Nucleocytoplasmic transport of macromolecules through the nuclear pores of Xenopus oocytes

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

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Full Text















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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C -) 0)C
.,- .C a) (-
4.->.r 0.
4-3 4-)
W Tl CM Io 0 0 (0
* * I I C 4-C C 0)
-3 04 I I -- C
0O~t.C UI
0 *cc= u
-o. .4(.e
CD3 4.)
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0 0 4J CL
00- M 0
(*) CD 4-
) CD J r -4 IDCD C0 ) UD w

'-4 *i 0e QO SL


-le -o
a) ~ ~ ~ ~ / lc) E)( JU Jf
S- *e- *r- *m -C m U


ao M = 0i 0a 4 0- L
EQ. Cr- m a- ai -

V/ 0 4c
Ln a-


C


0

lo:
*cc,
C)
(A

C)
u
0 .- -"
;m 4--) (D
.- 0 .
00 U/)
4-) IV
04- CD
I- 0 m


4-3

E

1- ^-
a) -
0.
r'-4
X .-
UJ ^


I







I


(
<




(
I


1

u

I


0


r
C
























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






S-
0
S..


a.
u
CL
5-

a.


"0 E
o
c,)
00
CV)


14- 4.
0 C.






U
C 0n
0 (D







*-,-
4) CI
o u
v-4r B0*-r









Co
41 41



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) C
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c 0



0



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> -0










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O C
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(aJ


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00
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Z f.-u
O IO
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*0>

Ot-3
0 ~ 0DO
0M .0J K 0









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


0.-
0)
5-
0


CD









u0)
4 -
EU



0)

U)











t-U
E
=L






in
4- Co










u 0
4-
IB0







a/)


4-)

0~


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


Tr i-crr en a cr



a) o T co












mo 4oo
CWo to 01 CMj 0
I--I 1--


x
4-
C)




3:
w
*-












CD
a)






C-
EU




0
!

0)

*%J






E
0


c


CM


Eu



4.J
0
I-






m
U)




a)
U







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EU






00




N4.)
$4-
4--
i- c-
0-
-F.4-'

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:
.Cc
00
ca

0
4-i


4-)
t0


C
0
C-)
a)
01
C



ca


a)


















0(

a
0)










10
to. CD

1-0
a)**


76





>1


CD

ci

s0













(D
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L
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CD
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o)


.-J
01










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i.


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>,:






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(C.


r--- I--
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r-4
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4r-)
















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cn
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4c
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1-,s
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0 3


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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
















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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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