amphibian oocytes and involve the
microinjection of either RNA- or protein-coated gold particles into the
nuclear or cytoplasmic compartment of the cells. The subsequent
intracellular distribution of the tracer particles and their relation to
the nuclear pores will be determined by electron microscopy.

CHAPTER II
TRANSLOCATION OF RNA-COATED GOLD PARTICLES
THROUGH THE NUCLEAR PORES OF OOCYTES
Introduction
The nuclear envelope in eukaryotic cells is the site of continual
macromolecular exchange between the nucleoplasm and the cytoplasm. Such
exchanges can occur either by passive diffusion or mediated transport
through the nuclear pores (Dingwall and Laskey, 1986). Microinjection
of various sized exogenous molecules has been used in previous studies
to determine the dimensions of the diffusion channels within the pores.
The channels were estimated to be 90 A in diameter in amphibian oocytes
(Paine et al., 1975), and are available for diffusion both into and out
of the nucleus (Paine, 1975). Evidence for protein transport across the
envelope was initially obtained for RN1 (Feldherr et al., 1983) and
nucleoplasmin (Dingwall et al., 1982), both of which are major
karyophilic proteins found in amphibian oocytes. The specific sites of
transport for nucleoplasmin have been identified by microinjecting
nucleoplasmin-coated gold particles into the cytoplasm of the oocytes
(Feldherr et al., 1984). It was found, by electron microscopy, that
transport of the colloidal particles occurred through channels, at least
200 A in diameter, located in the centers of the nuclear pores. Thus,
the regions of the pores available for transport are considerably larger
than those available for diffusion.
39

40
The exit of mRNA from isolated nuclei has been studied in a number
of laboratories (reviewed by Clawson et al., 1985). These
investigations have shown that efflux is a temperature-dependent,
energy-requiring process, indicating that some form of transport is
involved (Agutter, 1985a; Clawson et al., 1978). In support of this
view, isolated nuclear envelope preparations were found to contain
nucleoside triphosphatase activity which is affected by the same factors
(ionic composition, pH, etc.) that regulate RNA efflux (Agutter, 1985a;
Clawson et al., 1980; 1984). It has also been suggested, on the basis
of results obtained in several laboratories, that the poly(A) tail of
mRNA is involved in transport across the envelope (Agutter, 1985b; Bernd
et al., 1982).
The efflux of tRNA from the nucleus has been investigated by Zasloff
(1983) using i_n vivo procedures. He obtained evidence for a saturable,
carrier-mediated transport mechanism for tRNAmet in the amphibian
oocyte. De Robertis et al. (1982) have demonstrated that microinjected
5S RNA can migrate either into or out of the nucleus in Xenopus oocytes;
however, the mechanism of exchange was not determined.
It is generally believed that RNA transport occurs through the
nuclear pores. Indirect evidence supporting this view has been reviewed
by Franke and Scheer (1974); direct evidence was obtained by Stevens and
Swift (1966). These latter investigators observed that 400 A RNP
granules synthesized at the Balbiani rings in Chironomus salivary gland
cells exit through the pores. As the particles enter the pores they are
transformed into rod-like structures with diameters of about 200-250 A.

41
There is evidence that RNA is not freely diffusible within the
nucleus and cytoplasm but is associated with structural elements of the
nuclear matrix and cytoskeleton (Agutter, 1985a; Feldherr, 1980; Fey et
al., 1986; van Eekelen and van Venrooij, 1981). Based partly on these
findings, Agutter (1985a) postulated that the efflux of RNA occurs in
three stages by a solid-phase mechanism; first, transport to the nuclear
envelope along the nuclear matrix, second, translocation through the
pores, and third, transport along the cytoskeletal matrix.
The focus of the present investigation is on the second step of the
efflux process, i.e., translocation across the envelope. The main
objectives are; first, to localize and characterize the specific regions
of the nuclear pores involved in the translocation of different classes
of RNA-coated gold to the cytoplasm and, second, to determine whether
individual pores can function in both RNA efflux and protein uptake, or
whether separate classes of nuclear pores exist. In order to obtain the
resolution necessary to examine individual exchange sites, coated
colloidal gold particles are used as tracers. The particles are
microinjected into oocytes, and subsequently localized by electron
microscopy.
The results are summarized as follows: first, gold particles
coated with tRNA (met or phe), 5S RNA, or poly(A) are all translocated
into the cytoplasm through central channels, at least 230 A in diameter,
located within the nuclear pores. Particles stabilized with
nonphysiological polynucleotides are also translocated; however, the
number and distribution of the particles associated with the pores
varied for different coating agents. Second, approximately 97% of the

42
pores in Xenopus oocytes can function in the translocation of RNA-coated
gold. Third, the accumulation of particles in the pores is a saturable
process. Fourth, individual pores can function in both RNA efflux and
protein uptake. Control experiments ruled out the possibilities of
fixation artifacts and non-specific exchange processes.
Materials and Methods
Xenopus laevis were purchased from Xenopus 1 (Ann Arbor, Michigan)
and maintained as reported previously (Feldherr, 1975).
Nuceoplasmin isolation
Nucleoplasmin was isolated from a starting volume of 30 ml of
Xenopus ovaries. The isolation procedure was similar to that described
by Dingwall et al. (1982), with the exception that an anti-
nucleoplasmin IgG affinity column was substituted for the DEAE-
cellulose and phenyl sepharose columns. Polyclonal antibodies against
nucleoplasmin were generated in rabbits and affinity purified. A
cleared cell homogenate, obtained from the lysed oocytes (Feldherr et
al., 1984), was passed over an anti-nucleoplasmin affinity column, and
the column was washed free of nonbound proteins. Nucleoplasmin was
eluted in 1 ml fractions with 50 mM glycine-HCl buffer (pH 2.5) into
microcentrifuge tubes containing sufficient 1 M tris (pH 9.0) to
increase the pH to 7.5. The protein was monitored at 280 nm, and the
fractions containing nucleoplasmin were pooled and treated with
ammonium sulfate (55% saturated) overnight in the cold. The soluble
(NH4)2SO4 fraction was then dialyzed against a solution containing

43
0.05 M Tris and 0.05 M NaCl (pH 7.2), after which the nucleoplasmin was
precipitated in 80% alcohol and lyophilized as described previously
(Feldherr et al., 1984). Gel analysis was performed, as described by
Laemmeli (1970), to determine the purity of the preparation.
Gold preparation and stabilization
All glassware and solutions used in experiments involving RNA were
treated with 0.01% (vol/vol) diethyl pyrocarbonate and then autoclaved.
Colloidal gold particles were prepared by reducing chloroauric acid with
either trisodium citrate (Frens, 1973) or a saturated solution of white
phosphorus in ether (Feldherr, 1965). In this study, the trisodium
citrate method gave a particle distribution of 120-220 A in diameter,
whereas the phosphorus ether preparations ranged from either 20-50 A or
20-160 A, depending on the initial concentration of gold chloride used.
The gold sols were stabilized with tRNA (met or phe), 5S RNA,
poly(A) (3500 bases), poly(I) (500 bases), poly(dA) (500 bases),
nucleoplasmin, polyvinylpyrrolidone (PVP; 40 kd), polyglutamic acid,
ovalbumin, or bovine serum albumin (BSA). The RNAs were obtained from
Boehringer Mannheim Biochemicals (Indianapolis, IN) or Sigma Chemical
Company (St. Louis, M0). The purity of the RNAs was tested by running
the preparations on 10% polyacrylamide gels containing 8.3 M ultrapure
urea; ethidium bromide was used as a stain. Polyglutamic acid, BSA,
ovalbumin and PVP were purchased from Sigma Chemical Company.
In each instance, the minimum amount of coating agent required to
stabilize the particles, that is, prevent precipitation in 1% NaCl, was
determined as described earlier (Feldherr, 1984). Before stabilization,

44
tRNA, poly(A), poly(I), and poly(dA) were dissolved in 10 mM KC1, 7.2 mM
K2HPO4 and 4.8 mM KH2PO4 (pH 7.0). 5S RNA was rehydrated in 2 ml of
sterile ion-free water resulting in a salt concentration of 1 mM Tris-
HC1, 10 mM NaCl and 0.1 mM MgCl2 (pH 7.5). Nucleoplasmin, polyglutamic
acid, PVP, BSA, and ovalbumin were solubilized in a low ionic strength
buffer containing 7.2 mM K2HPO4 and 4.8 mM kH2P04 (pH 7.0).
Concentrations of coating agents and volumes used to stabilize the gold
preparations are given in Table I.
After stabilization, the 20-160 A preparations were centrifuged at
6,000g (at the bottom of the tube) at 4C for 10 minutes to remove any
large aggregates of gold. This step was not necessary for the 20-50 A
and 120-220 A fractions. Five to 7 ml of stabilized colloid were then
concentrated to 70-100 pi in Minicon concentrators (Amicon Corp.,
Danvers, MA). Finally, the samples were dialyzed against intracellular
medium consisting of 102 mM KC1, 11.1 mM NaCl, 7.2 mM K2HPO4, and 4.8 mM
KH2P04 (pH 7.0) for 3 h at 4C.
Injection
Frogs were anesthetized on ice for one hour and the ovaries
removed. Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually
defolliculated with watchmakers forceps and maintained in Ringer's
solution (Diberardino et al., 1977) at 22C. The defolliculated cells
were centrifuged at approximately 650g for 8-10 minutes as described
previously (Feldherr, 1980; Kressmann and Bernstiel, 1980). During
centrifugation, the nucleus migrates to a position just underneath the
plasma membrane at the animal pole and its outline can be visualized due

Table I Amounts of Coating Agent Required to Stabilize Gold Sols
mg of coating agent/ml of Size range of colloidal pi of solution needed to
Coating agent stabilizing solution particles stabilized (A) stabilize 1 ml of colloid
tRNAmet
0.2
20-160
200
20- 50
50
tRNAPhe
0.5
20-160
300
5S RNA
0.4
20-160
400
poly(A)
0.5
20-160
70
120-220
70
poly(dA)
0.25
20-160
350
poly(I)
0.5
20-160
400
Nucleoplasmin
0.1
120-220
60
PVP
0.1
20- 50
40
BSA
1.0
20- 50
60
Ovalbumin
1.0
20-160
250
Polyglutamic acid
10
20- 50
200
*These are average values, intended to serve as a guide. The exact amounts of coating agents
required for stabilization should be determined for each individual gold preparation.

46
to the displacement of pigment granules in the cortex. Nuclear
injections could then be accomplished. Calcium-free Ringer's was used
as an extracellular medium during injection to prevent possible
precipitations of the colloid as the micropipettes were introduced into
the cells. Immediately after injection the cells were returned to
complete Ringer's solution until subsequent fixation. The total
exposure to calcium-free Ringer's was less than 30 minutes. The tip
diameters of the micropipettes were 10-15 pm.
Electron microscopy and analysis
The cells were fixed using a procedure similar to that described by
Kalt and Tandler (1971). The oocytes were fixed initially overnight at
4C in 100 mM HEPES containing 3% glutaraldehyde (vol/vol), 2%
paraformaldehyde (wt/vol), 2.5% DMSO (vol/vol) and 1 mM CaCl2 (pH 7.2).
The nuclei were then dissected out with their surrounding cytoplasm,
post-fixed in 2% OSO4 (wt/vol) for one hour, and stained with 0.5%
p-phenylenediamine (wt/vol) in 70% acetone for 30 minutes. Finally, the
samples were dehydrated in a graded series of acetone and embedded in
Spurr's medium. Thin sections were cut on a Reichert microtome and
analyzed with a JE0L 100S electron microscope (JEOL USA, Cranford, NJ).
In the RNA efflux studies it was presumed that gold particles
located within the pores were in the process of translocation, and that
particles present in the cytoplasm just adjacent to the pores had
completed the translocation process. These areas will be referred to as
regions 1 and 2, respectively (see Fig. 1). Pores that contained one or
more particles in either or both of these regions were considered to be

47
Nucleoplasm
Cytoplasm
Figure 1. A schematic representation of the regions used for
electron microscopic analysis of the particle distribution
within the pores. It is assumed that the particles in region 1
are undergoing translocation and that particles in region 2 have
completed the process. The arrow which defines the outer limits
of region 2 represents a distance of approximately 500 A.

48
actively involved in efflux. To determine the functional diameter of
the transport channels, the size distribution of the particles in
regions 1 and 2 were obtained and compared with the size of the injected
particles (i.e., particles within the nucleus). To standardize the
results, pores were analyzed in regions where the gold concentration in
the adjacent nucleoplasm was about 100 particles/0.36pm^.
Negative staining procedures were used to estimate the overall
diameters of the particles, that is, the gold plus the adsorbed coat
material.
Results
Purity of stabilizing agents
Approximately 600 pg of nucleoplasmin was isolated from 30 ml of
ovary using affinity chromatography. SDS-polyacrylamide gels of the
isolated protein showed major bands with apparent molecular weights of
165 kd and 145 kd, and a minor band with a molecular weight of
approximately 33 kd. The gel pattern (not shown) was identical to that
obtained for nucleoplasmin isolated using DEAE and phenyl sepharose
columns (Feldherr et al., 1984).
On a 10% polyacrylamide gel containing urea, tRNA ran as a single
distinct band corresponding to approximately 70 nucleotides, whereas the
pattern obtained for 5S RNA contained one major band corresponding to
approximately 130 nucleotides and minor bands of smaller sizes, probably
representing breakdown products. Gel scans of the tRNA and 5S RNA
showed the major band in each sample to be >80% of the total RNA.

49
Nuclear injection of tRNA-, 5S RNA- and poly(A)-coated gold
It was determined initially that varying the amount of the
injectate from 8-20 nl (the RNA content ranged from 30-100 ng) had no
effect on the results. Routinely, 8 nl of colloid were injected. This
volume contained a sufficient number of particles for electron
microscopic analysis and minimized possible damage to the nucleus. The
oocytes were fixed 15 min, 1 h, or 6 h after injection. Eighteen to 20
cells were examined for each type of RNA at each time interval.
The results were essentially the same for 20-160 A particles
coated with tRNA (met or phe), 5S RNA, or poly(A). At all time
intervals, particles were distributed randomly throughout the
nucleoplasm, except for aggregates of gold that were occasionally
observed at the surface of the nucleoli. After 15 min and 1 h,
particles were associated with almost all of the nuclear pores.
Representative results for tRNA and 5S RNA are shown in Figs. 2 and 3,
respectively. Based on the assumption that the presence of gold
particles within the pores or in the adjacent cytoplasm (regions 1 and
2 in Fig. 1) is indicative of nucleocytoplasmic exchange, it was
concluded that over 97% of the pores were involved in translocation 15
min and 1 h after injection (see Table II). There was an obvious
decrease in the percentage of pores that contained particles in the 6 h
experiments, but these results were not quantitated. In all instances,
particles were observed in the cytoplasm beyond the immediate vicinity
of the pores (i.e., beyond region 2). However, even after 6 h the
cytoplasmic to nuclear concentration ratio was only 1:14.

Figure 2. tRNA-gold, nuclear injection 15 min experiment.
Gold particles (20-160 A) are observed evenly distributed throughout
the nucleus (N). Particles are seen within the centers of the majority
of the pores and also in the adjacent cytoplasm (C).
Bar, 0.2 pm.
Figure 3. 5S RNA-gold, nuclear injection 60 min experiment.
The tracer particles (20-160 A) show a similar distribution as
described in Figure 2. Particles are present within the pores
and the adjacent cytoplasm (C). N, nucleus. Bar, 0.2 pm.

51
N
t.-
* '
/>1 *' j * *i
3 sv C
v Wi A**

Table II Translocation of Gold Particles as a Function of the Coating Agent
No. of Particles
Experiment (1 h)Translocated*
% of Pores Active
Particles/Pore in Translocation*
Region
1 Region 2
Total
tRNA-gold
437
414
851
4.3
97%
5S RNA-gold
458
425
883
4.4
98%
Poly(A)-gold
309
479
788
3.9
98%
Poly(dA)-gold
334
144
478
2.4
98%
Poly(I)-gold
397
543
940
4.7
96%
PVP-gold**
3
5
8
0.04
4%
BSA-gold**
5
3
8
0.04
4%
Ovalbumin-gold
0
t-
9
11
0.05
6%
* Data based on
the analysis
of 200 pores
all within
equivalent
concentrations of
gold particles.
**The size of the gold particles in these experiments ranged from 20-50 A. In all
other experiments the particle sizes ranged from 20-160 A.

53
The size distributions of tRNA- and 5S RNA-coated particles present
within the nucleoplasm and also associated with the nuclear pores
(regions 1 and 2) are given in Table III. Since the thickness of the
RNA coat, in both cases, was estimated to be 15-20 A, the results
indicate that particles with an overall diameter of at least 170 A can
pass through the centers of the pores. Since larger particles could not
be stabilized with tRNA or 5S RNA, it was not possible to determine an
upper size limit for translocation using these coating agents. In
contrast, 120-220 A particles could be stabilized with poly(A). The
results obtained wtih poly(A)-gold are illustrated in Fig. 4. Despite
the fact that relatively few particles were injected into the nuclei,
gold was found in over 70% of the pores after 15 min and 1 h. The size
distribution of poly(A)-coated particles associated with the pores is
shown in Table III. It is apparent from the data, which is based on the
examination of 12 cells, that particles at least 230 A in diameter
(including the coat) are translocated through the pores.
Nuclear injection of poly(I)- and poly(dA)-coated gold
In addition to the studies performed with tRNA, 5S RNA and poly(A),
nuclei were also injected with poly(I)- or poly(dA)-coated particles.
The volumes injected, and the procedure used for analysis of these
nonphysiological tracers, were the same as described above. In these
experiments, the oocytes were fixed 1 h after injection, and 5 cells
were examined in each group. The results are shown in Table II.
Particles coated with either poly(I) or poly(dA) were translocated
through the centers of the pores; however, differences were observed in

Table III Size Distribution of Gold Particles Present in the Nuclei and Pores
Total No. % of Particles in Each Size Class*
Experiment of Particles 20-40 40-60 60-80 80-100 100-120 120-140 140-160 160-180 180-200 >200
(1 h) Measured A A A A A A A A A A
tRNAmet
Nuclei**
486
0.3
27.9
51.5
12.8
4.2
1.5
1.5
--
--
--
Pores
462
0.4
22.1
52.6
15.4
6.1
1.3
2.1


5S RNA
Nuclei
1110
3.2
35.8
48.4
10.0
1.8
0.45
0.36
--
--
--
Pores
773
2.6
37.0
48.4
10.3
1.3
0.25
0.15



Poly (A)
Nuclei
906





6.6
35.5
43.8
9.8
4.2
Pores
593
--




5.2
37.3
42.0
13.6
1.9
* Particle dimensions do not include the thickness of the coat. Negative staining indicated that the coat
thickness adds 30-40 A to the overall diameters of the particles.
** Particles present within the nuclei.
*** Total number of particles present within the pores and in the cytoplasm immediately adjacent to the
pores.

Figure 4. Poly(A)-gold, nuclear injection 60 min experiment.
Large gold particles (120-220 A) are observed extending through
the nulcear pores and are present in the adjacent cytoplasm (C)
N, nucleus. Bar, 0.2pm.
Figure 5. PVP-gold, nuclear injection 60 min experiment.
Gold particles are retained within the nucleoplasm and are rarely
seen associated with the envelope or within the nuclear pores.
C, cytoplasm. N, nucleus. Bar, 0.2 pm.

56

57
the numbers and distribution of the particles associated with these
structures. Compared with poly(dA)-coated gold, almost twice as many
poly(I)-coated particles were present in the pore areas and, in
addition, a higher proportion of these particles were located in
region 2. These results suggest that some polynucleotides are
translocated more efficiently than others.
Nuclear injection of BSA-, ovalbumin-, polyglutamic acid-, and PVP-
coated gold
To determine if the translocation of RNA-gold is a selective
process, nuclei were injected with gold fractions that had been
stabilized with the exogenous molecules PVP, BSA, or ovalbumin. The
volumes injected, and the experimental times (15 min, 1 h and 6 h), were
the same as those used in the RNA studies. The data are based on the
examination of 6-9 cells per time interval for each coating agent that
was used. The result of a 1 h nuclear PVP-gold injection is
illustrated in Fig. 5. Similar distributions were observed when
particles stabilized with BSA or ovalbumin were injected. It was
found, at all time intervals, that essentially all of the particles
coated with exogenous substances were retained within the nuclei and
less than 6% of the pores contained gold particles (Table II). It can
also be seen in Table II that the total number of control particles
translocated per 200 pores was only about 1% of the total number of
translocated RNA-coated particles. These results demonstrate that the
translocation of RNA-gold was due to the specific properties of the
adsorbed coat material.

58
To determine if the translocation of polynucleotide-coated
particles is due simply to a high negative charge density, gold
particles were coated with polyglutamic acid, which, like RNA, is a
polyanion. Since polyglutamic acid is not a highly effective
stabilizing agent, the colloid preparations that were injected were
relatively dilute. To compensate for this factor, tRNA-gold
preparations having an equivalent particle concentration were injected
in parallel experiments. The results, which are based on an analysis of
5 cells, are shown in Table IV. Tracer particles coated with
polyglutamic acid were observed in only 8% of the pores, compared with a
value of 83% obtained for tRNA-gold. These findings demonstrate that
the translocation of the RNA-coated particles is not simply a charge
effect.
Controls
Control experiments were performed on 6 oocytes to establish if the
results obtained for RNA-gold could be due to a redistribution of the
particles during fixation. In this study, the nuclei were injected with
tRNA-coated gold and the oocytes fixed within 10 seconds. The
particles near the site of injection were rarely observed within the
pores or in the adjacent cytoplasm. These data indicate that the
presence of RNA-gold within the pores at 15 min and 1 h intervals was
not a fixation artifact, but reflected an i_n vivo exchange process.
In order to ascertain whether the presence of excess RNA itself
could alter the properties of the pores, nuclei of 6 oocytes were
simultaneously injected with PVP-coated gold and 100 ng of soluble tRNA.

Table IV Translocation of Gold Particles Coated with Polyglutamic Acid
Experiment (1 h)
No. of Particles
Translocated
% of Pores Active
Parti cles/Pore in Translocation
Region 1
Region 2
Total
Polyglutamic
Acid-gold
21
2
23
tRNA-gold
175
164
339
8%
83%
*Data based on the analysis of 200 pores. In both instances the concentration of gold
particles (20-50 A) in the adjacent nucleoplasm was approximately 60 particles/0.36 pm2.

60
Fewer than 4% of the 200 pores analyzed were involved in translocation.
Thus, the injection of RNA in amounts greater than those normally used
in this investigation, have no apparent effect on the physical
properties of the pores.
Concentration dependence of RNA translocation
To determine the concentration dependence of the translocation
process, the gold distribution in the pores was analyzed in areas in
which different concentrations of colloid were present in the adjacent
nucleoplasm. These experiments were performed with tRNA-coated gold
particles, and the cells were fixed after 1 h. To ensure that an
appropriate concentration range was obtained, different dilutions of
colloid were injected. The amount of tRNA in the injectate varied from
6-30 ng. The results are shown in Table V, and are based on the
examination of 6 oocytes. It can be seen that a maximum number of
particles per pore is obtained at a concentration of 90
particles/0.36pm^. Above this concentration, no differences were
observed either in the numbers or distribution of the particles,
suggesting that the translocation process is saturable.
Double label experiment
The high percentage of pores involved in RNA translocation
suggested that each pore may be a bidirectional channel, capable of both
protein and RNA transport. To address this possibility directly, large
gold fractions (120-220 A) coated with nucleoplasmin and small gold
fractions (20-50 A) stabilized with tRNA were used. The tRNA-coated

Table V Concentration Dependence of tRNA-gold Translocation*
No. of Particles % of Pores Active
Particle No./0.36 pm^ Translocated Particles/Pore in Translocation
Region
1 Region 2
Total
30
34
84
118
1.2
61%
60
77
94
171
1.7
82%
90
159
197
356
3.6
98%
120
154
205
359
3.6
98%
190
188
174
362
3.6
98%
* Data based
on the analysis
of 100 pores
for each
gold particle
concentration
The size of
the gold particles ranged from 20-50
A, and the cells were fixed
1 h after injection.

62
particles were injected into the nucleus and nucleoplasmin-coated gold
was injected into the cytoplasm of the same cell. The sequence of the
injections varied in different experiments; however, in all instances
the interval between the first and second injection was 15 min, and the
oocytes were fixed after a total of 45 min. In all, 12 cells were
examined. The results, which were the same regardless of the injection
sequence, are illustrated in Fig. 6, a-d. Small RNA particles can be
observed immediately adjacent to the cytoplasmic surface of the pores
and large nucleoplasmin particles are present on the nuclear side of the
same pores. These distributions clearly demonstrate that individual
pores can function in both protein uptake and RNA efflux.
Cytoplasmic injections
The RNA-gold preparations [tRNA, 5S RNA and poly(A)] were injected
into the cytoplasm to determine if transport is reversible.
Approximately 40 nl of colloid (containing approximately 200 ng of RNA)
were introduced adjacent to the nuclear envelope in a total of 20
centrifuged cells. Similar distributions were obtained for all RNAs
used. In both 1 h and 6 h experiments, gold was seen within the
poresnear the site of injection, but relatively few particles were found
in the nucleoplasm. The nuclear to cytoplasmic concentration ratios
were approximately 0.01 after 1 h. The 5S RNA-gold results are
illustrated in Fig. 7. It should be pointed out that the number of
pores that contained gold varied considerably from cell to cell,
therefore, no effort was made to quantitate these results.

Figure 6. Double injection experiment 45 min.
tRNA-gold (20-50 A) was injected into the nucleus (N), and the adjacent
cytoplasm (C) was injected with nucleoplasmin-gold (120-220 A). The
large nucleoplasmin-coated particles can be seen just on the nuclear
sides of the pores and small RNA particles are present on the
cytoplasmic side. Bar, 0.1 pm.
Figure 7. 5S RNA-gold, cytoplasm injection 15 min experiment.
Gold particles are present in the cytoplasm (C) and can be seen
extending through the nuclear pores. Only a small percentage of
particles enter the nucleus (N). Bar, 0.1 pm.

64
**> ~
N

65
Discussion
Colloidal gold procedures have been used previously to identify and
characterize the regions of the pores that are involved in the transport
of karyophilic proteins across the nuclear envelope (Feldherr et al.,
1984). It was found that the transport channels are located in the
centers of the pores and have functional diameters of at least 200 A.
In this study, the same experimental approach was used to characterize
the pathways for RNA efflux and, in addition, to determine whether
individual pores have the capability of transporting both RNA and
protein.
The presence of RNA-coated gold particles within and along the
cytoplasmic surface of the nuclear pores provides direct evidence that
these tracers are translocated across the envelope at these sites.
Furthermore, almost all of the pores (97% or more) can function in the
translocation process. The biological significance of these findings;
however, is dependent on demonstrating that the pathways visualized for
the colloidal gold tracers are the same as those normally used for the
exchange of endogenous RNA. In this regard, we have attempted to
demonstrate that the translocation of RNA-gold through the nuclear
pores is both a selective and active process, and that the observed gold
distributions were not due to technical artifacts.
The fact that gold was not present in the pores of cells fixed 10
seconds after injection shows that accumulation in these structures is
not simply a result of the injection procedures per se or a
redistribution of the particles during fixation and processing for
electron microscopy. Control studies also demonstrated that the

66
injection of excess RNA does not alter the overall properties of the
pores.
Gold particles coated with synthetic polymers (PVP or polyglutamic
acid), exogenous proteins (BSA or ovalbumin) and even endogenous
karyophilic proteins (nucleoplasmin, Feldherr et al., 1984) are
essentially excluded from the pores after nuclear injection,
demonstrating that the translocation of RNA-gold is a selective process.
The data obtained with poly(I) and poly(dA) indicate that the capacity
for translocation is not necessarily restricted to physiologically
active RNAs but may be a general property of polynucleotides. The
chemical and/or physical characteristics of RNA that facilitate
translocation are not known, although a comparison of the results
obtained with poly(A) and poly(dA) suggests that the sugar moieties
could be involved. The high negative charge density of RNA does not
appear to be a major contributing factor since particles coated with
polyglutamic acid are largely excluded from the pores.
In view of the results obtained by Zasloff (1983), which showed
that the transport of labelled tRNA is markedly reduced by a single
substitution of G-to-U at position 57, one might expect a greater
degree of specificity for the translocation of polynucleotides. It
should be kept in mind, however, that Zasloff measured the overall
efflux of RNA whereas our results relate specifically to the
translocation step across the envelope. Thus, the decreased rate of
transport of the variant is not necessarily due to an effect on
translocation through the pores, but could be due to increased binding

67
within the nucleoplasm or changes in the rate of migration in the
cytoplasm.
As discussed in the introduction, there is evidence that
macromolecules larger than 90 A in diameter are unable to diffuse across
the nuclear envelope, whereas particles as large as 200 A in diameter,
which contain nuclear targeting signals, can be transported through
central channels in the pores. It was found in this study that RNA-
coated gold particles as large as 230 A in diameter (including the coat)
readily penetrate the pores. Since this far exceeds the upper limit for
diffusion, these results suggest that a transport process is involved.
Consistent with this interpretation is the finding that the accumulation
of RNA-gold in the pores is a saturable process. Saturation indicates
the presence of a carrier-mediated transport process. However,
saturation would also occur if the particles simply occupied all of the
available space within and adjacent to the pore channels. The latter
explanation, which would have little bearing on the mechanism of
exchange, is unlikely for two reasons. First, direct electron
microscopic examination of region 1 shows that these areas are not fully
occupied at saturation levels. Second, particles coated with
nucleoplasmin can reach more than twice the concentration in region 2,
than tRNA-coated gold (data not shown), demonstrating that the nature of
the coat material rather than the total number of tracer particles is
the determining factor.
Colloidal tracers, although well suited for localizing and
characterizing exchange sites, are not appropriate for detailed kinetic
studies, which require numerous time points and involve the analysis of

68
large samples. For this reason a comprehensive examination of the
temperature dependence of translocation was not attempted. However, a
cursory study was performed at 4C and at 21C, comparing the
accumulation and distribution of tRNA-coated gold particles in the
pores. After 15 minutes, the average number of particles per pore
(based on the analysis of 100 pores) at 21C and 4C was 3.8 and 1.5,
respectively. Furthermore, at 4C only 25% of the particles were
located in region 2, compared to 50% in cells kept at 21C. The
temperature effects are very likely influenced by two separate
processes, binding to receptors and movement through the pores; thus,
the results are difficult to interpret. Despite this problem, however,
the overall effect of temperature is greater than would be expected for
a physical process, such as diffusion, and is consistent with the view
that an energy requiring step is involved.
Since the translocation of gold particles through the pores is i)
dependent on the properties of the adsorbed RNA-coat, ii) likely
involved in a transport process and iii) not an artifact of the
technique, it is concluded that the pathways visualized for the
translocation of RNA-coated colloidal tracers are the same as those
normally used for endogenous RNA. These pathways are located in the
centers of the nuclear pores and have apparent functional diameters of
approximately 230 A. These results are consistent with those obtained
previously by Stevens and Swift (1966) for mRNA efflux in Chironomus
salivary gland cells.
It is not known whether tRNA and 5S RNA complex with specific
proteins prior to exiting the nucleus. There is evidence that the

69
poly(A) tails of mRNA bind different polypeptides in the nucleus and
cytoplasm (Baer and Kornberg, 1983; Sachs and Kornberg, 1985; Setyono
and Greenberg, 1981); however, it has not been determined if these
proteins are involved in transport.
The fact that over 97% of the pores are involved in RNA efflux,
combined with the earlier observation that the majority of the pores
contained nucleoplasmin-coated particles following cytoplasmic
injections (Feldherr et al., 1984), suggests that these pathways are
bifunctional. The double injection experiments provided direct evidence
supporting this view. Thus, it appears that the central channels within
the pores can function in the translocation of both RNA and protein.
However, this does not necessarily mean that the same molecular
mechanisms are employed.
Six hours after injection, the nuclear to cytoplasmic concentration
ratio of RNA-coated gold was found to be 14:1. Correcting for the
difference in the volumes of the two compartments, it is estimated that
no more than 36% of the particles entered the cytoplasm. Based on data
presented by Zasloff (1983, Table I) an equivalent amount of
radiolabel led tRNA would be expected to leave the nucleus in about 3 h.
There are several factors that could account for this difference.
First, the conformation of tRNA could be modified after adsorption to
the gold particle. In this regard, Tobian et al. (1985) found that
point mutations which alter the conformation of tRNA also decrease its
rate of transport to the cytoplasm. Second, since several tRNA
molecules are adsorbed to the gold particles (the exact number has not
been determined) the binding avidity to components within the nucleus,

70
pores or even the adjacent cytoplasm could be affected. Third, the size
of the tracers could influence the transport rates. Although RNA-coated
particles leave the nucleus at a reduced rate, it should be emphasized
that the properties of RNA required for translocation through the pores
are retained.
Zasloff (1983) and De Robertis et al. (1982) reported that
radiolabel1ed tRNA is unable to enter the nucleus following injection
into the cytoplasm; however, the exchange of 5S RNA across the envelope
does appear to be bidirectional (De Robertis et al., 1982). Gold
particles coated with tRNA, 5S RNA, or poly(A) were able to pass from
the cytoplasm into the nucleus, but exchange across the envelope was
greatly restricted. Even after 1 h the nuclear to cytoplasmic
concentration ratio was only about 0.01. Considering these low rates of
uptake, it was surprising to find that RNA-coated particles were
observed extending through the centers of the pores. These results
could be interpreted to mean that the translocation of RNA through the
pores is a reversible process, but that release into the nucleoplasm,
which could require a separate mechanism, might be a limiting factor.
However, before any definitive conclusion can be drawn it will be
necessary to obtain reliable quantitative data concerning the percentage
of pores that contain gold and additional information relating to the
nuclear uptake rates. Furthermore, it is not known whether
translocation is initiated by the adsorbed RNA itself or whether the
particles fortuitously bind karyophilic proteins which, in turn, induce
transport (Mattaj, 1986). Hopefully it will be possible to resolve some
of these questions using isolated nuclei.

CHAPTER III
THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE
OF TARGETING SIGNALS ON NUCLEAR UPTAKE
Introduction
The nuclear pore complex has been identified as the major, if not
the exclusive site for macromolecular diffusion and transport between
the nucleus and cytoplasm of eukaryotic cells (Feldherr et al., 1962;
Feldherr et al. 1984). Evidence for mediated protein transport was
determined intially for RN1 (Feldherr et al., 1983) and nucleoplasmin
(Dingwall et al., 1982), both of which are major karyophilic proteins
found in amphibian oocytes. From electron microscopic analysis of the
intracellular distribution of nucleoplasmin-coated gold particles, it
was established that the transport channels, located in the centers of
the pores, are at least 200 A in diameter (Feldherr, et al., 1984). In
contrast, the channel has a functional diameter of approximately 90 A
with respect to diffusion (Paine et al., 1975).
Several laboratories have utilized recombinant DNA methodology and
single amino acid substitutions to obtain probes useful for the
localization and characterization of transport signals that target
specific proteins to the nucleus. These approaches have been used to
study nuclear uptake of the following proteins; nucleoplasmin (Burglin
and De Robertis, 1987; Dingwall et al., 1987), simian virus (SV 40)
large T-antigen (Kalderon et al., 1984a,b), the yeast regulatory
proteins MATa2 (Hall et al., 1984) and GAL4 (Silver et al., 1984), yeast
71

72
histones 2A and 2B (Moreland et al., 1987), the yeast ribosomal protein
L3 (Moreland et al., 1985), polyoma large T-antigen (Richardson et al.,
1986), and the adenovirus Ela protein (Lyons et al., 1987). Although
there is no concensus signal, it appears that nuclear targeting is
dependent on short, basic amino acid sequences.
Of the proteins listed above, SV 40 large T-antigen has been
studied most extensively. Kalderon et al. (1984b) constructed hybrid
proteins by linking various amino acid sequences found in SV 40 large
T-antigen to the amino terminus of pyruvate kinase and found that the
shortest sequence capable of targeting the enzyme to the nucleus was
Pro-Lys-Lys^-Lys-Arg-Lys-Val. Transport is especially sensitive to a
point mutation at the Lys1^ position (Kalderon et al., 1984a; Lanford
et al., 1986). Amino acid mutations in the vicinity of the Lys1^
position reduces, but does not necessarily abolish transport of SV 40
large T-antigen into the nucleus. Roberts et al. (1987) have shown that
multiple copies of a partially defective signal can cooperate to enhance
nuclear localization.
Lanford et al. (1986) synthesized peptides that contained the SV 40
large T transport signal and cross-linked them to several carrier
proteins (ovalbumin, BSA, IgG, slgA, ferritin and IgM). When the
conjugates were injected into the cytoplasm of cultured cells, all but
IgM (m.w. 970 kd) entered the nucleus. Qualitative evidence, utilizing
indirect immunofluorescence, suggested that the number of signals per
carrier protein can affect the rate of uptake. Goldfarb et al. (1986)
found that BSA conjugated with SV 40 large T-antigen signals accumulates

73
in the nuclei of Xenopus oocytes with saturable uptake kinetics,
suggesting the involvement of a receptor-mediated process.
Nucleoplasmin, a 110 kd pentameric karyophilic protein (as
determined by sequence analysis), also has been used extensively to
study nuclear transport in oocytes (Dingwall et al., 1982; Feldherr et
al., 1984) and cultured cells (Schulz and Peters, 1986; Sugawa et al.,
1985). It has recently been determined that each monomeric subunit
contains 1 targeting signal. Furthermore, removal of one or more signal
domains by proteolytic cleavage markedly reduces the rate of nuclear
uptake (Dingwall et al., 1982). The region containing the signal has
been sequenced and although it is similar to the SV 40 targeting signal,
it is not identical (Dingwall, personal communication).
The data summarized above suggest that variations in the nuclear
targeting signal could significantly influence nucleocytoplasmic
exchange of proteins. To better understand the nature of these effects,
experiments were performed to determine how macromolecules containing
different signal sequences and different numbers of signals interact
with, and modulate the properties of the nuclear pores. Various size
colloidal gold particles were coated with 1) BSA conjugated with
different numbers of synthetic peptides containing the SV 40 targeting
signal, 2) BSA conjugated with inactive SV 40 signals, 3) large
T-antigen, or 4) nucleoplasmin. These tracers were microinjected into
oocytes and their distribution within the oocytes was later determined
by electron microscopy.
The data indicate that as the number of signals per molecule
increases, both the relative uptake of the tracer particles into the

74
nucleus and the functional size of the transport channels increase. The
SV 40 and nucleoplasmin targeting sequences varied in their ability to
facilitate transport. This could be related to differences in their
binding affinity for nuclear envelope (pore) receptors. Double
labelling experiments demonstrated that different targeting signals can
be recognized by and transported through the same pore. Control
experiments ruled out the possibilities of nonspecific exchange
processes.
Materials and Methods
Xenopus laevis were purchased from Xenopus I (Ann Arbor, Michigan)
and maintained as reported previously (Feldherr, 1975).
Colloidal gold coating agents
The synthesis of peptides containing the SV 40 large T-antigen
targeting signals and their conjugation to BSA was described in detail
by Lanford et al. (1986). In brief, the thirteen amino acid sequence
was synthesized on a glycyl-Merrifield resin (Merrifield, 1963) using a
Biosearch Sam Two (Biosearch, San Rafael, CA) automated peptide
synthesizer. The synthetic peptides were conjugated to BSA using the
heterobifunctional cross-linking agent m-maleimido benzoyl-N-
hydroxysuccimide ester (MBS; Pierce Chemical Co., Rockford, IL).
Dialysis in phosphate-buffered saline followed by repeated concentration
and dilution of the conjugates separated the unconjugated peptides from
the carrier proteins. By varying the molar ratios of the reactants,
different protein-peptide coupling ratios were obtained. Amino acid

75
analyses were performed with a Beckman Model 7300 analyzer (Beckman
Instruments Inc., Palo Alto, CA) to obtain a number-average ratio of
signal peptides to carrier protein BSA. The peptide to carrier protein
ratios obtained by SDS-PAGE were similar to those calculated by amino
acid analysis.
Large T-antigen was expressed in insect cells using the baculovirus
expression vector system (Luckow and Summers, 1988) and was purified by
immunoaffinity chromatography as described previously (Siamins and Lane,
1985). Nucleoplasmin was isolated using an anti-nucleoplasmin IgG
affinity column as described previously (see Chapter II, Materials and
Methods). BSA used in the control experiments was purchased from Sigma
Chemical Co. (St. Louis, MO).
The BSA conjugates used as coating agents are listed in Table VI.
The table shows the synthetic 13 amino acid sequence conjugated to BSA
and the average number of peptides per BSA molecule. The synthetic
peptide contains the seven amino acid sequence required for nuclear
localization of the SV 40 large-T antigen (Lanford et al., 1986). The
conjugates denoted BSA-WT represent preparations that contain active
nuclear targeting signals. In the BSA-cT conjugates, neutral asparagine
was substituted for the second lysine (equivalent to Lys12 in SV 40
large T-antigen) in the synthetic peptide (see Table VI). The resulting
signal is similar to the mutation present in the SV 40(cT)-3 mutant
(Lanford and Bute!, 1984), and is defective in transport. To obtain a
coating agent containing an average of 3 signals per carrier molecule,
the BSA-WT3 conjugate was diluted 3-fold with the BSA-cTy preparation
prior to stabilizing the gold particles. The use of BSA-cTy to dilute

Table VI Protein Preparations Used as Coating Agents
Coating Agent
BSA
BSA-WT5
BSA-WT8
BSA-WTn
Average No.
of Signals Sequence Conjugated to BSA
0
5
8
11
Cys-Gly-Tyr-Gly-Pro-Lys-Lys-Lys-Arq-Lys-Val-Gly-Gly
BSA-cTy
BSA-cT13
BSA-WTg + cT7*
Large T-antigen
Nucleoplasmin
7
13
3
1 per monomer
1 per monomer
Cys-Gly-Tyr-Gly-Pro-Lys-Asn-Lys-Arg-Lys-Val-Gly-Gly
The underlined sequence represents the active (WT preparations) and inactive (cT preparations)
SV 40 large T nuclear transport signal.
*The average number of signals obtained for this coating agent was performed by diluting
BSA-WT8 with BSA-cTy.

77
the active signal assured that the overall properties (shape and size)
of the particles were consistent with those of other tracers used in
this investigation. Purified SV 40 large T-antigen, which has 1 signal
per monomer, and nucleoplasmin, which has a different signal sequence,
also were used as coating agents. BSA alone was employed as an
additional control.
Preparation and stabilization of colloidal gold
The colloidal gold fractions that contained particles 20-50 A and
20-160 A in diameter were both prepared by reducing chloroauric acid
with a solution of white phosphorus in ether (Feldherr, 1965). 50-280 A
gold particles were obtained by adding 2.5 ml of 0.6% gold chloride to a
freshly prepared 20-160 A fraction, 1 ml of additional reducing agent
was then added, and the preparation was boiled for 2-3 minutes.
Fractions containing 120-280 A particles were prepared by reducing
chloroauric acid with trisodium citrate, as described previously (Frens,
1973). The 50-280 A and 120-280 A gold sols, were brought to pH 7.0
with 0.72 N K2C03.
Prior to stabilization, all of the coating agents were either
dialyzed against, or dissolved in, a low ionic strength buffer (7.2 mM
K2HPO4 and 4.8 mM KH2PO4, pH 7.0). The volumes of the different agents
required to stabilize 1 ml of the gold sols are listed in Table VII.
The procedure used to determine these volumes is outlined in Feldherr et
al. (1984).
After stabilization, the 20-160 A and 50-280 A preparations were
centrifuged at 2000g at 4C for 10 minutes to remove any aggregates of

Table VII Volumes of Coating Agent (ul) Required to Stabilize 1 ml of Gold Sol*
Fraction Size
Coating Agents
(cone.)
20-50 A
20-160 A
50-280
A 120-280 A
BSA
(1.0mg/ml)
60



BSA-WT5
(1.3mg/ml)
10
60
40

BSA-WTg
(0.6mg/ml)
30
150
150
20
BSA-WTn
(0.5mg/ml)

200

15
BSA-cTy
(1.4mg/ml)
20
80


BSA-cT13
(0.6mg/ml)


140

Large T-Ag
(0.4mg/ml)

400


Nucleoplasmin
(0.6mg/ml)

40
150
25
*These are average values,
coating agents required for
intended to serve as a guide. The
stabilization should be determined
exact amounts
for each
individual gold preparation.

79
gold. Centrifugation of the 20-50 A and 120-280 A fractions was not
necessary. In each instance, 5-7 ml of stabilized colloid were
concentrated to 70-100 ul in Minicon concentrators (Amicon Corp.,
Danvers, MA) and dialyzed against intracellular injection medium (102 mM
KC1, 11.1 mM NaCl, 7.2 mM K2HP04 and 4.8 mM KH2P04, pH 7.0) at 4C prior
to injection.
Injection
Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually
defolliculated in amphibian Ringer's solution and centrifuged at
approximately 650g for 8-10 minutes (Kressman and Birnstiel, 1980). The
cells were then microinjected with approximately 40 nl of gold sol at a
site adjacent to the nucleus and fixed at intervals of 15 min, 1 h, 6 or
20 h. The tip diameters of the micropipettes were 10-15 pm.
Electron microscopy and analysis
The cells were fixed for electron microscopy and prepared for
sectioning as described previously (see Chapter 2, Materials and
Methods). Relative nuclear uptake of gold particles stabilized with the
different coating agents was determined by counting particles in equal
and adjacent areas of the nucleus and cytoplasm close to the site of
injection. Yolk granules and mitochondria were excluded from the
analyses. The counts are reported as nuclear to cytoplasmic ratios
(N/C). The size distributions of particles present within the nucleus
and cytoplasm were determined by direct measurement from electron
micrograph negatives. The envelope to cytoplasm ratios were obtained by

80
comparing the number of particles associated with the cytoplasmic
surface of the envelope (i.e., at or within 650 A of the nuclear
surface) to the number of particles in an equal, randomly selected area
of cytoplasm.
Negative staining with 1% phosphotungstic acid was used to estimate
the thickness of the adsorbed protein coats.
By extrapolating from data published by De Roe et al. (1984) and
correcting for additional mass contributed by the peptides, estimates
were made of the number of BSA molecules adsorbed onto the surfaces of
different size gold particles. For example, particles with diameters of
35 A, 80 A, 140 A, and 180 A (the mean size of the 4 different gold
preparations injected) would have 2, 8, 24, and 39 molecules of BSA
conjugates, respectively. Knowing the number of BSA molecules adsorbed
and the synthetic peptide to carrier protein ratios, it was possible to
estimate the total number of SV 40 large T-antigen targeting signals on
the different size gold particles; however, the proportion of signal
actually available for transport (i.e., exposed signals) is not known.
Results
Cytoplasmic injections of the tracer particles
It was determined initially that microinjection of approximately
40 nl of stabilized gold adjacent to the nucleus delivered a sufficient
number of particles for electron microscopic analysis. The protein
content of the injectate ranged from 50-300 ng depending on the size of
the gold fraction and the specific coating agent used.

81
All preparations containing active transport signals (BSA-WT
conjugates, large T-antigen or nucleoplasmin) were translocated into the
nucleus through central channels located within the nuclear pores. In
the region of injection, the particles were uniformly distributed in the
cytoplasm; however, at longer time intervals, 6 and 20 h, the BSA-WT
conjugates occasionally formed aggregates. The reason for this is not
known, but it appeared to be dependent on the presence of active signals
since similar aggregates were not observed with BSA-cT conjugates. At
all time intervals, particles containing active transport signals were
present both within the pores and the nucleoplasm. With increasing time
there was a concomitant increase in the number of particles present in
the nucleus (data not shown). Gold coated with the BSA-cT conjugates or
BSA alone were essentially excluded from the pores and nucleoplasm.
These general distributions are illustrated in Figs. 8 and 9, which show
the results obtained 1 h after injecting particles coated with BSA-WT^
and BSA-CT7, respectively.
Relative uptake
Quantitative data, dependent on the specific properties of the
individual coating agents, were obtained 1 h after injection. This time
interval allowed for sufficient particle distribution within the
cytoplasm, and minimized possible loss or redistribution of soluble cell
components caused by the injection procedures (Miller et al, 1984). The
N/C ratios obtained at 1 h do not represent equilibrium values since
nuclear uptake was observed to increase with time, but reflect relative

Figure 8. BSA-WTij-gold. 1 h experiment.
Gold particles (20-160 A), near the site of injection, are observed
evenly distributed throughout the cytoplasm (C). Particles are seen
passing through the centers of the pores and also within the nucleus
(N). Bar, 0.1 pm.
Figure 9. BSA-cTygold 1 h experiment.
Tracer particles (20-50 A), near the site of injection, are distributed
evenly throughout the cytoplasm (C), but are rarely seen within the
nucleus (N) or along the envelope. Bar, 0.1 pm.

83

84
uptake of the tracer particles. Four to 6 cells were examined for each
coating agent within each size fraction.
The nuclear to cytoplasmic (N/C) ratios for different coating
agents are given in Table VIII. After 1 h, N/C ratios obtained with the
20-50 A fraction stabilized with BSA-WTg and BSA-WTg were 0.58 and 0.76,
respectively. The difference in these ratios, as determined by the
Student's t-test, is not statistically significant (p>0.25). When the
particle size was increased to 20-160 A or 50-280 A in diameter, the
differences in N/C ratios between the BSA-WT5 and BSA-WTg conjugates
were highly significant (p<0.002). In addition, BSA-WTg was more
effective in facilitating transport than large T-antigen which contains
1 signal per monomer (p<0.002). However, increasing the number of
signals from 8 (BSA-WTg) to 11 (BSA-WT^) did not significantly increase
the N/C ratio (p values obtained for the 20-160 A and 120-280 A
fractions were p>0.8 and 0.1>p>0.05, respectively).
The N/C ratio obtained for the BSA-WTg-cTy dilution (approximately
3 signals per BSA molecule) was significantly lower than that obtained
for the BSA-WTg conjugate (p<0.002); however, it was also significantly
lower than that observed for large T-antigen-gold (0.01 could be due to variation in the availability and/or spatial
distribution of the signal at the surface of the gold particles.
The N/C values for BSA-cTy, BSA-cTig-, and BSA-coated gold were
significantly lower than all gold preparations containing active nuclear
targeting signals (p<0.002).
Overall, it is concluded from these results that there is a direct
correlation between the number of transport signals and the relative

Table VIII N/C Ratios* 1 h
Coating Agents
20-50 A
20-160 A
50-280 A
120-260
BSA
0.009



BSA-cTy
0.01
0.009


BSA-cT13


0.006

bsa-wt8 +
BSA-CT7 (1:3 dilution)

0.035


BSA-WT5
0.58
0.18
0.06

bsa-wt8
0.76
0.79
0.38
0.14
BSA-WTn

0.80

0.24
Large T-Antigen

0.077


Nucleoplasmin

2.43
0.71
0.51
*N/C ratios were calculated from 500-3000 particle counts per data point.

86
uptake of particles into the nucleus. Furthermore, the data suggest
that as the size of the particles increases, more signals are required
for their transport across the envelope.
To compare the effectiveness of a different targeting signal,
parallel studies were performed with nucleoplasmin-gold. The relative
uptake of different size nucleoplasmin-coated particles by the nucleus
is shown in Table VIII. The N/C ratios determined for nucleoplasmin-
coated particles were significantly greater than those obtained for BSA-
WT5-, BSA-WTg-, or BSA-WT^-coated gold. In all instances, the
probability values were p<0.002. Since there are only 5 targeting
signals per nucleoplasmin molecule, these differences in uptake suggest
that the nucleoplasmin transport signal is more effective than the SV 40
large T-antigen targeting sequence, at least in oocytes.
Size distributions
In view of the above results, the size distributions of particles
in different regions of the oocytes were analyzed in more detail. The
nuclear and cytoplasmic size distributions determined for BSA-WT5 and
BSA-WTg 1 h after injection are given in Table IX and Fig. 10.
Seventeen percent of the BSA-WTg-coated particles that entered the
nucleus were larger than 157 A, compared to 5.6% for particles coated
with BSA-WT5. When particles larger than 185 A are compared, the
difference in uptake is almost 10-fold. The difference in the size of
the particles able to penetrate the pores is statistically significant
as determined by Chi-square analysis.

TABLE IX ~ Size Distribution of BSA-WT^-and WTfl-coated Particles in Injected Cells*
Experiment
LUl)
Total No.
of Particles
Measured
Percentage of Particles in Each Size Class#
45-73 73-101 101-129 129-157 157-185 185-213 213-241 241-269 >269
A A A A A A A A A
BSA-WT5
Nucleus
406
31.5
19.7
24.6
18.5
4.9
0.7
-
-
-
Cytoplasm
533
3.2
11.1
24.2
29.8
14.8
9.0
3.8
1.1
2.8
bsa-wt8
Nucleus
1018
14.3
20.3
24.6
23.8
10.1
4.8
1.7
0.4
_
Cytoplasm
808
5.2
13.1
25.0
28.2
14.7
7.4
3.6
1.7
0.
*These experiments were performed with 45-280 A gold particles and the mean size of the fraction was 140 A.
^The size of the gold particles does not include the thickness of the coating agent.

% of Total Nuclear Particles
40
45-73 73-101 101-129 129-157 157-185 185-213 213-241 241-269 >269
Particle Size (A)
Nuclear particle distributions: BSA-WTg and BSA-WTg
Figure 10.

89
When the number of signals per BSA molecule is increased beyond 8
there is no further increase in functional pore size. This is indicated
in Fig. 11, which compares the size distribution of BSA-WT8", BSA-WT^-
and nucleoplasmin-coated particles within the nucleus 1 h after
injecting a 120-280 A gold fraction. The sizes of the particles able to
pass through the pores did not vary significantly for the different
coating agents. A comparison of the cytoplasmic distributions to the
nuclear distributions, as shown in Table X, demonstrates that particles
larger than 230 A (average of the size class), do not readily penetrate
the pores, regardless of the coating agent. These results demonstrate
that the maximum size particle able to enter the nucleus is
approximately 260 A in diameter. This value includes the thickness of
the coat material, which adds about 30 A to the overall particle
diameter.
In contrast, the large T-antigen data given in Table XI indicate
that particles larger than 90 A were not detected in the nucleus after
1 h. The size data obtained for the BSA-WT8-CT7 dilutional experiment
(Table XI) gave similar results, although a few particles larger than 90
A were present in the nucleus.
Overall, analysis of the size distributions of particles in the
nucleus and cytoplasm indicate a direct relationship between the
functional dimensions of nuclear pores and the number of active SV 40
large T targeting signals per BSA molecule.

% of Total Nuclear Particles
60
50 -
30 -
BSA-WT-8
BSA-WT-11
Nucleoplasmin
20 -
10 ^
120-145
145-170
170-195
195-220
1
220-245 >245
40 -
Particle Size (A)
Figure 11. Nuclear particle distributions: BSA-WTg, BSA-WT^, and nucleoplasmin
o

TABLE X Size Distribution of BSA-WTr-, WTii~ and Nucleoplasmin-coated Particles in Injected Cells*
Experiment
(1 h)
Total No.
of Particles
Measured
Percentage of Particles in Each Size Class#
120-145 145-170 170-195 195-220 220-245 >245
A A A A A A
BSA-WT8
Nucleus
371
3.2
21.6
55.5
18.6
1.1
--
Cytoplasm
556
2.9
16.5
51.3
21.9
5.9
1.4
BSA-WTn
Nucleus
358
4.2
21.5
53.6
19.6
1.1

Cytoplasm
557
3.2
14.7
50.6
22.8
7.0
1.6
Nucleoplasmin
Nucleus
514
3.5
18.9
55.6
19.0
2.4

Cytoplasm
525
3.2
16.4
49.3
20.4
7.2
3.4
*These experiments were performed with 120-260 A gold particles and the mean size of the fraction
was 180 A.
#The size of the gold particles does not include the thickness of the coating agent.

TABLE XI ~ Size Distribution of BSA-WTfl-cT7- and Large T-Ag-coated Particles in Injected Cells*
Total No.
Percentage of Particles
in Each Size
Class#
Experiment
(1 h)
of Particles
Measured
10-30 30-50 50-70
AAA
70-90
A
90-110
A
>110
A
Large T-Ag
Nucleus 386
Cytoplasm 600
BSA-WTg-cTy (1:3 dilution)
Nucleus 574
Cytoplasm 621
22.8 49.0 26.2
16.2 30.3 39.3
26.0 40.1 27.4
11.6 23.7 37.6
2.0
8.8 3.7 1.7
5.2 0.7 0.2
16.7 7.1 3.6
*These experiments were performed with 20-120 A gold particles and the mean size of the
fraction was 60 A.
#The size of the gold particles does not include the thickness of the coating agent.

93
Accumulation of tracers along the nuclear envelope
Although the nuclear size distributions were the same for particles
coated with the BSA conjugates (WT8 or WT^) or nucleoplasmin, there was
a significant difference in the relative uptake of the gold as a
function of the coating agent, indicating that not all transport signals
are equally effective. The results shown in Fig. 12 and Table XII
suggest that the effectiveness of specific signals could be related to
their ability to bind to the nuclear envelope.
Figure 12 illustrates the intracellular distributions of 50-280 A
gold particles coated with BSA-WT5 (12a), BSA-WTg (12b) and
nucleoplasmin (12c). Similar amounts of colloid were injected into each
oocyte. Progressing from 12a to 12c, there is an increase in the number
of particles present both within the nucleus and along the envelope.
The relationship between the accumulation of particles along the
envelope and relative uptake is shown in Table XII. The envelope to
cytoplasm ratios were obtained by comparing the number of particles
associated with the envelope to the number of particles in an equal,
randomly selected area of cytoplasm. It is evident that a direct
relationship exists between the number of particles associated with the
envelope and nuclear uptake. A similar relationship between uptake and
binding was obtained with the 120-280 A particle fractions, coated with
BSA-WTg, BSA-WT11, or nucleoplasmin (data not shown).
Coinjection of BSA-WTr- and nucleoplasmin-coated particles
To determine if different targeting signals can be transported
through the same pore, small particles (20-50 A), coated with BSA-WTg,

Figure 12. Accumulation of tracers along the nuclear envelope 1 h
experiment.
A comparison of the intracellular distributions of 50-280 A gold
particles coated with BSA-WT5 (12a), BSA-WTg (12b) and nucleoplasmin
(12c). Similar amounts of colloid were injected in each oocyte.
Progressing from a to c, there is an increase in the number of
particles present both within the nucleus (N) and along the nuclear
envelope. C, cytoplasm. Bar, 0.2 pm.

95
N

Table XII Envelope-Associated Particles
Coating Agent
Envelope:cytoplasm Ratios*
N/C Ratio
BSA-WT5
1.2
0.06
bsa-wt8
2.7
0.35
Nucleoplasmin
6.3
0.71
*These ratios were obtained 1 h after injection as described in the text.

97
and large particles (120-280 A), coated with nucleoplasmin, were
injected simultaneously into the cytoplasm. As seen in Figs. 13a and
13b, small particles and large particles are located either within or
adjacent to the nuclear surface of the same nuclear pore. To control
for nonspecific binding of the small particles to the large particles,
or possible exchange of coat material during the injection procedure,
experiments were also performed in which small gold particles were
coated with BSA-cTy and mixed with the large nucleoplasmin-gold
particles prior to injection. Electron microscopic analysis of this
experiment, shown in Fig. 13c, indicates that only large particles are
present in the pores and the nucleus while the small tracer particles
are retained in the cytoplasm. These experiments demonstrate that
individual pores can recognize and translocate different nuclear
targeting signals.
Discussion
By stabilizing colloidal gold particles with BSA conjugated with
synthetic peptides that contained either active or inactive SV 40 large
T-antigen nuclear transport signals, it was possible to prepare a range
of electron microscopic tracers that varied in both size and signal
content. Electron microscopic analysis of the intracellular
distributions of the tracer particles following microinjection into
oocytes led to the following conclusions. First, BSA conjugates
containing active signals are transported into the nucleus through
central channels located within the pores. Second, both the functional
size of the channels and relative nuclear uptake increase as the signal

Figure 13. Coinjection of gold particles coated with BSA-conjugates
and nucleoplasmin 1 h experiments.
13a and 13b, large nucleoplasmin-coated particles (120-280 A) and small
BSA-WT8-coated particles (20-50 A) can both be seen within the pore
region or just at the nuclear surface of the pores. 13c, large
nucleoplasmin-coated particles are seen entering the nucleus (N) while
small BSA-cTycoated particles are retained in the cytoplasm (C).
Bar, 0.1 pm.

99
N

100
number per gold particle increases. Furthermore, the maximum size of
the transport channel is estimated to be 260 A in diameter. Control
experiments, utilizing BSA conjugated with inactive signals,
demonstrated that differences in the uptake of gold particles coated
with different BSA-WT conjugates were due to variations in the number of
active signals and not to nonspecific factors such as alterations in
particle size and charge.
Nucleoplasmin-coated gold was used as a stabilizing agent to
compare the effectiveness of a different nuclear targeting signal. It
was found that nucleoplasmin, BSA-WTg, and BSA-WT^ all had similar
effects on the functional size of the transport channel, even though
nucleoplasmin has fewer signals than either of the conjugates. However,
the relative uptake of nucleoplasmin-coated gold was significantly
greater than that observed for particles coated with the BSA-WT
conjugates. The fact that nucleoplasmin-gold accumulated along the
nuclear surface to a greater degree than other tracers suggests that the
relative effectiveness of different targeting sequences might be related
to their binding affinity for transport receptors.
The possibility that binding might be an important step in the
transport process was originally suggested by Feldherr et al. (1984),
and was based on the observation that nucleoplasmin-gold particles
accumulate at the surface of the pores during translocation. Other data
in support of this view are the kinetic studies by Goldfarb et al.
(1986) which demonstrate that the nuclear uptake of BSA conjugated with
large T targeting signals is saturable and, therefore, likely to be a
receptor-mediated process. Furthermore, Newmeyer and Forbes (1988)

101
obtained evidence that transport involves two separate events; the first
is binding to the pores, which is signal sequence dependent, and the
second is translocation into the nucleus, which is ATP dependent.
Overall, the data obtained in this study are consistent with the view
that transport occurs through the pores by a gated process. I would
suggest that a 90 A channel is normally present within the pores
allowing for the diffusion of smaller macromolecules into and out of the
nucleus (Paine, 1975). However, in response to an appropriate transport
signals, the dimensions of the channel can increase in size to
accommodate the uptake of transportable (nondiffusive) macromolecules.
The results obtained with nucleoplasmin and the BSA conjugates
indicate that the extent of channel dilation might be variable and
dependent on the number of simultaneous interactions between signals and
receptors. Thus, the degree to which the channels are dilated is likely
to be modulated by a combination of two factors, 1) the number of
transport signals available and 2) the binding affinity of the signals
for the receptors. According to this model, a small number of high
affinity signals might be as effective in regulating the size of the
transport channel as a larger number of low affinity signals.
In evaluating the effect of signal number on the translocation
process, it should be kept in mind that endogenous karyophilic proteins
would also contribute to the total pool of transport signals. The
degree to which endogenous proteins might influence the uptake of the
tracer particles cannot be determined at this time.
Coinjection of different size gold particles, coated with proteins
containing different nuclear targeting signals (BSA-WT and

102
nucleoplasmin), demonstrated that individual pores are capable of
recognizing and transporting different nuclear targeting signals. In a
study to show the bidirectional capability of the nuclear pores, it was
demonstrated that individual pores can transport both protein and RNA
(Chapter II, Results). Whether all transport signals can be recognized
by each pore has yet to be determined.

CHAPTER IV
SUMMARY AND PROSPECTUS
Summary of Results
In the present study, microinjection of colloidal gold particles
coated with different RNAs or proteins was used to establish
morphological criteria concerning the nucleocytoplasmic exchange of
macromolecules j_n vivo. The results from the injection of RNA-coated
particles (Chapter II) can be summarized as follows: first, central
channels located within the nuclear pores are visualized as the major,
if not exclusive, site for RNA translocation into the cytoplasm.
Second, poly(A)-coated particles, at least 230 A in diameter, can
penetrate the envelope. Third, most, if not all, of the nuclear pores
have the ability to transport the RNA tracer particles. Finally,
individual pores can be bifunctional, i.e., they can recognize and
transport both RNA and protein. The results presented in chapter III
have established how variations in the number and amino acid sequence of
protein transport signals can affect protein uptake. First, it was
found that particles coated with BSA conjugated with synthetic peptides
that contained the SV 40 large T-antigen transport signal translocate
into the nucleus through the centers of the nuclear pores. It was then
demonstrated that variations in the number and sequence of protein
transport signals can affect both the functional size of the transport
channel and the relative uptake of particles into the nucleus. The
103

104
maximum functional size of the transport channel was estimated to be 260
A in diameter. Furthermore, it was found that individual pores can
recognize and transport proteins containing different targeting signals.
The ramifications of these results are discussed in relation to how the
pores might regulate nucleocytoplasmic exchange and thus, be intimately
involved in the modulation of cellular activity.
The selectivity of the nucleocytoplasmic exchange process can occur
at the level of the nuclear envelope, within the nucleus or cytoplasm.
Since the nuclear pores represent the major sites of exchange for
macromolecules, their role in regulating the exchange process is of
special interest.
Nuclear Envelope Selectivity
As discussed in Chapter I, the patent size of the nuclear pore can
determine the size of the molecule which can diffuse into and out of the
nucleus, with the rate of uptake or efflux being inversely related to
size. This mode of selection by the envelope can be considered one
level of specificity since any molecule that is larger than the
diffusion channel (90-120 A in diameter) tends to be excluded from the
nucleus, unless a specific transport process is involved.
Several lines of evidence have clearly established that a selection
process exists for the transport of proteins into the nucleus. It has
been demonstrated for a number of nuclear proteins (Chapter I,
endogeneous macromolecules), that information which specifies selective
nuclear entry resides in a region of their primary sequence.
Presumably, the interaction of the targeting signal with the cytoplasmic

105
surface of the nuclear pore most likely involves the specific
recognition of the signal by a pore receptor. This interaction provides
the cell with an additional level of control since binding allows for
the selection of transportable molecules. The removal or alteration of
these targeting signals by either proteolytic cleavage or DNA
methodology results in the protein retaining its cytoplasmic location.
The results in Chapters II and III indicate that translocation of
gold particles coated with RNA, nucleoplasmin, or the BSA-WT-conjugates
occurs through the centers of the pores and not along the periphery
which indicates the involvement of common pathways. The translocation
of the tracers was found to be a selective process. In all cases,
particles coated with synthetic polymers (PVP or polyglutamic acid) or
exogenous macromolecules (BSA or ovalbumin) were virtually excluded from
the pores, demonstrating that the RNA- and BSA-WT-coated gold
translocation is due to the presence of the coating agent. In addition,
the size of the particles, up to 260 A in diameter, that penetrate the
pore region far exceeds the upper limit of diffusion, suggesting that an
active transport process is involved.
It was demonstrated that most, if not all, of the nuclear pores are
capable of transporting different classes of RNA. Similar results were
obtained after cytoplasmic injection of gold particles coated with
proteins containing active targeting signals. Visualization of the
nuclear envelope in the region of injection indicated that a large
percentage of the pores contained protein-coated gold particles. In
addition, the double injection experiments showed that individual pores
are capable of translocating both RNA-coated and protein-coated gold

106
particles and they can also recognize and transport proteins containing
different nuclear targeting signals. Based upon these results it is
suggested that nuclear pores have a broad range of specificity and
distinct functional classes of pores might be nonexistent.
Since the signals from large T-antigen cross-linked to BSA and
different RNAs are capable of translocating through the same nuclear
pores as nucleoplasmin, it is of interest whether they all use the same
mechanism for translocation. It is not known if the RNAs complex with
proteins which subsequently initiate their transport or whether RNA
itself has a putative transport signal. Since BSA-WT- and
nucleoplasmin-coated particles can translocate through the same pore it
remains to be determined if there are different receptors for different
transport signals within individual pores. Although a concensus
sequence for protein transport signals does not exist, signal sequences
usually contain a short stretch of basic amino acids. In this respect,
nucleoplasmin and large T-antigen share partial homology.
It is clear that specificity observed for the nucleocytoplasmic
exchange of endogeneous macromolecules cannot be accounted for entirely
by the properties of the pores. Thus, it is likely that selectivity of
proteins and RNA designated for transport can also occur within either
the nuclear or cytoplasmic compartments. For example, mRNA must be
released from the intranuclear matrix with the hydrolysis of ATP prior
to efflux (Schroder et al., 1987). The presence of poly(A) tails on
mRNA might be part of the selection process; however, poly(A)mRNA also
must somehow be selected since histone mRNA is not polyadenylated. In
addition, many of the proteins can be bound within the cytoplasm or

107
nucleus, thus being denied access to the pores and preventing transport.
Therefore, the cell can have molecules with active transport signals;
however, they may be inaccessible for transport due to selective binding
away from the pores or specific masking of the signal.
Dynamic Aspects of the Transport Channels
In addition to localizing the pathway for exchange, microinjection
of the tracers provided a method for analyzing the morphological and
functional characteristics of the transport channel. After injection of
a gold fraction ranging from 50-280 A in diameter, the largest particles
capable of penetrating the pores were 230 A. When including the size of
the coat material (approximately 30 A), the maximum functional size of
the transport channel was estimated to be 260 A in diameter. Since it
is unlikely that a single endogenous protein would have as high a number
of transport signals as the BSA-WTg and BSA-WT^ conjugates, 8 and 11
signals respectively, it is of interest to consider the possible
biological significance for a channel of this size. First, it is likely
that several endogenous molecules are continually being transported into
and out of the nucleus and simultaneous exchange through the pores might
require a full dilation of the transport channel. A second requirement
for the 260 A translocation channel is the transport of large ribosomal
components and mRNPs that are synthesized in the nucleus and pass
through the pores into the cytoplasm. Stevens and Swift (1966) and
Skoglund et al. (1983) have shown that a large mRNP particle,
approximately 260 A in diameter, passes through the nuclear pores of

108
Chironomus salivary gland cells. These results provide direct evidence
for the requirement of a 260 A transport channel.
Two possible mechanisms were explored to determine how the cell and
the pore complex might regulate the exchange of proteins across the
nuclear envelope. Since the number of synthetic peptides per carrier
protein could be altered and the SV 40 large T-antigen signal was not
homologous to the nucleoplasmin transport signal, the effect of
variations in signal number and sequence on protein transport through
the pores was studied.
At the surface of the pore, the interaction between the signal and
its receptor sets up another point of regulatory control by the nuclear
envelope. As shown in chapter III, the functional size of the transport
channel and relative uptake is dependent on both the nature of the
signal and number of targeting signals per molecule. The results
indicate a direct relationship between signal number and the size of the
tracer particles that can penetrate the nuclear pores. The functional
size of the channel would be dependent on the total number of available
signals accessible to the pore at a given time.
In addition to signal number, the affinity of the signal for its
receptor could affect the uptake of coated particles. A comparison of
uptake and accumulation of tracers at the nuclear surface suggests that
the nucleoplasmin signal has a higher affinity for the pore receptors
than the large T-antigen signal. This could explain why nucleoplasmin-
coated gold, which has a fewer number of signals than BSA-WTg and BSA-
WT^, is transported more efficiently than the active BSA-conjugates.
Lanford et al., (1988) studied the effects of amino acid substitutions

109
within the synthetic peptide signal on protein transport. They found
the rate of transport decreased when different basic amino acids were
substituted in the lysine 128 position, and that transport activity was
abolished when neutral asparagine was used. Based on these findings, I
would propose that a change of amino acids within the signal sequence
can alter the affinity of the signal for the receptor, resulting in
slower uptake.
Proposed Model
The transport channel is not a fixed structure since it can
fluctuate between 90 A (patent diffusion channel) and 260 A (maximum
size of the transport channel) in diameter, the functional diameter
being dependent on the number of binding events between receptors and
transport signals. There appear to be at least two factors involved in
the variability of the transport channel: the signal number and
affinity. From this data, I would propose that the mechanism for
protein transport through the nuclear pores of oocytes occurs by a gated
process. Normally, in an unactivated state, the channel within the pore
would be 90 A in diameter and available for diffusion into and out of
the nucleus. However, activation by transport signals would result in
the dilation of the transport channel to an extent which is directly
related to the number of active signals bound to the receptors at or
within the pore complex. Furthermore, a small number of high affinity
binding signals might be as effective in regulating the size of the
transport channel as a larger number of low affinity signals.
Alternatively, the dilation of the pore might be an all or none process

110
rather than a channel opening incremently. Although the protein uptake
data favors the latter, the two mechanisms cannot be distinguished at
this time.
Consistent with the gating model are the results presented by
Newmeyer and Forbes (1988) and Richardson et al. (1988). They show that
the transport of proteins across the envelope i_n vitro can be
experimentally separated into at least two steps. The first step is
the binding of the proteins to the cytoplasmic pore surface which
involves the specific recognition of the transport signal by the nuclear
pore complex. Binding to this region is signal sequence dependent, thus
again demonstrating selectivity by the pore complex, and ATP
independent. The second step is the ATP dependent translocation through
the pores.
According to the model, multiple interactions need occur prior to
distention of the transport channel. If so, then multiple copies of
receptors should reside in the pore complex. Components speculated to
be part of the translocation machinery are the group of glycoproteins
localized to the pore complex by Snow et al. (1987), Finlay et al.
(1967), and Davis and Blobel (1986). Although these glycoproteins have
not been demonstrated to function as receptors, they are present in
multiple copies within each pore complex, consistent with the gating
model. Furthermore, in an attempt to define the role of the
glycoproteins in protein transport it was shown that WGA binds to the
pore region and inhibits the uptake of nucleoplasmin in synthetic nuclei
(Finlay et al., 1987) and cultured cells (Yoneda et al., 1987), but does
not block the diffusion channel.

Ill
There are several implications of the model with regard to
regulation of nucleocytoplasmic exchange. The dilation of the pores
would control exchange in accordance to the related needs of the cell
and provide the cell with an efficient method for exchange. In
addition, the model suggests that endogenous molecules available for
transport can act collectively to open the transport channels. The
factors involved in regulating pore activation, signal number and
affinity, are supportive of this model. To increase the rate of uptake,
a single protein could have multiple signals (i.e., nucleoplasmin) or
proteins might aggregate to increase their overall signal number,
possibly like histones. Furthermore, proteins containing different
targeting signals might have differential binding affinities for pore
receptors which, in turn, might modulate their rate of uptake. Finally,
the complete activation of the pores is large enough for the transfer of
ribosomal subunits and RNP particles.
Future Trends
One question that remains is whether the protein transport signal
is vectorial. In a cursory study, nuclear injection of the protein
conjugates was performed to determine if the signal for protein uptake
is bidirectional, i.e., capable of transport into and out of the
nucleus. One and six hours after nuclear injection, particles coated
with the BSA-conjugates were not observed in either the pores or the
cytoplasm (data not presented) indicating that transport signal is
vectorial; however, the experiments are not definitive. It is possible
that the gold particles were bound specifically or nonspecifically

112
within the nucleus due to the signals or the BSA, thus making the gold
conjugates inaccessible to the pores. Interestingly, the signal region
of large T-antigen is not the same as the DNA binding domain and
preliminary results suggest that BSA alone is not concentrated within
the nucleus; thus, the signal might be vectorial in nature. One
possible reason for the nuclear retention of the conjugates is that the
signal becomes masked upon nuclear entry which then causes the efflux
process to be inactive. The best evidence for the masking or unmasking
of transport signals is the snRNA associated proteins binding to snRNAs
to form snRNPs prior to nuclear entry (Mattaj and De Robertis, 1985).
Although the complex may be diffusable within its cellular compartment,
masking of the transport signal can be considered one form of binding
that blocks bidirectional transport.
Another explanation to support vectorial transport is the
asymmetric distribution of transport receptors. Snow et al. (1987) have
indicated that one of their antibodies, specific for a glycoprotein,
labels almost exclusively the nucleoplasmic side of the pore complex.
Although this glycoprotein is not known to function as a receptor, its
asymmetric distribution is consistent with the view of vectorial
transport. Until more is known about the intranuclear interactions of
the tracer particles, it cannot be concluded if the uptake signals are
unidirectional. The use of resealed nuclear envelope vesicles (see
chapter I), which contain no intranuclear components, could be one
method for studying this question.
Additional mechanisms that the cell could use to regulate exchange
of macromolecules across the envelope include: i) changing the

113
permeability properties of the nuclear pores and/or ii) altering the
number of pores per envelope. It has been shown in amoebae that the
nuclear pores have some variability with respect to diffusion that
occurs during different stages of the cell cycle (Feldherr, 1966). In
addition, it was demonstrated that there are changes in the functional
properties of the pores during different metabolic states of the amoebae
and the differences in uptake were not due to a change in pore number or
size (Feldherr, 1971).
Maul et al. (1972) have demonstrated that pore formation in HeLa
cells during the cell cycle is biphasic. They found two significant
increases in pore formation during mitosis, the first being within an
hour after division and the second, shortly after the beginning of
S phase. Furthermore, they found that the number of pores per nucleus
can vary with a change in cellular activity. Thus, the cell, in
principle, can regulate the rate of accumulation by changing the number
of pores available for nucleocytoplasmic exchange.
In contrast to diffusion (see above), a question that remains to be
investigated is whether the transport properties of the pores change
significantly during the cell cycle and/or during changes in cellular
activity. Dreyer et al. (1985) have suggested that the temporal
accumulation of different proteins can be due to the nature and number
of the transport signals. However, as mentioned above, pore variability
can be dependent on the physiological state of the cell. Since the
proteins do not undergo posttranslational modification and they
distribute differently at various times throughout development, they
might be responding to changes in the envelope. At present, it is not

114
known if the nuclear envelope transport properties are altered during
changes in cellular activities. These results can also be explained by
de novo synthesis of specific carriers which subsequently result in a
shift in a molecule's partitioning between cytoplasm and nucleus
although no carriers for nuclear transport have been identified.
What is the underlying molecular mechanism required for the
movement of macromolecules through the length of the transport channel?
Berrios et al. (1983) have identified an ATPase polypeptide associated
with nuclear-envelope enriched fractions. The molecular mechanisms for
the actual translocation has been postulated to be a contractile force
provided by this ATPase activity which is a myosin-like heavy chain
polypeptide localized in the nuclear periphery (Berrios and Fischer,
1986). However, it remains to be determined if the ATPase activity
localizes predominantly in the pore complexes. Schindler and Jiang
(1986) used the fluorescence redistribution after photobleaching
technique to study factors that influence the flux rates of dextrans
across the nuclear envelope of isolated nuclei. They found that the
addition of anti-actin antibodies markedly reduced the rate of nuclear
entry of the labelled dextran. Furthermore, anti-myosin antibodies
significantly blocked the ATP stimulatory effects on the dextran flux
rate. From these results, it was postulated that the contractile
proteins, actin and myosin, are part of the pore complex and involved in
the opening and closing of the pores. The problem is that these results
have not been verified i_n vivo, and it is known that nuclear isolation
can significantly alter the properties of the pores.

115
Isolation of receptors involved in the translocation process
remains an essential step to understanding the nucleocytoplasmic
transport of macromolecules. Additionally, it is equally important to
determine if there are different receptors in individual pores or
whether there are common receptors for the transport of different
protein signal sequences and/or putative RNA transport signals. After
identification of receptors an important area that remains to be
investigated is the binding affinities of different transport signals as
the affinity of signals is most likely, in part, a mechanism that the
cell can use to regulate nucleocytoplasmic exchanges.

APPENDIX
Top View
Side View
A schematic representation of the nuclear pore complex based on
data from Franke's (1974) and Unwin and Milligan's (1982) model. G,
annular globules; S, spokes; C, central granule.
116

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BIOGRAPHICAL SKETCH
Steven I. Dworetzky was born December 19, 1959 in New Rochelle, New
York. He graduated from high school in 1977. Following high school, he
attended Skidmore College in Saratoga Springs, New York, and earned a
Bachelor of Arts degree in biology/chemistry in 1981. In September 1981
he entered graduate school at the University of Florida in the
Department of Anatomy. In the summer of 1984, he successfully completed
an eight week course entitled Cell Physiology: Cellular and Molecular
Biology at the Marine Biological Laboratory in Woods Hole,
Massachusetts. He completed the requirements for the degree of Doctor
of Philosophy in April 1988 and accepted a postdoctoral research
position at the University of Massachusetts Medical Center in
Shrewsbury, Massachusetts. He is a member of the American Society for
Cell Biology.
130

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Anatomy and Cell Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Robert y. Cohen
Associate Professor of Biochemistry
and Molecular Biology
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Christopher M. West
Associate Professor
Cell Biology
of Anatomy and
I certify that I have read this study and that
conforms to acceptable standards of scholar^y-brese
adequate, in scope and quality, as a dissertation
Doctor of Philosophy.
in my opinion it
tion and is fully
the degree of
>ary S. Steii
Professor of/'Biochemistry and
Molecular Biology
This dissertation was
College of Medicine and to
partial fulfillment of the
Philosophy.
submitted to the Graduate Faculty of the
the Graduate School and was accepted as
requirements for the degree of Doctor of
Dean, College of Medicine
/vVcx-
Dean,
August, 1988



LIST OF ABBREVIATIONS
a
A
ATP
Arg
3
BSA
cT
DMSO
DNA
G
3H
h
HEPES
125!
IgG
kd
M
min
ml
mM
A1 pha
Angstrom
Adenosine-51-triphosphate
Arginine
Beta
Bovine serum albumin
Mutant large T-antigen
Dimethyl sulfoxide
Deoxyribonucleic acid
Guanosine
Tritiated
Hour(s)
N-2-Hydroxyethylpiperazine-N1-2-
Ethanesulfuric acid
Iodinated
Immunoglobulin
Kilodaltons
Molar concentration
Minute(s)
Mi 11 i 1iter(s)
Mi 11imolar
ix


35
binding sites that increase in affinity by a kinase-dependent
phosphorylation reaction. The actual role these components play in the
in vivo translocation of mRNA across the envelope has not yet been
clearly defined; however, poly(A)+mRNA efflux models have been proposed
by Agutter (1985a) and Schroder et al. (1988).
The model proposed by Agutter (1985a) integrates the components
involved in RNA translocation: NTPase, poly(A) binding sites, protein
kinases and phosphohydrolase, to explain nucleocytoplasmic transport of
poly(A)+mRNA. The following scheme is based on Agutter's model (1985a).
The NTPase exists in a complex with the poly(A) binding site. A protein
kinase uses MgATP as a substrate to phosphorylate the poly(A) binding
site. Binding of nuclear poly(A)+mRNA to the [phosphorylated binding
site-NTPase] complex stimulates dephosphorylation by the
phosphohydrolase. MgATP is also a substrate for NTPase, the binding of
MgATP to the NTPase of the [poly(A)+mRNA-poly(A) binding site-NTPase]
complex causes the displacement of poly(A)+mRNA, thus facilitating the
translocation of mRNA. The NTPase then hydrolyses ATP and the [NTPase-
poly(A) binding site] complex is available again to start another cycle.
At least two cytosolic proteins, 34 kd and 58 kd, with an affinity
for poly(A) can stimulate mRNA efflux j_n vitro (Moffett and Webb, 1981,
1983; Schroder et al., 1986b). The 34 kd protein, in the presence of
poly(A), stimulates the NTPase activity to a greater extent than poly(A)
by itself. In the absence of poly(A), p34 has no effect on kinase or
phosphohydrolase activity. The 34 kd protein is thought to enhance
NTPase activity by increasing the affinity of poly(A) to the poly(A)
binding site in the phosphorylated nuclear envelope. Schroder et al.


46
to the displacement of pigment granules in the cortex. Nuclear
injections could then be accomplished. Calcium-free Ringer's was used
as an extracellular medium during injection to prevent possible
precipitations of the colloid as the micropipettes were introduced into
the cells. Immediately after injection the cells were returned to
complete Ringer's solution until subsequent fixation. The total
exposure to calcium-free Ringer's was less than 30 minutes. The tip
diameters of the micropipettes were 10-15 pm.
Electron microscopy and analysis
The cells were fixed using a procedure similar to that described by
Kalt and Tandler (1971). The oocytes were fixed initially overnight at
4C in 100 mM HEPES containing 3% glutaraldehyde (vol/vol), 2%
paraformaldehyde (wt/vol), 2.5% DMSO (vol/vol) and 1 mM CaCl2 (pH 7.2).
The nuclei were then dissected out with their surrounding cytoplasm,
post-fixed in 2% OSO4 (wt/vol) for one hour, and stained with 0.5%
p-phenylenediamine (wt/vol) in 70% acetone for 30 minutes. Finally, the
samples were dehydrated in a graded series of acetone and embedded in
Spurr's medium. Thin sections were cut on a Reichert microtome and
analyzed with a JE0L 100S electron microscope (JEOL USA, Cranford, NJ).
In the RNA efflux studies it was presumed that gold particles
located within the pores were in the process of translocation, and that
particles present in the cytoplasm just adjacent to the pores had
completed the translocation process. These areas will be referred to as
regions 1 and 2, respectively (see Fig. 1). Pores that contained one or
more particles in either or both of these regions were considered to be


123
Kalderon, D., W.D. Richardson, A.F. Markham, and A.E. Smith. 1984a.
Sequence requirements for nuclear localization of simian virus 40
large-T antigen. Nature 311:33-38.
Kalderon, D., B.L. Roberts, W.D. Richardson, and A.E. Smith. 1984b. A
short amino acid sequence able to specify nuclear location. Cell
39:499-509.
Kalt, M.R., and B. Tandler. 1971. A study of fixation of early
amphibian embryos for electron microscopy. J. Ultrastruct. Res.
36:633-645.
Kanno, Y., and W.R. Loewenstein. 1963. A study of the nucleus and
cell membrane of oocytes with an intra-cellular electrode. Exp.
Cell Res. 31:149-166.
Kim, S.D., G.J. Quigley, F.L. Suddath, A. McPherson, D. Sneden, J.J.
Kim, J. Weinzierl, and A. Rich. 1973. Three-dimensional structure
of yeast phenylalanine transfer RNA; Folding of the polynucleotide
chain. Science (Wash.) 179:285-288.
Kleinschmidt, J.A., C. Dingwall, G. Maier, and W.W. Franke. 1986.
Molecular characterization of a karyophilic, histone-binding
protein: cDNA cloning, amino acid sequence and expression of
nuclear proteins N1/N2 of Xenopus laevis. EMBO J. 5:3547-3552.
Kohen, E., G. Siebert, and C. Kohen. 1971. Transfer of metabolites
across the nuclear membrane: A microfluorometric study. Hoppe-
Seyler's Z. Physiol. Chem. 352:927-937.
Kressman, A., and M.L. Birnstiel. 1980. Surrogate genetics in the
frog oocyte. In_ Transfer of Cell Constituents into Eukaryotic
Cells. J.E. Celis, A. Graessmann, and A Loyter, editors. Plenum
Press, New York. 383-407.
Laemmli, U.K. 1970. Cleavage of structural proteins during the
assembly of the head of bacteriophage T4. Nature (Lond.) 227:680-
685.
Lam, K.S, and C.B. Kasper. 1979. Selective phosphorylation of a
nuclear envelope polypeptide by an endogenous protein kinase.
Biochem. 18:307-311.
Lanford, R.E., and J.S. Butel. 1980. Biochemical characterization of
nuclear and cytoplasmic forms of SV 40 tumor antigens encoded by
parental and transport-defective mutant SV40-adenovirus T hybrid
viruses. Virology 105:314-327.
Lanford, R.E., and J.S. Butel. 1984. Construction and
characterization of an SV40 mutant defective in nuclear transport
of T antigen. Cel 1 37:801-813.


10
Small molecules
Permeability of the nuclear envelope to small molecules was studied
by microinjecting sucrose (Horowitz, 1972), inulin (Horowitz and Moore,
1974), and the non-metabolizable amino acid a-aminoisobutyric acid (AIB)
(Frank and Horowitz, 1975) into oocytes. All of these small molecules
rapidly crossed the envelope such that standard flux measurements could
not accurately define the rate of movement, suggesting that the nuclear
envelope is of equivalent permeability to an equal thickness of
cytoplasm. To better define the uptake kinetics of small molecules,
Kohen et al. (1971) used high resolution microfluorimetry to quantitate
rapid movement across the envelope. They investigated the permeability
of glycolytic intermediates. Their results demonstrate that the
metabolites move across the nuclear envelope with a 35 millisecond
delay, indicating that for small molecules the nuclear envelope does not
constitute a permeability barrier. Thus, similar to ions, it is likely
that small molecules migrate into the nucleus through the nuclear pores.
Exogenous macromolecules
The nuclear uptake of radio-labelled or fluorescein-labelled
exogenous molecules has been investigated by microinjecting these
tracers into the cytoplasm of cells and following their intracellular
distribution with time. Colloidal gold particles and ferritin, both of
which can be visualized with the electron microscope, have also been
used to study nuclear permeability. Microinjection of these exogenous
tracers provides a method to better understand some of the physical
characteristics of the nuclear envelope with respect to diffusion.


CHAPTER III
THE EFFECTS OF VARIATIONS IN THE NUMBER AND SEQUENCE
OF TARGETING SIGNALS ON NUCLEAR UPTAKE
Introduction
The nuclear pore complex has been identified as the major, if not
the exclusive site for macromolecular diffusion and transport between
the nucleus and cytoplasm of eukaryotic cells (Feldherr et al., 1962;
Feldherr et al. 1984). Evidence for mediated protein transport was
determined intially for RN1 (Feldherr et al., 1983) and nucleoplasmin
(Dingwall et al., 1982), both of which are major karyophilic proteins
found in amphibian oocytes. From electron microscopic analysis of the
intracellular distribution of nucleoplasmin-coated gold particles, it
was established that the transport channels, located in the centers of
the pores, are at least 200 A in diameter (Feldherr, et al., 1984). In
contrast, the channel has a functional diameter of approximately 90 A
with respect to diffusion (Paine et al., 1975).
Several laboratories have utilized recombinant DNA methodology and
single amino acid substitutions to obtain probes useful for the
localization and characterization of transport signals that target
specific proteins to the nucleus. These approaches have been used to
study nuclear uptake of the following proteins; nucleoplasmin (Burglin
and De Robertis, 1987; Dingwall et al., 1987), simian virus (SV 40)
large T-antigen (Kalderon et al., 1984a,b), the yeast regulatory
proteins MATa2 (Hall et al., 1984) and GAL4 (Silver et al., 1984), yeast
71


LIST OF FIGURES
Figure Page
1. A schematic representation of regions 1 and 2 47
2. tRNA-gold, nuclear injection 51
3. 5S RNA-gold, nuclear injection 51
4. Poly(A)-gold, nuclear injection 56
5. PVP-gold, nuclear injection 56
6. Double injection experiment 64
7. 5S RNA-gold, cytoplasmic injection 64
8. BSA-WTn-gold 83
9. BSA-cT7-gold 83
10. Nuclear particle distributions: BSA-WTg and BSA-WT5 ... 88
11. Nuclear particle distributions: BSA-WTg, BSA-WT^ and
nucleoplasmin 90
12. Accumulation of tracers along the nuclear envelope .... 95
13. Coinjection of gold particles coated with BSA-conjugates
and nucleoplasmin 99


% of Total Nuclear Particles
40
45-73 73-101 101-129 129-157 157-185 185-213 213-241 241-269 >269
Particle Size (A)
Nuclear particle distributions: BSA-WTg and BSA-WTg
Figure 10.


95
N


65
Discussion
Colloidal gold procedures have been used previously to identify and
characterize the regions of the pores that are involved in the transport
of karyophilic proteins across the nuclear envelope (Feldherr et al.,
1984). It was found that the transport channels are located in the
centers of the pores and have functional diameters of at least 200 A.
In this study, the same experimental approach was used to characterize
the pathways for RNA efflux and, in addition, to determine whether
individual pores have the capability of transporting both RNA and
protein.
The presence of RNA-coated gold particles within and along the
cytoplasmic surface of the nuclear pores provides direct evidence that
these tracers are translocated across the envelope at these sites.
Furthermore, almost all of the pores (97% or more) can function in the
translocation process. The biological significance of these findings;
however, is dependent on demonstrating that the pathways visualized for
the colloidal gold tracers are the same as those normally used for the
exchange of endogenous RNA. In this regard, we have attempted to
demonstrate that the translocation of RNA-gold through the nuclear
pores is both a selective and active process, and that the observed gold
distributions were not due to technical artifacts.
The fact that gold was not present in the pores of cells fixed 10
seconds after injection shows that accumulation in these structures is
not simply a result of the injection procedures per se or a
redistribution of the particles during fixation and processing for
electron microscopy. Control studies also demonstrated that the


56


51
N
t.-
* '
/>1 *' j * *i
3 sv C
v Wi A**


112
within the nucleus due to the signals or the BSA, thus making the gold
conjugates inaccessible to the pores. Interestingly, the signal region
of large T-antigen is not the same as the DNA binding domain and
preliminary results suggest that BSA alone is not concentrated within
the nucleus; thus, the signal might be vectorial in nature. One
possible reason for the nuclear retention of the conjugates is that the
signal becomes masked upon nuclear entry which then causes the efflux
process to be inactive. The best evidence for the masking or unmasking
of transport signals is the snRNA associated proteins binding to snRNAs
to form snRNPs prior to nuclear entry (Mattaj and De Robertis, 1985).
Although the complex may be diffusable within its cellular compartment,
masking of the transport signal can be considered one form of binding
that blocks bidirectional transport.
Another explanation to support vectorial transport is the
asymmetric distribution of transport receptors. Snow et al. (1987) have
indicated that one of their antibodies, specific for a glycoprotein,
labels almost exclusively the nucleoplasmic side of the pore complex.
Although this glycoprotein is not known to function as a receptor, its
asymmetric distribution is consistent with the view of vectorial
transport. Until more is known about the intranuclear interactions of
the tracer particles, it cannot be concluded if the uptake signals are
unidirectional. The use of resealed nuclear envelope vesicles (see
chapter I), which contain no intranuclear components, could be one
method for studying this question.
Additional mechanisms that the cell could use to regulate exchange
of macromolecules across the envelope include: i) changing the


41
There is evidence that RNA is not freely diffusible within the
nucleus and cytoplasm but is associated with structural elements of the
nuclear matrix and cytoskeleton (Agutter, 1985a; Feldherr, 1980; Fey et
al., 1986; van Eekelen and van Venrooij, 1981). Based partly on these
findings, Agutter (1985a) postulated that the efflux of RNA occurs in
three stages by a solid-phase mechanism; first, transport to the nuclear
envelope along the nuclear matrix, second, translocation through the
pores, and third, transport along the cytoskeletal matrix.
The focus of the present investigation is on the second step of the
efflux process, i.e., translocation across the envelope. The main
objectives are; first, to localize and characterize the specific regions
of the nuclear pores involved in the translocation of different classes
of RNA-coated gold to the cytoplasm and, second, to determine whether
individual pores can function in both RNA efflux and protein uptake, or
whether separate classes of nuclear pores exist. In order to obtain the
resolution necessary to examine individual exchange sites, coated
colloidal gold particles are used as tracers. The particles are
microinjected into oocytes, and subsequently localized by electron
microscopy.
The results are summarized as follows: first, gold particles
coated with tRNA (met or phe), 5S RNA, or poly(A) are all translocated
into the cytoplasm through central channels, at least 230 A in diameter,
located within the nuclear pores. Particles stabilized with
nonphysiological polynucleotides are also translocated; however, the
number and distribution of the particles associated with the pores
varied for different coating agents. Second, approximately 97% of the


127
Riedel, N., and H. Fasold. 1987. Preparation and characterization of
nuclear-envelope vesicles from rat liver nuclei. Biochem. J.
241:203-212.
Roberts, B.L., W.D. Richardson, and A.E. Smith. 1987. The effect of
protein context on nuclear location signal function. Cel 1 50:465-
475.
Sachs, A.B., and R.D. Kornberg. 1985. Nuclear polyadenylate-binding
protein. Mol. Cel 1. Biol. 5:1993-1996.
Schatz, G., and R.A. Butow. 1983. How are proteins imported into
mitochondria? Cell 32:316-318.
Scheer, U. 1970. The ultrastructure of the nuclear envelope of
amphibian oocytes: A reinvestigation. III. Actinomycin-induced
decrease in central granules within the pores. J. Cell Biol. 45:
445-449.
Schindler, M., and L.W. Jiang. 1986. Nuclear actin and myosin as
control elements in nucleocytoplasmic transport. J. Cell Biol.
102:859-862.
Schroder, H.C., M. Bachmann, B. Diehl-Seifert, and W.E.G. Muller.
1988. Transport of mRNA from nucleus to cytoplasm. Progress in
Nuc. Acid Res. Mol. Biol. 34:89-142.
Schroder, H.C., M. Rottmann, M. Bachmann, and W.E.G. Muller. 1986a.
Purification and characterization of the major nucleoside
triphosphatase from rat liver nuclear envelopes. J. Biol. Chem.
261:663-668.
Schroder, H.C., M. Rottmann, M. Bachmann, W.E.G. Muller, A.R. McDonald,
and P.S. Agutter. 1986b. Proteins from rat liver cytosol which
stimulate mRNA transport: Purification and interactions with the
nuclear envelope mRNA translocation system. Eur. J. Biochem.
159:51-59.
Schroder, H.C., D. Trolltsch, U. Friese, M. Bachmann, and W.E.G.
Muller. 1987. Mature mRNA is selectively released from the
nuclear matrix by an ATP/dATP-dependent mechanism sensitive to
topoisomerase inhibitors. J. Biol. Chem. 262:8917-8925.
Schulz, B., and R. Peters. 1986. Intracellular transport of a
karyophilic protein, hi Nucleocytoplasmic Transport. R. Peters
and M. Trendelenburg, editors. Springer-Verlag, Berlin. 171-184.
Setyono, B., and J.R. Greenberg. 1981. Proteins associated with
poly(A) and other regions of mRNA and hnRNA molecules as
investigated by crosslinking. Cel 1 24:775-783.


42
pores in Xenopus oocytes can function in the translocation of RNA-coated
gold. Third, the accumulation of particles in the pores is a saturable
process. Fourth, individual pores can function in both RNA efflux and
protein uptake. Control experiments ruled out the possibilities of
fixation artifacts and non-specific exchange processes.
Materials and Methods
Xenopus laevis were purchased from Xenopus 1 (Ann Arbor, Michigan)
and maintained as reported previously (Feldherr, 1975).
Nuceoplasmin isolation
Nucleoplasmin was isolated from a starting volume of 30 ml of
Xenopus ovaries. The isolation procedure was similar to that described
by Dingwall et al. (1982), with the exception that an anti-
nucleoplasmin IgG affinity column was substituted for the DEAE-
cellulose and phenyl sepharose columns. Polyclonal antibodies against
nucleoplasmin were generated in rabbits and affinity purified. A
cleared cell homogenate, obtained from the lysed oocytes (Feldherr et
al., 1984), was passed over an anti-nucleoplasmin affinity column, and
the column was washed free of nonbound proteins. Nucleoplasmin was
eluted in 1 ml fractions with 50 mM glycine-HCl buffer (pH 2.5) into
microcentrifuge tubes containing sufficient 1 M tris (pH 9.0) to
increase the pH to 7.5. The protein was monitored at 280 nm, and the
fractions containing nucleoplasmin were pooled and treated with
ammonium sulfate (55% saturated) overnight in the cold. The soluble
(NH4)2SO4 fraction was then dialyzed against a solution containing


DEDICATED TO MY GRANDPARENTS, LEON AND FRANCES DWORETZKY


16
selectively retained within the nucleus, by binding to a nondiffusible
substrate. Second, the exchange of some or all of the endogenous
proteins, through the pores, can occur by facilitated uptake. This mode
of exchange is governed by the intrinsic properties of the molecule.
Following translocation across the envelope, accumulation in the nucleus
can occur by selective binding or by another mechanism for irreversible
transport.
It has been reported that many proteins accumulate in the nucleus
against a concentration gradient--e.g., for example, histones (Gurdon,
1970) and N1/N2 (De Robertis et al., 1978)--attain nuclear to
cytoplasmic ratios of 115 and 120, respectively. Austerberry and Paine
(1982) used cryomicrodissection and two-dimensional electrophoresis to
measure quantitatively the intracellular distribution of 90 proteins in
the living cell. Their results demonstrated that at least 35 proteins
within the nucleus have nuclear to cytoplasmic ratios greater than 10,
which suggests some form of selective retention. Additional evidence to
support nuclear binding comes from the experiments performed by Feldherr
and Ogburn (1980). When the permeability properties of the envelope are
altered, as shown by its failure to exclude labelled BSA, most of the
endogenous nuclear proteins still accumulate within the nucleus (see
below).
Bonner (1975b) studied the migration of iji vivo labelled endogenous
proteins into nuclei of amphibian oocytes. After injection of labelled
nuclear proteins into the cytoplasm of unlabelled oocytes, the
radioactivity was found to concentrate in the nucleus. If labelled
cytoplasmic proteins were injected into the cytoplasm of unlabelled


2
Morphology
The discovery of the nucleus is documented as early as 1833. Fifty
years later, light microscopists began speculating that a membrane
surrounded the nucleus. It was Chambers and Fell (1931) who
microdissected cells and demonstrated the existence of a nuclear
envelope. With the use of the electron microscope, Callan and Tomlin
(1950) performed ultrastructural studies on manually isolated oocyte
nuclei and confirmed the existence of an envelope surrounding the
nucleus. They described it as a double membrane structure, with a
porous outer membrane adjacent to the cytoplasm and a continuous inner
membrane adjacent to the nucleoplasm.
With advances in the resolution of the electron microscope and
improvements in fixation procedures, the nuclear envelope and pore
structure became the subject of intense morphological studies. The
envelope and associated structures can be visualized with the electron
microscope by a variety of techniques including i) i_n situ fixation
followed by ultrathin sectioning, ii) negative staining of isolated
envelopes, iii) freeze-etching, and iv) high-resolution scanning
microscopy. One of the first detailed studies utilizing transmission
electron microscopy was performed by Watson (1955, 1959). He analyzed
nuclei from a wide variety of cell types and found that the inner and
outer membranes fuse at regular intervals along the envelope resulting
in the formation of nonmembranous regions ("holes"), ranging from 800-
1200 A in diameter, referred to as the nuclear pores. These pores form
channels that represent potential sites of interaction between the
nucleoplasm and cytoplasm. Since then, laboratories have used the


22
identified to have weak homology with targeting signals from the yeast
MATa2 protein. It was postulated from these results that each tail
domain might have four targeting signals, with a total of 20 signals per
pentamer. Further experimentation on the tail domain has now
demonstrated that each tail contains only 1 signal sequence consisting
of approximately 14 amino acids and though it is similar to the SV 40 T-
antigen targeting signal, it is not homologous (Dingwall, personal
communication 1988).
The list of proteins containing nuclear targeting signals continues
to be expanded. In a search to find a conserved sequence, several
targeting signals have been compared to SV 40 large T-antigen. Protein
signals that have similar amino acid sequences include nucleoplasmin
(Dingwall et al., 1987), polyoma virus large T-antigen (Richardson et
al., 1986), SV 40 VP1 (Wychowski et al., 1986), and Xenopus laevis N1/N2
protein (Kleinschmidt et al., 1986). Nuclear localization sequences
from other proteins showing little homology to SV 40 large T-antigen
include the adenovirus Ela protein (Lyons et al., 1987), the yeast
proteins L3 (Moreland et al., 1985) and MATa2 (Hall et al., 1984), and
GAL4 (Silver et al., 1984).
The translocation of proteins directly across intracellular
membranes has been well documented for endoplasmic reticulum (Warren and
Dobberstein, 1978) and mitochondria (Schatz and Butow, 1983). Proteins
destined for either organelle contain a signal sequence, which is
cleaved after entry. Protein import into the endoplasmic reticulum
occurs by a cotranslational mechanism; however, this mechanism is
unlikely for nuclear uptake since cytoplasmically injected proteins,


69
poly(A) tails of mRNA bind different polypeptides in the nucleus and
cytoplasm (Baer and Kornberg, 1983; Sachs and Kornberg, 1985; Setyono
and Greenberg, 1981); however, it has not been determined if these
proteins are involved in transport.
The fact that over 97% of the pores are involved in RNA efflux,
combined with the earlier observation that the majority of the pores
contained nucleoplasmin-coated particles following cytoplasmic
injections (Feldherr et al., 1984), suggests that these pathways are
bifunctional. The double injection experiments provided direct evidence
supporting this view. Thus, it appears that the central channels within
the pores can function in the translocation of both RNA and protein.
However, this does not necessarily mean that the same molecular
mechanisms are employed.
Six hours after injection, the nuclear to cytoplasmic concentration
ratio of RNA-coated gold was found to be 14:1. Correcting for the
difference in the volumes of the two compartments, it is estimated that
no more than 36% of the particles entered the cytoplasm. Based on data
presented by Zasloff (1983, Table I) an equivalent amount of
radiolabel led tRNA would be expected to leave the nucleus in about 3 h.
There are several factors that could account for this difference.
First, the conformation of tRNA could be modified after adsorption to
the gold particle. In this regard, Tobian et al. (1985) found that
point mutations which alter the conformation of tRNA also decrease its
rate of transport to the cytoplasm. Second, since several tRNA
molecules are adsorbed to the gold particles (the exact number has not
been determined) the binding avidity to components within the nucleus,


36
(1988) suggest that the 58 kd protein inhibits kinase activity by
promoting poly(A) binding to unphosphorylated envelopes, which prevents
the down-regulation of NTPase activity by the kinase.
The third step in the overall RNA transport is the involvement of
the cytoplasm. Although it is established that much of the mRNA is
bound to the cytoskeleton (Jeffery, 1982), it is not clear how the RNA
finds its final destination. The proteins associated with poly(A)+mRNA
in the cytoplasm are different from those that are bound to poly(A)
within the nucleus (Baer and Kornberg, 1983; Sachs and Kornberg, 1985;
Setyono and Greenberg, 1981). It is not known if these proteins are
involved in the transport of RNAs within the cytoplasm or serve as
anchors to cytoskeletal elements. In this regard, it is necessary to
further understand the functional role of the cytoplasmic proteins
associated with RNAs.
Ribonucleoproteins (RNPs) have been visualized exiting the nucleus
through the nuclear pores by electron microscopic cytochemical analysis
(Stevens and Swift, 1966). In a more recent study, Skoglund et al.
(1983) studied the formation and transport of an hnRNP particle, a
transcription product from the Balbiani ring genes in Chironomus. Their
results, by using the Miller spread technique, describe the growth of a
100 A wide filament into a large 400-500 A granule. Prior to export,
these mature RNP particles undergo conformational changes that result in
the formation of rod-shaped structures with an average length of 1350 A
and diameters of 250-300 A that pass through the centers of the nuclear
pores. Even though these RNPs undergo a shape change, they are still


67
within the nucleoplasm or changes in the rate of migration in the
cytoplasm.
As discussed in the introduction, there is evidence that
macromolecules larger than 90 A in diameter are unable to diffuse across
the nuclear envelope, whereas particles as large as 200 A in diameter,
which contain nuclear targeting signals, can be transported through
central channels in the pores. It was found in this study that RNA-
coated gold particles as large as 230 A in diameter (including the coat)
readily penetrate the pores. Since this far exceeds the upper limit for
diffusion, these results suggest that a transport process is involved.
Consistent with this interpretation is the finding that the accumulation
of RNA-gold in the pores is a saturable process. Saturation indicates
the presence of a carrier-mediated transport process. However,
saturation would also occur if the particles simply occupied all of the
available space within and adjacent to the pore channels. The latter
explanation, which would have little bearing on the mechanism of
exchange, is unlikely for two reasons. First, direct electron
microscopic examination of region 1 shows that these areas are not fully
occupied at saturation levels. Second, particles coated with
nucleoplasmin can reach more than twice the concentration in region 2,
than tRNA-coated gold (data not shown), demonstrating that the nature of
the coat material rather than the total number of tracer particles is
the determining factor.
Colloidal tracers, although well suited for localizing and
characterizing exchange sites, are not appropriate for detailed kinetic
studies, which require numerous time points and involve the analysis of


I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Professor of Anatomy and Cell Biology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is fully
adequate, in scope and quality, as a dissertation for the degree of
Doctor of Philosophy.
Robert y. Cohen
Associate Professor of Biochemistry
and Molecular Biology
I certify that I
conforms to acceptable
adequate, in scope and
Doctor of Philosophy.
have read this study and that in my opinion it
standards of scholarly presentation and is fully
quality, as a dissertation for the degree of
Christopher M. West
Associate Professor
Cell Biology
of Anatomy and
I certify that I have read this study and that
conforms to acceptable standards of scholar^y-brese
adequate, in scope and quality, as a dissertation
Doctor of Philosophy.
in my opinion it
tion and is fully
the degree of
>ary S. Steii
Professor of/'Biochemistry and
Molecular Biology
This dissertation was
College of Medicine and to
partial fulfillment of the
Philosophy.
submitted to the Graduate Faculty of the
the Graduate School and was accepted as
requirements for the degree of Doctor of
Dean, College of Medicine
/vVcx-
Dean,
August, 1988


38
numbers of synthetic nuclear targeting signals on protein uptake and the
functional size of the transport channel. It will also be determined
whether individual pores have the capability to transport proteins
containing different nuclear targeting sequences.
Many of the previous nucleocytoplasmic exchange experiments were
based on quantitative and qualitative studies performed with
fluorescein- or radio-labelled molecules; however, there are certain
characteristics of the exchange sites that cannot be studied using these
type of tracer molecules. In this study, colloidal gold particles will
be used to identify and characterize the sites for macromolecular
exchange.
Colloidal gold tracers have some unique properties that make them
ideal for this type of experimentation. First, gold particles with
different diameters can be easily prepared. Second, the surface
properties of the particles can be modified by the adsorption of
different molecules and the particles will then acquire the
characteristics of their coating agents. Third, tracer particles can be
localized precisely to specific cell structures by electron microscopy.
The following study will focus primarily on nucleocytoplasmic
transport of macromolecules, especially with regard to the
characteristics of the exchange sites located within the nuclear pores.
The experiments are performed on amphibian oocytes and involve the
microinjection of either RNA- or protein-coated gold particles into the
nuclear or cytoplasmic compartment of the cells. The subsequent
intracellular distribution of the tracer particles and their relation to
the nuclear pores will be determined by electron microscopy.


120
Dingwall, C., and R.A. Laskey. 1986. Protein import into the cell
nucleus. Ann. Rev. Cell Biol. 2:367-90.
Dingwall, C., S.V. Sharnick, and R.A. Laskey. 1982. A polypeptide
domain that specifies migration of nucleoplasmin into the nucleus.
Cell 30:449-458.
Dreyer, C., R. Stick, and P. Hausen. 1986. Uptake of oocyte nuclear
proteins by nuclei of Xenopus embryos. XU Nucleocytoplasmic
Transport. R. Peters and M. Trendelenburg, editors. Springer-
Verlag, Berlin. 143-157.
Dumont, J.N. 1972. Oogenesis in Xenopus laevis (Daudin). I. Stages
of oocyte development in laboratory maintained animals. J.
Morphol. 136:153-179.
Dwyer, N., and G. Blobel. 1976. A modified procedure for the
isolation of a pore complex-lamina fraction from rat liver nuclei.
J. Cell Biol, 70:581-591.
Earnshaw, W.C., B.M. Honda, R.A. Laskey, and O.J. Thomas. 1980.
Assembly of nucleosomes: the reaction involving laevis
nucleoplasmin. Cel 1 21:373-383.
Feldherr, C.M. 1962. The nuclear annuli as pathways for nucleo
cytoplasmic exchanges. J. Cell Biol. 14:65-72.
Feldherr, C.M. 1965. The effect of the electron-opaque pore material
on exchanges through the nuclear annuli. J. Cell Biol. 25:43-53.
Feldherr, C.M. 1966. Nucleocytoplasmic exchanges during cell
division. J. Cell Biol. 31:199-203.
Feldherr, C.M. 1971. Evidence for changes in nuclear permeability
during different physiological states. Tissue and Cell 3:1-8.
Feldherr, C.M. 1975. The uptake of endogenous proteins by oocyte
nuclei. Exp. Cell Res. 93:411-419.
Feldherr, C.M. 1980. Ribosomal RNA synthesis and transport following
disruption of the nuclear envelope. Cell Tissue Res. 205:157-162.
Feldherr, C.M., R.J. Cohen, and J.A. Ogburn. 1983. Evidence for
mediated protein uptake by amphibian oocyte nuclei. J. Cell Biol.
96:1486-1490.
Feldherr, C.M., E. Kallenbach, and N. Schultz. 1984. Movement of a
karyophilic protein through the nuclear pores of oocytes. J. Cell
Biol. 99:2216-2222.


81
All preparations containing active transport signals (BSA-WT
conjugates, large T-antigen or nucleoplasmin) were translocated into the
nucleus through central channels located within the nuclear pores. In
the region of injection, the particles were uniformly distributed in the
cytoplasm; however, at longer time intervals, 6 and 20 h, the BSA-WT
conjugates occasionally formed aggregates. The reason for this is not
known, but it appeared to be dependent on the presence of active signals
since similar aggregates were not observed with BSA-cT conjugates. At
all time intervals, particles containing active transport signals were
present both within the pores and the nucleoplasm. With increasing time
there was a concomitant increase in the number of particles present in
the nucleus (data not shown). Gold coated with the BSA-cT conjugates or
BSA alone were essentially excluded from the pores and nucleoplasm.
These general distributions are illustrated in Figs. 8 and 9, which show
the results obtained 1 h after injecting particles coated with BSA-WT^
and BSA-CT7, respectively.
Relative uptake
Quantitative data, dependent on the specific properties of the
individual coating agents, were obtained 1 h after injection. This time
interval allowed for sufficient particle distribution within the
cytoplasm, and minimized possible loss or redistribution of soluble cell
components caused by the injection procedures (Miller et al, 1984). The
N/C ratios obtained at 1 h do not represent equilibrium values since
nuclear uptake was observed to increase with time, but reflect relative


9
35S0=4> leucine-^C, and alanine-^C were shown by Naora et al. (1962)
to pass readily across the nuclear envelope.
To measure precisely the intracellular distribution of Na+, Horowitz
and Fenichel (1970) utilized ultra-low temperature autoradiographic
techniques. They described at least three intracellular Na+ fractions:
a cytoplasmic fast fraction, a cytoplasmic slow fraction, and a nuclear
fast fraction. The fast fractions were thought to represent freely
diffusible Na+, whereas the slow fraction is interpreted to be Na+ bound
to nondiffusible elements within the cytoplasm. This slow diffusion
fraction could account for the higher concentration of the Na+ ion found
within the cytoplasm. Therefore, the asymmetric ion concentrations can
be accounted for by two factors: first, the differences in water
content between the nucleus and cytoplasm, and second, possible binding
in the cytoplasm.
Another method for studying the permeability of the nuclear
membrane to ions is by measuring the envelope's electrical resistance.
Inserting intracellular microelectrodes into the nucleus and cytoplasm
of amphibian oocytes, Kanno and Loewenstein (1963) found no appreciable
resistance, indicating that ions are able to diffuse freely across the
envelope. Comparing the results from oocytes, the flux measurements and
electrical resistance data are in good agreement.
The results presented above suggest that the nuclear envelope is
not a barrier against ion diffusion. Since the nuclear envelope does
not impede ion passage and ion transport by the nuclear envelope is not
indicated (Century et al., 1970), it is likely that ions pass through
the aqueous channel formed by the nuclear pores.


48
actively involved in efflux. To determine the functional diameter of
the transport channels, the size distribution of the particles in
regions 1 and 2 were obtained and compared with the size of the injected
particles (i.e., particles within the nucleus). To standardize the
results, pores were analyzed in regions where the gold concentration in
the adjacent nucleoplasm was about 100 particles/0.36pm^.
Negative staining procedures were used to estimate the overall
diameters of the particles, that is, the gold plus the adsorbed coat
material.
Results
Purity of stabilizing agents
Approximately 600 pg of nucleoplasmin was isolated from 30 ml of
ovary using affinity chromatography. SDS-polyacrylamide gels of the
isolated protein showed major bands with apparent molecular weights of
165 kd and 145 kd, and a minor band with a molecular weight of
approximately 33 kd. The gel pattern (not shown) was identical to that
obtained for nucleoplasmin isolated using DEAE and phenyl sepharose
columns (Feldherr et al., 1984).
On a 10% polyacrylamide gel containing urea, tRNA ran as a single
distinct band corresponding to approximately 70 nucleotides, whereas the
pattern obtained for 5S RNA contained one major band corresponding to
approximately 130 nucleotides and minor bands of smaller sizes, probably
representing breakdown products. Gel scans of the tRNA and 5S RNA
showed the major band in each sample to be >80% of the total RNA.


mol wt
Molecular weight
N
Normal concentration
N/C
Nuclear to Cytoplasmic
ng
Nanogram
ni
Nanoliter(s)
NTPase
Nucleotide triphosphatase
OSO4
Osmium tetroxide
P
Probability
PAGE
Polyacrylamide gel electrophoresis
Poly(A)
Polyadenylic acid
Poly d(A)
Poly-deoxy-adenylic acid
Poly(I)
Polyinosinic acid
Poly(U)
Polyuridylic acid
Pro
Proline
PVP
Polyvinylpyrrolidone
RNA
Ribonucleic acid
hnRNA
Heteronuclear ribonucleic acid
mRNA
Messenger ribonucleic acid
rRNA
Ribosomal ribonucleic acid
tRNA
Transfer ribonucleic acid
RNP
Ribonucleoprotein
s
Second(s)
SDS
Sodium dodecyl sulfate
SV 40
Simian virus 40
T-antigen
Tumor antigen
U
Uridine
X


118
Berezney, R. 1980. Fractionation of the nuclear matrix. I. Partial
separation into matrix protein fibrils and a residual
ribonucleoprotein fraction. J. Cell Biol. 85:641-650.
Bernd, A., H.C. Schroder, R.K. Zahn, and W.E.G. Muller. 1982.
Modulation of the nuclear-envelope nucleoside triphosphatase by
poly(A)-rich mRNA and by microtubule protein. Eur. J. Biochem.
129:43-49.
Berrios, M., G. Blobel, and P.A. Fischer. 1983. Characterization of
an ATPase/dATPase activity associated with the Drosophila nuclear
matrix-pore complex-lamina fraction. Identification of the
putative enzyme polypeptide by direct ultraviolet photoaffinity
labeling. J. Biol. Chem. 258:4548-4555.
Berrios, M., and P.A. Fisher. 1986. A myosin heavy chain-like
polypeptide is associated with the nuclear envelope in higher
eukaryotic cells. J. Cell Biol, 103:711-724.
Bonner, W.M. 1975a. Protein migration into nuclei. I. Frog oocyte
nuclei in vivo accumulate microinjected histones, allow entry to
small proteins, and exclude large proteins. J. Cell Biol. 64:421-
430.
Bonner, W.M. 1975b. Protein migration into nuclei. II. Frog oocyte
nuclei accumulate a class of microinjected oocyte nuclear proteins
and exclude a class of microinjected oocyte cytoplasmic proteins.
J. Cell Biol. 64:431-437.
Burglin, T.R., and E.M. De Robertis. 1987. The nuclear migration
signal of Xenopus laevis nucleoplasmin. EMBO J. 6:2617-2625.
Butel, J.S., M.J. Guentzel, and F. Rapp. 1969. Variants of
defective simian papovirus 40 (PARA) characterized by cytoplasmic
localization of simian papovirus 40 tumor antigen. J. Virol.
4:632-641.
Callan, H.G., and S.G. Tomlin. 1950. Experimental studies on
amphibian oocyte nuclei. I. Investigation of the structure of the
nuclear membrane by means of the electron microscope. Proc. Roy.
Soc. London B137:367-378.
Century, T.J., I.R. Fenichel, and S.B. Horowitz. 1970. The
concentrations of water, sodium and potassium in the nucleus and
cytoplasm of amphibian oocytes. J. Cell Sci. 7:5-13.
Chambers, R., and B. Fell. 1931. Micro-operations on cells in
tissue cultures. Proc. Roy. Soc. London. B109:380-403.


105
surface of the nuclear pore most likely involves the specific
recognition of the signal by a pore receptor. This interaction provides
the cell with an additional level of control since binding allows for
the selection of transportable molecules. The removal or alteration of
these targeting signals by either proteolytic cleavage or DNA
methodology results in the protein retaining its cytoplasmic location.
The results in Chapters II and III indicate that translocation of
gold particles coated with RNA, nucleoplasmin, or the BSA-WT-conjugates
occurs through the centers of the pores and not along the periphery
which indicates the involvement of common pathways. The translocation
of the tracers was found to be a selective process. In all cases,
particles coated with synthetic polymers (PVP or polyglutamic acid) or
exogenous macromolecules (BSA or ovalbumin) were virtually excluded from
the pores, demonstrating that the RNA- and BSA-WT-coated gold
translocation is due to the presence of the coating agent. In addition,
the size of the particles, up to 260 A in diameter, that penetrate the
pore region far exceeds the upper limit of diffusion, suggesting that an
active transport process is involved.
It was demonstrated that most, if not all, of the nuclear pores are
capable of transporting different classes of RNA. Similar results were
obtained after cytoplasmic injection of gold particles coated with
proteins containing active targeting signals. Visualization of the
nuclear envelope in the region of injection indicated that a large
percentage of the pores contained protein-coated gold particles. In
addition, the double injection experiments showed that individual pores
are capable of translocating both RNA-coated and protein-coated gold


79
gold. Centrifugation of the 20-50 A and 120-280 A fractions was not
necessary. In each instance, 5-7 ml of stabilized colloid were
concentrated to 70-100 ul in Minicon concentrators (Amicon Corp.,
Danvers, MA) and dialyzed against intracellular injection medium (102 mM
KC1, 11.1 mM NaCl, 7.2 mM K2HP04 and 4.8 mM KH2P04, pH 7.0) at 4C prior
to injection.
Injection
Late stage 5 and stage 6 oocytes (Dumont, 1972) were manually
defolliculated in amphibian Ringer's solution and centrifuged at
approximately 650g for 8-10 minutes (Kressman and Birnstiel, 1980). The
cells were then microinjected with approximately 40 nl of gold sol at a
site adjacent to the nucleus and fixed at intervals of 15 min, 1 h, 6 or
20 h. The tip diameters of the micropipettes were 10-15 pm.
Electron microscopy and analysis
The cells were fixed for electron microscopy and prepared for
sectioning as described previously (see Chapter 2, Materials and
Methods). Relative nuclear uptake of gold particles stabilized with the
different coating agents was determined by counting particles in equal
and adjacent areas of the nucleus and cytoplasm close to the site of
injection. Yolk granules and mitochondria were excluded from the
analyses. The counts are reported as nuclear to cytoplasmic ratios
(N/C). The size distributions of particles present within the nucleus
and cytoplasm were determined by direct measurement from electron
micrograph negatives. The envelope to cytoplasm ratios were obtained by


23
extracted from the nucleus, can accumulate back into the nucleus.
Mitochondrial proteins have signal sequences that target them into
specific regions of the mitochondria and cross the membrane(s) by a
posttranslational mechanism. However, no evidence exists that nuclear
transport occurs by a posttranslational process directly across the
membrane, although it still remains a possibility.
An elegant approach to study the exchange sites of a known
transportable nuclear protein was performed by coating colloidal gold
particles with nucleoplasmin (Feldherr et al., 1984). Their results
demonstrate that nucleoplasmin-coated gold enter the nucleus through
channels located in the centers of the pores. Particles, ranging in
size from 50-200 A, were able to penetrate the pore region, thus
indicating that the functional size of the transport channel is
different than the size of the diffusion channel. The transported
particles are nondeformable and spherical, which argues against
structural deformation of nucleoplasmin prior to transport.
Furthermore, the particles are markedly larger than the diffusion
channel, which suggests the involvement of a selective uptake process.
In vitro uptake
The use of an i_n vitro assay system to investigate protein
transport has both advantages and disadvantages. The drawbacks are,
first, changes in the permeability characteristics of isolated nuclei.
Peters (1983) measured the permeability of single isolated liver nuclei
to dextrans using fluorescent microphotolysis and estimated the pore
diameter to be 112-118 A, which is 2-8 A larger than pores from i_n vivo


99
N


53
The size distributions of tRNA- and 5S RNA-coated particles present
within the nucleoplasm and also associated with the nuclear pores
(regions 1 and 2) are given in Table III. Since the thickness of the
RNA coat, in both cases, was estimated to be 15-20 A, the results
indicate that particles with an overall diameter of at least 170 A can
pass through the centers of the pores. Since larger particles could not
be stabilized with tRNA or 5S RNA, it was not possible to determine an
upper size limit for translocation using these coating agents. In
contrast, 120-220 A particles could be stabilized with poly(A). The
results obtained wtih poly(A)-gold are illustrated in Fig. 4. Despite
the fact that relatively few particles were injected into the nuclei,
gold was found in over 70% of the pores after 15 min and 1 h. The size
distribution of poly(A)-coated particles associated with the pores is
shown in Table III. It is apparent from the data, which is based on the
examination of 12 cells, that particles at least 230 A in diameter
(including the coat) are translocated through the pores.
Nuclear injection of poly(I)- and poly(dA)-coated gold
In addition to the studies performed with tRNA, 5S RNA and poly(A),
nuclei were also injected with poly(I)- or poly(dA)-coated particles.
The volumes injected, and the procedure used for analysis of these
nonphysiological tracers, were the same as described above. In these
experiments, the oocytes were fixed 1 h after injection, and 5 cells
were examined in each group. The results are shown in Table II.
Particles coated with either poly(I) or poly(dA) were translocated
through the centers of the pores; however, differences were observed in


89
When the number of signals per BSA molecule is increased beyond 8
there is no further increase in functional pore size. This is indicated
in Fig. 11, which compares the size distribution of BSA-WT8", BSA-WT^-
and nucleoplasmin-coated particles within the nucleus 1 h after
injecting a 120-280 A gold fraction. The sizes of the particles able to
pass through the pores did not vary significantly for the different
coating agents. A comparison of the cytoplasmic distributions to the
nuclear distributions, as shown in Table X, demonstrates that particles
larger than 230 A (average of the size class), do not readily penetrate
the pores, regardless of the coating agent. These results demonstrate
that the maximum size particle able to enter the nucleus is
approximately 260 A in diameter. This value includes the thickness of
the coat material, which adds about 30 A to the overall particle
diameter.
In contrast, the large T-antigen data given in Table XI indicate
that particles larger than 90 A were not detected in the nucleus after
1 h. The size data obtained for the BSA-WT8-CT7 dilutional experiment
(Table XI) gave similar results, although a few particles larger than 90
A were present in the nucleus.
Overall, analysis of the size distributions of particles in the
nucleus and cytoplasm indicate a direct relationship between the
functional dimensions of nuclear pores and the number of active SV 40
large T targeting signals per BSA molecule.


8
methodology has been used to probe structural features of proteins that
are specifically targeted to the nucleus.
Ion permeability
Ions are known to interact with chromatin and activate specific
genes. During mitosis, meiosis, DNA synthesis, and hormonal
stimulation, ionic changes are occurring within the cell; thus, it is
important to understand intracellular ion distributions and how the
envelope might regulate their exchange between the nucleus and
cytoplasm.
Earlier permeability studies were qualitative in nature and based
on the structural or colloidal changes that occurred within isolated
nuclei following incubation in different salt solutions. It was
concluded from these studies that both cations and anions can rapidly
penetrate the nuclear envelope.
The use of radioactive isotopes as tracer molecules provided a new
method to give precise data on the permeability of the envelope.
Abelson and Duryee (1949) incubated individual oocytes in ^4Na+ and
later studied the intracellular ion distribution by quick freezing and
radioautography. The results demonstrated that the nuclear envelope in
amphibian oocytes was readily permeable to ^Na+. in addition to these
findings, the nucleus, at equilibrium, contained twice as much
radioactive tracer per unit volume as the cytoplasm. The measurement of
water content in both compartments indicated the nucleus to have a
higher percentage of water than the cytoplasm, which could account for
the difference in Na+ concentrations. Other molecules, ^K+, ^pg^


104
maximum functional size of the transport channel was estimated to be 260
A in diameter. Furthermore, it was found that individual pores can
recognize and transport proteins containing different targeting signals.
The ramifications of these results are discussed in relation to how the
pores might regulate nucleocytoplasmic exchange and thus, be intimately
involved in the modulation of cellular activity.
The selectivity of the nucleocytoplasmic exchange process can occur
at the level of the nuclear envelope, within the nucleus or cytoplasm.
Since the nuclear pores represent the major sites of exchange for
macromolecules, their role in regulating the exchange process is of
special interest.
Nuclear Envelope Selectivity
As discussed in Chapter I, the patent size of the nuclear pore can
determine the size of the molecule which can diffuse into and out of the
nucleus, with the rate of uptake or efflux being inversely related to
size. This mode of selection by the envelope can be considered one
level of specificity since any molecule that is larger than the
diffusion channel (90-120 A in diameter) tends to be excluded from the
nucleus, unless a specific transport process is involved.
Several lines of evidence have clearly established that a selection
process exists for the transport of proteins into the nucleus. It has
been demonstrated for a number of nuclear proteins (Chapter I,
endogeneous macromolecules), that information which specifies selective
nuclear entry resides in a region of their primary sequence.
Presumably, the interaction of the targeting signal with the cytoplasmic


Table IV Translocation of Gold Particles Coated with Polyglutamic Acid
Experiment (1 h)
No. of Particles
Translocated
% of Pores Active
Parti cles/Pore in Translocation
Region 1
Region 2
Total
Polyglutamic
Acid-gold
21
2
23
tRNA-gold
175
164
339
8%
83%
*Data based on the analysis of 200 pores. In both instances the concentration of gold
particles (20-50 A) in the adjacent nucleoplasm was approximately 60 particles/0.36 pm2.


29
with a core of five common proteins. Functional roles that have been
identified for a few of the U-snRNAs include polyadenylation,
intranuclear transport, and splicing of hnRNA.
To investigate intracellular RNA transport within intact cells,
De Robertis et al. (1982) injected radiolabelled RNA from HeLa cells
into the cytoplasm of Xenopus oocytes. The results demonstrate that
snRNAs can migrate from the cytoplasm into the nucleus and concentrate
there 30- to 60-fold. It was also established that HeLa snRNAs complex
with oocyte proteins in the cytoplasm before their migration into the
nucleus. Zeller et al. (1983) analyzed the distribution of snRNAs and
their associated binding proteins during oogenesis and early
development. Mature Xenopus oocytes and embryos prior to gastrulation
are known to contain an excess of snRNA binding proteins (De Robertis et
al., 1982) and less U1 and U2 snRNAs than previtellogenic oocytes. In
embryos, the midblastula transition (Newport and Kirschner, 1982) marks
the onset of a burst in snRNA synthesis and the migration of snRNA
binding proteins into the nucleus. Injection of labelled snRNA into the
cytoplasm of mature oocytes can prematurely displace the cytoplasmic
binding proteins into the nucleus, suggesting that movement of the
proteins requires the formation of the snRNPs. To determine whether
nuclear accumulation occurs only when snRNA and its binding proteins are
complexed together, Mattaj and De Robertis (1985) used site-directed
mutagenesis to delete the protein binding site on 112 snRNA. As a result
of the snRNA's inability to bind its cytoplasmic proteins, it becomes
unable to enter the nucleus. It is suggested that the formation of the
snRNP within the cytoplasm might unmask a cryptic targeting signal which


11
The pores, from a morphological standpoint, would appear to be a
likely pathway for movement into and out of the nucleus. In support of
this view, Feldherr (1962) injected colloidal gold particles coated with
polyvinylpyrrolidone (PVP) into the cytoplasm of the amoebae Chaos chaos
and localized the distribution of the tracers by electron microscopy.
After 1-2 minutes, gold particles were observed in the centers of the
pores and 24 hours after injection, gold particles were found to
accumulate in the nucleus. Injection of different size gold fractions,
20-50 A and 20-160 A in diameter, showed that small gold particles
localized within the nucleus after 3 min, in contrast to the larger gold
fraction which had negligible uptake after 10 min. These results
indicate that the rate of movement across the nuclear envelope is
inversely proportional to the molecular size of the tracer. After
longer time intervals, it was determined for Amoeba proteus that PVP-
coated gold particles up to 120 A can penetrate the pores of the nuclear
envelope, whereas in Chaos chaos, particles as large as 140 A can enter
the nucleus (Feldherr, 1965). It is important to note that the maximum
size of the particles able to penetrate the envelope is smaller than the
800 A internal diameter of the pores. Overall, it was concluded from
these studies that pores can serve as pathways for nucleocytoplasmic
exchange but can restrict the movement of large molecules since the
entire area of the pore is not available for free exchange.
Gurdon (1970) studied permeability properties of the nuclear
envelope by injecting ^5¡_iabelled histone (10,000-20,000 kd) and
bovine serum albumin (67,500 kd), into amphibian oocytes. After 50
minutes, it was demonstrated by autoradiographic analysis that the


108
Chironomus salivary gland cells. These results provide direct evidence
for the requirement of a 260 A transport channel.
Two possible mechanisms were explored to determine how the cell and
the pore complex might regulate the exchange of proteins across the
nuclear envelope. Since the number of synthetic peptides per carrier
protein could be altered and the SV 40 large T-antigen signal was not
homologous to the nucleoplasmin transport signal, the effect of
variations in signal number and sequence on protein transport through
the pores was studied.
At the surface of the pore, the interaction between the signal and
its receptor sets up another point of regulatory control by the nuclear
envelope. As shown in chapter III, the functional size of the transport
channel and relative uptake is dependent on both the nature of the
signal and number of targeting signals per molecule. The results
indicate a direct relationship between signal number and the size of the
tracer particles that can penetrate the nuclear pores. The functional
size of the channel would be dependent on the total number of available
signals accessible to the pore at a given time.
In addition to signal number, the affinity of the signal for its
receptor could affect the uptake of coated particles. A comparison of
uptake and accumulation of tracers at the nuclear surface suggests that
the nucleoplasmin signal has a higher affinity for the pore receptors
than the large T-antigen signal. This could explain why nucleoplasmin-
coated gold, which has a fewer number of signals than BSA-WTg and BSA-
WT^, is transported more efficiently than the active BSA-conjugates.
Lanford et al., (1988) studied the effects of amino acid substitutions


107
nucleus, thus being denied access to the pores and preventing transport.
Therefore, the cell can have molecules with active transport signals;
however, they may be inaccessible for transport due to selective binding
away from the pores or specific masking of the signal.
Dynamic Aspects of the Transport Channels
In addition to localizing the pathway for exchange, microinjection
of the tracers provided a method for analyzing the morphological and
functional characteristics of the transport channel. After injection of
a gold fraction ranging from 50-280 A in diameter, the largest particles
capable of penetrating the pores were 230 A. When including the size of
the coat material (approximately 30 A), the maximum functional size of
the transport channel was estimated to be 260 A in diameter. Since it
is unlikely that a single endogenous protein would have as high a number
of transport signals as the BSA-WTg and BSA-WT^ conjugates, 8 and 11
signals respectively, it is of interest to consider the possible
biological significance for a channel of this size. First, it is likely
that several endogenous molecules are continually being transported into
and out of the nucleus and simultaneous exchange through the pores might
require a full dilation of the transport channel. A second requirement
for the 260 A translocation channel is the transport of large ribosomal
components and mRNPs that are synthesized in the nucleus and pass
through the pores into the cytoplasm. Stevens and Swift (1966) and
Skoglund et al. (1983) have shown that a large mRNP particle,
approximately 260 A in diameter, passes through the nuclear pores of


122
Gerace, L.C., A. Blum, and G. Blobel. 1978. Immunocytochemical
localization of the major polypeptides of the nuclear pores
complex-lamina fraction. Interphase and mitotic distribution.
J. Cell Biol. 79:546-566.
Gerace, L., C. Comeau, and M. Benson. 1984. Organization and
modulation of nuclear lamina structure. J. Cell Sci. Suppl. 1:137-
160.
Gerace, L., Y. Ottaviano, and C. Kondor-Koch. 1982. Identification of
a major polypeptide of the nuclear pore. J. Cell Biol. 95:826-
837.
Giese, G., and F. Wunderlich. 1983. In vitro ribosomal
ribonucleoprotein transport. Temperature-induced 'graded
unlocking' of nuclei. J. Biol. Chem. 131-135.
Goldfarb, D.S., J. Gariepy, G. Schoolnik, and R.D. Kornberg. 1986.
Synthetic peptides as nuclear localization signals. Nature
322:641-644.
Goldstein, L., and W. Plaut. 1955. Direct evidence for nuclear
synthesis of cytoplasmic ribose nucleic acid. Proc. Natl. Acad.
Sci. 41:874-880.
Gurdon, J.B. 1970. Nuclear transplantation and the control of gene
activity in animal development. Proc. Roy. Soc. Lond. B176:303-
314.
Hall, M.N., L. Hereford, and I. Herskowitz. 1984. Targeting of E.
coli [3-gal actosi dase to the nucleus in yeast. Cel 1 36:1057-1065.
Harris, J.R. 1978. The biochemistry and ultrastructure of the nuclear
envelope. Biochem. Biophy. Acta. 515:55-104.
Horowitz, S.B. 1972. The permeability of the amphibian oocyte
nucleus,_i_n situ. J. Cell Biol. 54:609-625.
Horowitz, S.B., and I.R. Fenichel. 1970. Analysis of sodium transport
in the amphibian oocyte by extractive and radioautographic
techniques. J. Cell Biol. 47:120-131.
Horowitz, S.B., and L.C. Moore. 1974. The nuclear permeability,
intracellular distribution, and diffusion of inulin in the
amphibian oocyte. J. Cell Biol. 60:405-415.
Jeffery, W.R. 1982. Messenger RNA in the cytoskeletal framework:
Analysis by i_n situ hybridization. J. Cell Biol. 95:1-7.


Figure 12. Accumulation of tracers along the nuclear envelope 1 h
experiment.
A comparison of the intracellular distributions of 50-280 A gold
particles coated with BSA-WT5 (12a), BSA-WTg (12b) and nucleoplasmin
(12c). Similar amounts of colloid were injected in each oocyte.
Progressing from a to c, there is an increase in the number of
particles present both within the nucleus (N) and along the nuclear
envelope. C, cytoplasm. Bar, 0.2 pm.


CHAPTER I
A REVIEW OF NUCLEAR ENVELOPE PERMEABILITY:
DIFFUSION AND TRANSPORT
Introduction
The formation of the nuclear envelope around the cell's genetic
material is one of the major evolutionary differences between
prokaryotes and eukaryotes. The nucleus, site of both RNA and DNA
synthesis, contains the genetic information needed for phenotypic
expression. The cytoplasm, on the other hand, is the site of protein
synthesis, which requires certain RNAs that are exported from the
nucleus. Furthermore, the nucleus is dependent on proteins synthesized
in the cytoplasm. Therefore, cell growth, division, differentiation,
and maintenance require a continual exchange of information between both
the cytoplasm and nucleus. To better understand cellular function, it
is important to determine the factors that control the rate of movement
between the two compartments and the types of molecules that are
exchanged. For this reason, knowledge of the nuclear envelope's
structure and permeability properties is essential in understanding the
interrelationships that exist between the nucleus and cytoplasm of the
eukaryotic cel 1.
In the first part of this chapter an ultrastructural description of
the nuclear envelope is provided, followed by a review of permeability
experiments which have helped to elucidate the role of the envelope in
regulating nucleocytoplasmic exchanges.
1


24
nuclei (see exogenous molecules section). According to the model of
Paine et al. (1975), a small change in pore diameter can have a marked
effect on flux rates. Second, after isolation of oocyte nuclei under
aqueous conditions, 95% of the proteins is lost with a half-time of
250 s (Paine et al., 1983). Third, assuming some nuclear proteins are
transported along cytoskeletal elements to the nuclear surface, the
isolation procedure removes the nucleus from its cytoplasmic anchorage,
thus removing a possible step in transport. In light of these
disadvantages, the results from the i_n vitro experiments must be
carefully interpreted.
An i_n vitro system is advantageous to study protein uptake since
the steps in the process can be easily dissected and experimentally
manipulated. In vitro assays can be used: i) to characterize
components needed for translocation, ii) to determine inhibitors of
protein transport, and iii) to determine if protein transport is ATP-
dependent.
Forbes et al. (1983) have shown that bacteriophage DNA
microinjected into Xenopus eggs can form nucleus-like structures.
Addition of DNA from either bacteriophage lambda or Xenopus to an
extract from Xenopus eggs results in the formation of synthetic nuclei
that have morphological similarities to intact nuclei (Newmeyer et al.,
1986). These synthetic nuclei have a double membrane envelope, however,
not all possess nuclear pore complexes. The reconstituted nuclei, in
conjunction with labelled nucleoplasmin, have been used to investigate
aspects of the protein transport process. Newmeyer et al. (1986)
demonstrated that nucleoplasmin uptake is ATP- and temperature-


77
the active signal assured that the overall properties (shape and size)
of the particles were consistent with those of other tracers used in
this investigation. Purified SV 40 large T-antigen, which has 1 signal
per monomer, and nucleoplasmin, which has a different signal sequence,
also were used as coating agents. BSA alone was employed as an
additional control.
Preparation and stabilization of colloidal gold
The colloidal gold fractions that contained particles 20-50 A and
20-160 A in diameter were both prepared by reducing chloroauric acid
with a solution of white phosphorus in ether (Feldherr, 1965). 50-280 A
gold particles were obtained by adding 2.5 ml of 0.6% gold chloride to a
freshly prepared 20-160 A fraction, 1 ml of additional reducing agent
was then added, and the preparation was boiled for 2-3 minutes.
Fractions containing 120-280 A particles were prepared by reducing
chloroauric acid with trisodium citrate, as described previously (Frens,
1973). The 50-280 A and 120-280 A gold sols, were brought to pH 7.0
with 0.72 N K2C03.
Prior to stabilization, all of the coating agents were either
dialyzed against, or dissolved in, a low ionic strength buffer (7.2 mM
K2HPO4 and 4.8 mM KH2PO4, pH 7.0). The volumes of the different agents
required to stabilize 1 ml of the gold sols are listed in Table VII.
The procedure used to determine these volumes is outlined in Feldherr et
al. (1984).
After stabilization, the 20-160 A and 50-280 A preparations were
centrifuged at 2000g at 4C for 10 minutes to remove any aggregates of


32
Overall, tRNA, 5S RNA, and rRNA are generally believed to
translocate into the cytoplasm through the nuclear pores. However, much
of the evidence is circumstantial and there are no definitive data to
indicate that the pores serve as the major pathways for the efflux of
these different classes of RNA.
Messenger RNA
Heterogeneous nuclear RNA is processed and undergoes extensive
posttranscriptional modifications before mature mRNA can be transported
into the cytoplasm. Unspliced mRNA is retained in the nucleus and only
mature mRNA appears in the efflux supernatant from isolated nuclei iji
vitro or in the cytoplasm of intact cells, suggesting that processing is
one of the many requirements necessary for export. The movement of
mature mRNA, from its site(s) of transcription and processing, to the
nuclear envelope is the first step involved in export (see above).
Agutter (1985a) has suggested that mRNA might not be freely diffusible
throughout the cells. To determine whether RNA is preferentially
associated with the nuclear matrix, nuclei were treated with nonionic
detergents, high salt, EDTA, and urea. It was shown that greater than
90% of the nuclear RNA was tightly associated with the insoluble nuclear
matrix (Berezney, 1980; van Eekelen and van Venrooij, 1981). Release of
the mature ovalbumin mRNA but not the immature mRNA from the nuclear
matrix occurs in the presence of ATP and does not require hydrolysis of
the phosphodiester bond (Schroder et al., 1987). This selective
detachment from the matrix might be another level of control in
regulating the transport of mRNA. Overall, the migration of mature mRNA


49
Nuclear injection of tRNA-, 5S RNA- and poly(A)-coated gold
It was determined initially that varying the amount of the
injectate from 8-20 nl (the RNA content ranged from 30-100 ng) had no
effect on the results. Routinely, 8 nl of colloid were injected. This
volume contained a sufficient number of particles for electron
microscopic analysis and minimized possible damage to the nucleus. The
oocytes were fixed 15 min, 1 h, or 6 h after injection. Eighteen to 20
cells were examined for each type of RNA at each time interval.
The results were essentially the same for 20-160 A particles
coated with tRNA (met or phe), 5S RNA, or poly(A). At all time
intervals, particles were distributed randomly throughout the
nucleoplasm, except for aggregates of gold that were occasionally
observed at the surface of the nucleoli. After 15 min and 1 h,
particles were associated with almost all of the nuclear pores.
Representative results for tRNA and 5S RNA are shown in Figs. 2 and 3,
respectively. Based on the assumption that the presence of gold
particles within the pores or in the adjacent cytoplasm (regions 1 and
2 in Fig. 1) is indicative of nucleocytoplasmic exchange, it was
concluded that over 97% of the pores were involved in translocation 15
min and 1 h after injection (see Table II). There was an obvious
decrease in the percentage of pores that contained particles in the 6 h
experiments, but these results were not quantitated. In all instances,
particles were observed in the cytoplasm beyond the immediate vicinity
of the pores (i.e., beyond region 2). However, even after 6 h the
cytoplasmic to nuclear concentration ratio was only 1:14.


114
known if the nuclear envelope transport properties are altered during
changes in cellular activities. These results can also be explained by
de novo synthesis of specific carriers which subsequently result in a
shift in a molecule's partitioning between cytoplasm and nucleus
although no carriers for nuclear transport have been identified.
What is the underlying molecular mechanism required for the
movement of macromolecules through the length of the transport channel?
Berrios et al. (1983) have identified an ATPase polypeptide associated
with nuclear-envelope enriched fractions. The molecular mechanisms for
the actual translocation has been postulated to be a contractile force
provided by this ATPase activity which is a myosin-like heavy chain
polypeptide localized in the nuclear periphery (Berrios and Fischer,
1986). However, it remains to be determined if the ATPase activity
localizes predominantly in the pore complexes. Schindler and Jiang
(1986) used the fluorescence redistribution after photobleaching
technique to study factors that influence the flux rates of dextrans
across the nuclear envelope of isolated nuclei. They found that the
addition of anti-actin antibodies markedly reduced the rate of nuclear
entry of the labelled dextran. Furthermore, anti-myosin antibodies
significantly blocked the ATP stimulatory effects on the dextran flux
rate. From these results, it was postulated that the contractile
proteins, actin and myosin, are part of the pore complex and involved in
the opening and closing of the pores. The problem is that these results
have not been verified i_n vivo, and it is known that nuclear isolation
can significantly alter the properties of the pores.


102
nucleoplasmin), demonstrated that individual pores are capable of
recognizing and transporting different nuclear targeting signals. In a
study to show the bidirectional capability of the nuclear pores, it was
demonstrated that individual pores can transport both protein and RNA
(Chapter II, Results). Whether all transport signals can be recognized
by each pore has yet to be determined.


TABLE IX ~ Size Distribution of BSA-WT^-and WTfl-coated Particles in Injected Cells*
Experiment
LUl)
Total No.
of Particles
Measured
Percentage of Particles in Each Size Class#
45-73 73-101 101-129 129-157 157-185 185-213 213-241 241-269 >269
A A A A A A A A A
BSA-WT5
Nucleus
406
31.5
19.7
24.6
18.5
4.9
0.7
-
-
-
Cytoplasm
533
3.2
11.1
24.2
29.8
14.8
9.0
3.8
1.1
2.8
bsa-wt8
Nucleus
1018
14.3
20.3
24.6
23.8
10.1
4.8
1.7
0.4
_
Cytoplasm
808
5.2
13.1
25.0
28.2
14.7
7.4
3.6
1.7
0.
*These experiments were performed with 45-280 A gold particles and the mean size of the fraction was 140 A.
^The size of the gold particles does not include the thickness of the coating agent.


LIST OF TABLES
Table Page
I Amounts of Coating Agent Required to Stabilize Gold Sols. 45
II Translocation of Gold Particles as a Function of the
Coating Agent 52
III Size Distribution of Gold Particles Present in the Nuclei
and Pores 54
IV Translocation of Gold Particles Coated with Polyglutamic
Acid 59
V Concentration Dependence of tRNA-gold Translocation .... 61
VI Protein Preparations Used as Coating Agents 76
VII Volumes of Coating Agent (pi) Required to Stabilize 1 ml
of Gold Sol 78
VIII N/C ratios 1 h 85
IX Size Distribution of BSA-WTg- and WTg-coated Particles
in Injected Cells 87
X Size Distribution of BSA-WTg-, WT^i- and Nucleoplasmin-
coated Particles in Injected Cells 91
XI Size Distribution of BSA-WTg-cTy- and Large T-Ag-coated
Particles in Injected Cells 92
XII Envelope-Associated Particles 96
vi i i


34
kinetic properties, substrate specificities, and sensitivities to
inhibitors (Agutter et al., 1976; Clawson et al., 1978, 1980). One
additional line of evidence that mRNA and NTPase are interrelated is the
inhibition of mRNA efflux by antibodies against pore complex-lamina
components that affect NTPase activity (Schroder et al.; unpublished
results).
The effects of poly(A) on NTPase activity were observed initially
by Agutter et al. (1977) on isolated, intact nuclear envelopes. They
showed that addition of poly(A) or poly(G) to the media could enhance
NTPase activity. Furthermore, Bernd et al. (1982) demonstrated the
NTPase activity to be markedly stimulated by either poly(A)+mRNA or a
homopolymer of poly(A) at least 15-20 bases long. Additional evidence
comes from experiments by Riedel and Fasold (1987). They prepared
resealed nuclear-envelope vesicles that retain NTPase activity and found
that both poly(A) and mRNA can stimulate the NTPase activity associated
with these vesicles. It is suggested from these data that poly(A) can
modulate NTPase activity and possibly regulate the translocation of
mRNA.
It has been demonstrated that protein kinases and phosphohydrolases
are present in the nuclear envelope (Lam and Kasper, 1979; Smith and
Wells, 1983; Steer et al., 1979a, 1979b). McDonald and Agutter (1980)
have studied the effects of polyribonucleotide binding on the
phosphorylation and dephosphorylation of nuclear envelope proteins and
found that the same polyribonucleotides that stimulate NTPase, inhibit
protein kinase activity and stimulate one of the phosphohydrolases.
They also found the nuclear envelope to contain a population of poly(A)


80
comparing the number of particles associated with the cytoplasmic
surface of the envelope (i.e., at or within 650 A of the nuclear
surface) to the number of particles in an equal, randomly selected area
of cytoplasm.
Negative staining with 1% phosphotungstic acid was used to estimate
the thickness of the adsorbed protein coats.
By extrapolating from data published by De Roe et al. (1984) and
correcting for additional mass contributed by the peptides, estimates
were made of the number of BSA molecules adsorbed onto the surfaces of
different size gold particles. For example, particles with diameters of
35 A, 80 A, 140 A, and 180 A (the mean size of the 4 different gold
preparations injected) would have 2, 8, 24, and 39 molecules of BSA
conjugates, respectively. Knowing the number of BSA molecules adsorbed
and the synthetic peptide to carrier protein ratios, it was possible to
estimate the total number of SV 40 large T-antigen targeting signals on
the different size gold particles; however, the proportion of signal
actually available for transport (i.e., exposed signals) is not known.
Results
Cytoplasmic injections of the tracer particles
It was determined initially that microinjection of approximately
40 nl of stabilized gold adjacent to the nucleus delivered a sufficient
number of particles for electron microscopic analysis. The protein
content of the injectate ranged from 50-300 ng depending on the size of
the gold fraction and the specific coating agent used.


125
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ATP is required for nucleoplasmin accumulation. EMBO J. 5:501-510.
Newport, J., and M. Kirschner. 1982. A major developmental transition
in early Xenopus embryos: I. Characterization and timing of
cellular changes at the midblastula stage. Cel 1 30:675-686.


ACKNOWLEDGEMENTS
The studies presented in this dissertation could not have been
performed without the guidance and assistance of Dr. Carl Feldherr. He
served as an excellent role model for conducting scientific research of
the highest caliber. I would like to express my gratitude to him for
allowing me to work in his laboratory throughout the past three years.
Special thanks are extended to Dr. Robert Cohen for serving as a
committee member and providing insight to the biophysical aspects
associated with my research. I would like to thank Drs. C. West and G.
Stein for serving as members of my committee and providing critical
review of the manuscripts submitted for publication.
I am grateful to Dr. Robert Lanford at the Southwest Foundation
for Biomedical Research in San Antonio, Texas, for providing all of the
BSA-conjugates that were used to study the effects of signal number on
protein uptake.
The work presented in Chapter II is reproduced from the Journal of
Cell Biology, 1988, volume 106, number 3, pages 575-584 by copyright
permission of The Rockefeller University Press.
I would like to thank Denifield Player for advice concerning
techniques in electron microscopy and Linda Mobley for typing of
manuscripts and other pertinent material throughout my graduate career.


70
pores or even the adjacent cytoplasm could be affected. Third, the size
of the tracers could influence the transport rates. Although RNA-coated
particles leave the nucleus at a reduced rate, it should be emphasized
that the properties of RNA required for translocation through the pores
are retained.
Zasloff (1983) and De Robertis et al. (1982) reported that
radiolabel1ed tRNA is unable to enter the nucleus following injection
into the cytoplasm; however, the exchange of 5S RNA across the envelope
does appear to be bidirectional (De Robertis et al., 1982). Gold
particles coated with tRNA, 5S RNA, or poly(A) were able to pass from
the cytoplasm into the nucleus, but exchange across the envelope was
greatly restricted. Even after 1 h the nuclear to cytoplasmic
concentration ratio was only about 0.01. Considering these low rates of
uptake, it was surprising to find that RNA-coated particles were
observed extending through the centers of the pores. These results
could be interpreted to mean that the translocation of RNA through the
pores is a reversible process, but that release into the nucleoplasm,
which could require a separate mechanism, might be a limiting factor.
However, before any definitive conclusion can be drawn it will be
necessary to obtain reliable quantitative data concerning the percentage
of pores that contain gold and additional information relating to the
nuclear uptake rates. Furthermore, it is not known whether
translocation is initiated by the adsorbed RNA itself or whether the
particles fortuitously bind karyophilic proteins which, in turn, induce
transport (Mattaj, 1986). Hopefully it will be possible to resolve some
of these questions using isolated nuclei.


57
the numbers and distribution of the particles associated with these
structures. Compared with poly(dA)-coated gold, almost twice as many
poly(I)-coated particles were present in the pore areas and, in
addition, a higher proportion of these particles were located in
region 2. These results suggest that some polynucleotides are
translocated more efficiently than others.
Nuclear injection of BSA-, ovalbumin-, polyglutamic acid-, and PVP-
coated gold
To determine if the translocation of RNA-gold is a selective
process, nuclei were injected with gold fractions that had been
stabilized with the exogenous molecules PVP, BSA, or ovalbumin. The
volumes injected, and the experimental times (15 min, 1 h and 6 h), were
the same as those used in the RNA studies. The data are based on the
examination of 6-9 cells per time interval for each coating agent that
was used. The result of a 1 h nuclear PVP-gold injection is
illustrated in Fig. 5. Similar distributions were observed when
particles stabilized with BSA or ovalbumin were injected. It was
found, at all time intervals, that essentially all of the particles
coated with exogenous substances were retained within the nuclei and
less than 6% of the pores contained gold particles (Table II). It can
also be seen in Table II that the total number of control particles
translocated per 200 pores was only about 1% of the total number of
translocated RNA-coated particles. These results demonstrate that the
translocation of RNA-gold was due to the specific properties of the
adsorbed coat material.


Table I Amounts of Coating Agent Required to Stabilize Gold Sols
mg of coating agent/ml of Size range of colloidal pi of solution needed to
Coating agent stabilizing solution particles stabilized (A) stabilize 1 ml of colloid
tRNAmet
0.2
20-160
200
20- 50
50
tRNAPhe
0.5
20-160
300
5S RNA
0.4
20-160
400
poly(A)
0.5
20-160
70
120-220
70
poly(dA)
0.25
20-160
350
poly(I)
0.5
20-160
400
Nucleoplasmin
0.1
120-220
60
PVP
0.1
20- 50
40
BSA
1.0
20- 50
60
Ovalbumin
1.0
20-160
250
Polyglutamic acid
10
20- 50
200
*These are average values, intended to serve as a guide. The exact amounts of coating agents
required for stabilization should be determined for each individual gold preparation.


Table V Concentration Dependence of tRNA-gold Translocation*
No. of Particles % of Pores Active
Particle No./0.36 pm^ Translocated Particles/Pore in Translocation
Region
1 Region 2
Total
30
34
84
118
1.2
61%
60
77
94
171
1.7
82%
90
159
197
356
3.6
98%
120
154
205
359
3.6
98%
190
188
174
362
3.6
98%
* Data based
on the analysis
of 100 pores
for each
gold particle
concentration
The size of
the gold particles ranged from 20-50
A, and the cells were fixed
1 h after injection.


119
Clawson, G.A., C.M. Feldherr, and E.A. Smuckler. 1985.
Nucleocytoplasmic RNA transport. Mol. Cell. Biochem. 67:87-99.
Clawson, G.A., D.S. Friend, and E.A. Smuckler. 1984. Localization of
nucleoside triphosphatase activity to the inner nuclear envelope and
associated heterochromatin. Exp. Cell Res. 155:310-314.
Clawson, G.A., J. James, C.H. Woo, D.S. Friend, D. Moody, and
E.A. Smuckler. 1980. Pertinence of nuclear envelope nucleoside
triphosphatase activity to ribonucleic acid transport. Biochem.
19:2748-2756.
Clawson, G.A., M. Koplitz, B. Castler-Schechter, and E.A.Smuckler.
1978. Energy utilization and RNA transport: Their interdependence.
Biochem. 17:3747-3752.
Clawson, G.A., M. Koplitz, D.E. Moody, and E.A. Smuckler. 1980.
Effects of thioacetamide treatment on nuclear envelope
nucleoside triphosphatase activity and transport of RNA from
rat liver nuclei. Cancer Res. 40:75-79.
Clawson, G.A., C.H. Woo, J. Button, and E.A. Smuckler. 1984.
Photoaffinity labeling of the major nucleosidetriphosphatase of rat
liver nuclear envelope. Biochem. 23:3501-3507.
Davis, L.I., and G. Blobel. 1986. Identification and characterization
of a nuclear pore complex protein. Cel 1 45:699-709.
De Robertis, E.M., S. Lienhard, and R.F. Parisot. 1982. Intracellular
transport of microinjected 5S and small nuclear RNAs. Nature
295:572-577.
De Robertis, E.M., R.F. Longthorne, and J.B. Gurdon. 1978.
Intracellular migration of nuclear proteins in Xenopus oocytes.
Nature 272:254-256.
De Roe, C., P.J. Courtoy, J. Quintart, and P. Baudhuin. 1984.
Molecular aspects of the interactions between proteins and
colloidal gold. J. Cell Biol. 99:57a.
Diberardino, M.A., N.J. Hoffner, and M.B. Matilsky. 1977. Methods of
studying nucleocytoplasmic exchange of nonhistone proteins in
embryos. Ijn Methods in Cell Biology. Chromatin and Chromosomal
Protein Research. D.M. Prescott, editor. Vol. XVI. Academic
Press, New York, 141-165.
Dingwall, C., S.M. Dilworth, S.J. Black, S.E. Kearsey, L.S. Cox, and
R.A. Laskey. 1987. Nucleoplasmin cDNA sequence reveals
polyglutamic acid tracts and a cluster of sequences homologous to
putative nuclear localization signals. EMBO J. 6:69-74.


97
and large particles (120-280 A), coated with nucleoplasmin, were
injected simultaneously into the cytoplasm. As seen in Figs. 13a and
13b, small particles and large particles are located either within or
adjacent to the nuclear surface of the same nuclear pore. To control
for nonspecific binding of the small particles to the large particles,
or possible exchange of coat material during the injection procedure,
experiments were also performed in which small gold particles were
coated with BSA-cTy and mixed with the large nucleoplasmin-gold
particles prior to injection. Electron microscopic analysis of this
experiment, shown in Fig. 13c, indicates that only large particles are
present in the pores and the nucleus while the small tracer particles
are retained in the cytoplasm. These experiments demonstrate that
individual pores can recognize and translocate different nuclear
targeting signals.
Discussion
By stabilizing colloidal gold particles with BSA conjugated with
synthetic peptides that contained either active or inactive SV 40 large
T-antigen nuclear transport signals, it was possible to prepare a range
of electron microscopic tracers that varied in both size and signal
content. Electron microscopic analysis of the intracellular
distributions of the tracer particles following microinjection into
oocytes led to the following conclusions. First, BSA conjugates
containing active signals are transported into the nucleus through
central channels located within the pores. Second, both the functional
size of the channels and relative nuclear uptake increase as the signal


62
particles were injected into the nucleus and nucleoplasmin-coated gold
was injected into the cytoplasm of the same cell. The sequence of the
injections varied in different experiments; however, in all instances
the interval between the first and second injection was 15 min, and the
oocytes were fixed after a total of 45 min. In all, 12 cells were
examined. The results, which were the same regardless of the injection
sequence, are illustrated in Fig. 6, a-d. Small RNA particles can be
observed immediately adjacent to the cytoplasmic surface of the pores
and large nucleoplasmin particles are present on the nuclear side of the
same pores. These distributions clearly demonstrate that individual
pores can function in both protein uptake and RNA efflux.
Cytoplasmic injections
The RNA-gold preparations [tRNA, 5S RNA and poly(A)] were injected
into the cytoplasm to determine if transport is reversible.
Approximately 40 nl of colloid (containing approximately 200 ng of RNA)
were introduced adjacent to the nuclear envelope in a total of 20
centrifuged cells. Similar distributions were obtained for all RNAs
used. In both 1 h and 6 h experiments, gold was seen within the
poresnear the site of injection, but relatively few particles were found
in the nucleoplasm. The nuclear to cytoplasmic concentration ratios
were approximately 0.01 after 1 h. The 5S RNA-gold results are
illustrated in Fig. 7. It should be pointed out that the number of
pores that contained gold varied considerably from cell to cell,
therefore, no effort was made to quantitate these results.


84
uptake of the tracer particles. Four to 6 cells were examined for each
coating agent within each size fraction.
The nuclear to cytoplasmic (N/C) ratios for different coating
agents are given in Table VIII. After 1 h, N/C ratios obtained with the
20-50 A fraction stabilized with BSA-WTg and BSA-WTg were 0.58 and 0.76,
respectively. The difference in these ratios, as determined by the
Student's t-test, is not statistically significant (p>0.25). When the
particle size was increased to 20-160 A or 50-280 A in diameter, the
differences in N/C ratios between the BSA-WT5 and BSA-WTg conjugates
were highly significant (p<0.002). In addition, BSA-WTg was more
effective in facilitating transport than large T-antigen which contains
1 signal per monomer (p<0.002). However, increasing the number of
signals from 8 (BSA-WTg) to 11 (BSA-WT^) did not significantly increase
the N/C ratio (p values obtained for the 20-160 A and 120-280 A
fractions were p>0.8 and 0.1>p>0.05, respectively).
The N/C ratio obtained for the BSA-WTg-cTy dilution (approximately
3 signals per BSA molecule) was significantly lower than that obtained
for the BSA-WTg conjugate (p<0.002); however, it was also significantly
lower than that observed for large T-antigen-gold (0.01 could be due to variation in the availability and/or spatial
distribution of the signal at the surface of the gold particles.
The N/C values for BSA-cTy, BSA-cTig-, and BSA-coated gold were
significantly lower than all gold preparations containing active nuclear
targeting signals (p<0.002).
Overall, it is concluded from these results that there is a direct
correlation between the number of transport signals and the relative


20
protein localizing in the nucleus. After narrowing down a region of the
protein necessary for nuclear localization, oligonucleotide-directed
mutagenesis can be used to map out the essential amino acid sequence
necessary for targeting. Overall, these approaches have been used to
study an array of proteins that are targeted to specific organelles:
endoplasmic reticulum, mitochondria, nuclei, and peroxisomes.
The simian virus (SV) 40 large T-antigen targeting signal has been
the most extensively studied sequence that confers nuclear localization.
Large T-antigen, during lytic infection, is responsible for the
regulation of viral transcription and replication. The protein has 708
amino acids and in the monomeric form (can be tetrameric) has a
molecular weight of 94 kd. The size of this protein suggests that it
cannot enter the nucleus through the pores by simple diffusion, although
large T-antigen is found predominantly within the nucleoplasm. Butel et
al. (1969) described a large T-antigen from a mutant SV 40 hybrid virus
that was localized in the cytoplasm of an infected cell. Comparison of
the sequence analysis of wild type large T (WT) to the mutant large T-
antigen (cT) revealed the amino acid at the 128 position was changed
from lysine to asparagine (Lanford and Butel, 1984). The mutant large
T-antigen was found to be soluble in the cytoplasm and capable of
binding DNA (Lanford and Butel, 1980). Thus, the failure to accumulate
in the nucleus was not caused by its inability to either diffuse
throughout the cytoplasm or bind DNA, but caused by a mutation in a
critical region of the protein sequence that is required for nuclear
localization.


93
Accumulation of tracers along the nuclear envelope
Although the nuclear size distributions were the same for particles
coated with the BSA conjugates (WT8 or WT^) or nucleoplasmin, there was
a significant difference in the relative uptake of the gold as a
function of the coating agent, indicating that not all transport signals
are equally effective. The results shown in Fig. 12 and Table XII
suggest that the effectiveness of specific signals could be related to
their ability to bind to the nuclear envelope.
Figure 12 illustrates the intracellular distributions of 50-280 A
gold particles coated with BSA-WT5 (12a), BSA-WTg (12b) and
nucleoplasmin (12c). Similar amounts of colloid were injected into each
oocyte. Progressing from 12a to 12c, there is an increase in the number
of particles present both within the nucleus and along the envelope.
The relationship between the accumulation of particles along the
envelope and relative uptake is shown in Table XII. The envelope to
cytoplasm ratios were obtained by comparing the number of particles
associated with the envelope to the number of particles in an equal,
randomly selected area of cytoplasm. It is evident that a direct
relationship exists between the number of particles associated with the
envelope and nuclear uptake. A similar relationship between uptake and
binding was obtained with the 120-280 A particle fractions, coated with
BSA-WTg, BSA-WT11, or nucleoplasmin (data not shown).
Coinjection of BSA-WTr- and nucleoplasmin-coated particles
To determine if different targeting signals can be transported
through the same pore, small particles (20-50 A), coated with BSA-WTg,


17
oocytes, radioactivity was concentrated in the cytoplasm although some
labelled protein entered the nucleus. From these results, Bonner
suggested the existence of three classes of proteins: N proteins, found
predominantly in the nucleus; C proteins, found predominantly in the
cytoplasm; and B proteins, found in equivalent amounts in both the
nucleus and cytoplasm. Feldherr (1975) measured the rate of nuclear
uptake for endogenous proteins that were labelled with tritiated amino
acids i_n vivo. His results demonstrated that newly synthesized nuclear
proteins, ranging from 94,000 to 150,000 kd, were three times more
concentrated within the nucleus as compared to the cytoplasm after 6 h.
The rates of accumulation for endogenous proteins of this size were
greater than expected in comparison to the results obtained for
exogenous proteins of similar size.
To better understand the role of the nuclear envelope in regulating
nucleocytoplasmic exchange of endogenous proteins, Feldherr and
Pomerantz (1978) altered the diffusion barrier by using glass needles to
puncture holes in the envelope and then measured the uptake of labelled
nuclear proteins. The results, as determined by one-dimensional gel
electrophoresis, showed no qualitative differences in the accumulation
of nuclear proteins in punctured nuclei as compared to nuclei from
nonpunctured control oocytes. Feldherr and Ogburn (1980) further
studied the mechanism of selecting nuclear proteins by first disrupting
the nuclear envelope and then using two-dimensional gel analysis,
fluorography, and double-labelling techniques to determine intracellular
distributions. Analysis of over 300 nuclear polypeptides showed less
than five percent of the proteins varied between experimentally


BIOGRAPHICAL SKETCH
Steven I. Dworetzky was born December 19, 1959 in New Rochelle, New
York. He graduated from high school in 1977. Following high school, he
attended Skidmore College in Saratoga Springs, New York, and earned a
Bachelor of Arts degree in biology/chemistry in 1981. In September 1981
he entered graduate school at the University of Florida in the
Department of Anatomy. In the summer of 1984, he successfully completed
an eight week course entitled Cell Physiology: Cellular and Molecular
Biology at the Marine Biological Laboratory in Woods Hole,
Massachusetts. He completed the requirements for the degree of Doctor
of Philosophy in April 1988 and accepted a postdoctoral research
position at the University of Massachusetts Medical Center in
Shrewsbury, Massachusetts. He is a member of the American Society for
Cell Biology.
130


13
histones, lysozyme, and trypsin inhibitor, and the neutral protein,
myoglobin.
To determine if exogenous macromolecules exit the nucleus, Paine
(1975) injected ovalbumin, horseradish peroxidase, myoglobin, and
cytochrome c directly into the nucleoplasm of Chironomus salivary gland
cells. The results showed that the fluorescein-labelled molecules enter
and leave the nucleus with similar kinetics, indicating that
nucleocytoplasmic exchange is bidirectional and the rate of movement
across the envelope is dependent on 1) the restrictions imposed by the
diameter of the diffusion channel and 2) the size of the molecule.
To estimate the patent diameter of the nuclear pores in amphibian
oocytes, Paine et al. (1975) injected tritiated dextrans with known
hydrodynamic radii of 12.0, 23.3, and 35.5 A. Dextrans are uncharged
polymers of D-glycopyranose and available in a range of molecular sizes.
To determine the intracellular localization of the tracers, the cells
were quickly frozen at different time intervals, sectioned, and
autoradiographed. Comparison of the grain profiles for all three
tracers showed that the nuclear envelope is less permeable to larger
dextrans, supporting the view that the envelope can limit the rate of
entry of larger macromolecules. The relationship between the size of
the dextrans and their rates of entry suggested that the nuclear
envelope had a sieving effect due to its porous nature. Developing a
mathematical model from the rates of uptake, diffusional coefficients,
and the length of the channel, the authors estimated the patent pore
diameter to be 90 A.


115
Isolation of receptors involved in the translocation process
remains an essential step to understanding the nucleocytoplasmic
transport of macromolecules. Additionally, it is equally important to
determine if there are different receptors in individual pores or
whether there are common receptors for the transport of different
protein signal sequences and/or putative RNA transport signals. After
identification of receptors an important area that remains to be
investigated is the binding affinities of different transport signals as
the affinity of signals is most likely, in part, a mechanism that the
cell can use to regulate nucleocytoplasmic exchanges.


Ill
There are several implications of the model with regard to
regulation of nucleocytoplasmic exchange. The dilation of the pores
would control exchange in accordance to the related needs of the cell
and provide the cell with an efficient method for exchange. In
addition, the model suggests that endogenous molecules available for
transport can act collectively to open the transport channels. The
factors involved in regulating pore activation, signal number and
affinity, are supportive of this model. To increase the rate of uptake,
a single protein could have multiple signals (i.e., nucleoplasmin) or
proteins might aggregate to increase their overall signal number,
possibly like histones. Furthermore, proteins containing different
targeting signals might have differential binding affinities for pore
receptors which, in turn, might modulate their rate of uptake. Finally,
the complete activation of the pores is large enough for the transfer of
ribosomal subunits and RNP particles.
Future Trends
One question that remains is whether the protein transport signal
is vectorial. In a cursory study, nuclear injection of the protein
conjugates was performed to determine if the signal for protein uptake
is bidirectional, i.e., capable of transport into and out of the
nucleus. One and six hours after nuclear injection, particles coated
with the BSA-conjugates were not observed in either the pores or the
cytoplasm (data not presented) indicating that transport signal is
vectorial; however, the experiments are not definitive. It is possible
that the gold particles were bound specifically or nonspecifically


Finally, and most importantly, I wish to express my utmost
gratitude to Barbara Ludwig. She was a constant source of moral
support and encouragement throughout the trials and tribulations of my
graduate education. In addition, I would like to thank my parents for
their words of encouragement and steadfast support.
IV


37
too large to pass through the pores by passive diffusion, suggesting
that some facilitated mechanism is a requirement for export.
Statement of Research Topic
It has been demonstrated that there are selective mechanisms which
facilitate the passage of certain endogenous macromolecules through the
nuclear pores. The mechanism and selectivity of the pore by which
transport occurs is not known; for example, does translocation proceed
through the pores by a gated mechanism, or does transport occur through
a fixed channel? How does the nuclear targeting signal interact with
the pore region to initiate translocation? Do different nuclear
transport signals use the same pore for uptake? What effects do
variations in the number and sequence of targeting signals have on
nuclear uptake? In relation to RNA transport, what is the precise
location for the translocation of different classes of RNA to the
cytoplasm? Are there specific functional classes of pores that are
involved in RNA transport? or do all pores have the capability to
translocate both RNAs and proteins?
To better understand the role of the nuclear envelope in regulating
nucleocytoplasmic exchange, experiments were designed to elucidate
characteristics of the macromolecular transport process. The data
obtained will focus on the translocation of RNAs. More specifically,
analysis will include the proportion of functional pores, the precise
location and size of the transport channels, and the multifunctional
capability of individual pores (i.e., both protein uptake and RNA
efflux). In addition, data will be obtained for the effect of different


18
punctured oocytes and nonpunctured controls. It was concluded from
these results that nuclear binding plays a major role in regulating the
nucleocytoplasmic distribution of endogenous proteins, and that the
envelope does not function as a rate-limiting barrier.
It is apparent from the above studies that many of the nuclear
proteins accumulate in the nucleus at significantly higher rates than
exogenous molecules of similar size. As discussed previously, exogenous
proteins larger than 45 kd (ovalbumin) do not readily enter the nucleus;
however, endogenous proteins larger than 90 kd accumulate quite rapidly.
Evidence for facilitated transport was obtained by Feldherr et al.
(1983) for RN1, a 148 kd nuclear protein, in the oocytes of Rana
pipiens. After microinjection or i_n vivo labelling of RN1, the kinetic
results demonstrated rapid accumulation within the nucleoplasm.
Theoretical estimates of the diffusion rate of a 148 kd protein through
the 90 A channel indicated that the rate of uptake could not be
accounted for by simple diffusion through the pores and subsequent
binding. Thus, in this instance some form of mediated transport is
apparently required for rapid accumulation.
Recent research has shown that signal sequences of some nuclear
proteins are required to target macromolecules to the nucleus. Two
approaches have been used to study the polypeptide domains that contain
these sequences. The first approach is the partial digestion of a
protein followed by analysis of the fragments capable of accumulating in
the nucleus and the second approach employs DNA methodology.
The first approach has been successfully employed to study
nucleoplasmin, a 110 kd karyophilic protein in Xenopus oocytes, which


26
Goldstein and Plaut (1955) obtained direct evidence in Amoeba
proteus for the transfer of RNA to the cytoplasm by first labelling the
RNA and then transplanting the labelled nucleus to an unlabelled
recipient cell. As shown by autoradiography, the number of grains
within the cytoplasm increased with time. In addition, no radioactivity
was observed in the unlabelled recipient cell nucleus. Ribonuclease
digestion was performed to show that the radioactivity was associated
with the RNA. Due to the size of the RNA, it was postulated that simple
diffusion was not the mechanism involved in RNA efflux.
The movement of RNA from nucleus to cytoplasm is likely to be a
three-step process, involving 1) migration from its site of
transcription to the inner surface of the nuclear envelope, 2)
translocation across the envelope, presumably through the nuclear pores,
and 3) movement from the outer surface of the nuclear envelope to its
final destination within the cytoplasm. The first two steps have been
the focus of intense investigation in an attempt to understand the
mechanism(s) involved in RNA transport. The final step of directing RNA
to its destination within the cytoplasm is not as well characterized as
steps 1 and 2. In addition, Agutter (1985a) has suggested that RNA
efflux occurs by a solid-state process; that is, movement occurs along
structural elements both within the nucleus and cytoplasm.
Transfer RNA
The structure of tRNA and its involvement in protein synthesis have
been the subject of many investigations. Transfer RNAs are small,
compact molecules. In the crystallized state, yeast tRNAP^e has a


Ml
nm
Val
vol/vol
WGA
WT
wt/vol
Microliter(s)
Micron(s)
Valine
Volume/Volume
Wheat germ agglutinin
Wild type large T-antigen
Weight/Volume
XI


Table XII Envelope-Associated Particles
Coating Agent
Envelope:cytoplasm Ratios*
N/C Ratio
BSA-WT5
1.2
0.06
bsa-wt8
2.7
0.35
Nucleoplasmin
6.3
0.71
*These ratios were obtained 1 h after injection as described in the text.