The biosynthesis of mitochondrial ribosomal proteins

MISSING IMAGE

Material Information

Title:
The biosynthesis of mitochondrial ribosomal proteins
Physical Description:
Book
Creator:
Schieber, Gretchen Lyn, 1955-
Publication Date:

Record Information

Rights Management:
All applicable rights reserved by the source institution and holding location.
Resource Identifier:
oclc - 10266766
System ID:
AA00020012:00001

Table of Contents
    Title Page
        Page i
    Acknowledgement
        Page ii
    Table of Contents
        Page iii
        Page iv
    List of Tables
        Page v
    List of Figures
        Page vi
    Abbreviations
        Page vii
        Page viii
    Abstract
        Page ix
        Page x
    Introduction
        Page 1
        Page 2
        Page 3
        Page 4
        Page 5
        Page 6
        Page 7
        Page 8
        Page 9
        Page 10
        Page 11
        Page 12
        Page 13
        Page 14
        Page 15
    Materials and methods
        Page 16
        Page 17
        Page 18
        Page 19
        Page 20
        Page 21
        Page 22
        Page 23
        Page 24
        Page 25
        Page 26
        Page 27
        Page 28
        Page 29
        Page 30
        Page 31
        Page 32
    Results
        Page 33
        Page 34
        Page 35
        Page 36
        Page 37
        Page 38
        Page 39
        Page 40
        Page 41
        Page 42
        Page 43
        Page 44
        Page 45
        Page 46
        Page 47
        Page 48
        Page 49
        Page 50
        Page 51
        Page 52
        Page 53
        Page 54
        Page 55
        Page 56
        Page 57
        Page 58
        Page 59
        Page 60
        Page 61
        Page 62
    Discussion
        Page 63
        Page 64
        Page 65
        Page 66
        Page 67
        Page 68
        Page 69
        Page 70
        Page 71
        Page 72
        Page 73
        Page 74
        Page 75
        Page 76
        Page 77
        Page 78
        Page 79
    References
        Page 80
        Page 81
        Page 82
        Page 83
        Page 84
        Page 85
        Page 86
        Page 87
        Page 88
        Page 89
        Page 90
    Biographical sketch
        Page 91
        Page 92
        Page 93
Full Text













THE BIOSYNTHESIS OF MITOCHONDRIAL
RIBOSOAL PROTEINS













By

GRETCHEN LYN SCHIEBER


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


1983













ACKOWLEEMENTS


In my many years in graduate school several people have had an

influence on my understanding and appreciation of both science and

life. I would like to acknowledge their contribution to this

document.

In Dr. O'Brien's laboratory I have had the honor to work with

Bill Cattaneo, Robert Cheatham, Susan Collingwood, Mary Conde, Gary

Daniels, Jane Edwards, Terry Harville, Robert Hessler, Carolann

Johnson, Maria Kucharska, Mark Lijewski, Vince LiCata, Mietek

Piatyszek, Sal Pietromonaco, Joyce Plugge, Bobby Sheehan, and John

Turse. Dr. Nancy Denslow deserves special mention for her never

ending support, optimism, and scientific discussion.

The members of the department have all affected my graduate

training. I would like to note the useful discussion and advice and

the extended support of my supervisory committee, Dr. .Charles Allen,

Dr. Peter McGuire, and Dr. Parker Small. My committee chairperson,

Dr. Thomas O'Brien, has, perhaps, taught me more than even he

realizes. He has made an invaluable contribution to this work.

My brother and sister-in-law, Marc and Marsha, and especially my

parents, Don and Dorothy Schieber, have been a tireless source of

encouragement. They have provided the intellectual and emotional

foundation for this work.

To all of you, my profound thanks.














TABLE OF CONTENTS


ACKNOWLEDGEMENTS ............................................... ii
LIST OF TABLES ................................................ v
LIST OF FIGURES ............................................... vi
ABBREVIATIONS .................................................. vii
ABSTRACT ....................................................... ix
INTRODUCTION ................................................ ... 1
Background ............................................. 1
The Nucleo-Cytoplasmic Protein Synthetic Campartment and
Mitochondrial Import .......................... .. ...... 1
The Mitochondrial Gemcme .................. .......... 4
Mitochondrial Ribosomes ................................. 9
Mitochondrial Ribosome Biosynthesis ..................o 12
Objectives of this Work .......................... ....... 14
MATERIALS AND MET'HODS ............... ................. ......... 16
Materials ................................................... 16
Experiments Using Mitochondria Isolated from Bovire
Liver .............................. ... ... ..... ...... 18
Preparation of Bovine Liver Mitochondria ............... 18
Conditions for Mitochondrial Incubation ............. 19
Experiments Using Cells Grown in Tissue Culture ........... 21
Conditions for Incorporation of Radiolabeled
Methionine ................ ........................ 21
Preparation of Mitochondria from Cultured Cells ........ 25
Isolation of Mitochondrial Ribosomes ................... 26
Assay for Inhibitor Response .......................... 28
Inmmnunologic Methods ..................................... 29
Preparation of Antisera ......................... ........... 29
Preparation of Immune Precipitates ..................... 30
Electrophoretic Methods ................................o 31


iii








Table of Contents continued


RESULTS ................................................ ........ 33
Experiments with Isolated Bovine Mitochondria ............. 33
Synthesis of Protein by Isolated Bovine Mitochondria.... 33
Analysis of Radiolabeled Mitochondrial Products by
T.D-Dimensional PAGE ............................... 35
Experiments with Cells in Culture ......................... 38
Mitoribosomal Proteins from Cells Labeled in the Absence
of Cytoplasmic Protein Synthesis ................... 38
Mitoribosomal Proteins from Cells Labeled in the Absence
of Mitochondrial Protein Synthesis ................. 42
Effect of Inhibitors .................................. 56
Precursors of Mitoribosomal Proteins ...................... 60
DISCUSSION ..................................................... 63
Protein Synthesis in Isolated Mitochondria ................ 64
Protein Synthesis in Cultured Bovine Cells ................ 67
Pools of Unassembled Mitoribosomal Proteins ............... 75
Implications for Mitochondrial Evolution and Biogenesis ... 77
REFERE CES ................. .................................9.... 80
BIOGRAPHICAL SKETCH ............................................ 91














LIST OF TABLES


1. Reading Frames of Mmmalian Mitochondrial DNA ............. 7

2. Solutions Cited .................o ...................... 17

3. In Vitro Mitochondrial Incubation Mixture ................. 20

4. Synthesis of Defined Mitoribosomal Proteins in the Presence
of Chloramphemicol ............................................ .. .. ... 52














LIST OF FIGURES


1. Electropherograms of the Proteins of the Bovine
Mitochondrial Ribosome .................................... 10

2. Schematic diagrams of the Proteins of the Bovine
Mitochondrial Ribosome ......................................... 11

3. Schematic representation of the labeling protocols for MDBK
cells ..................................................... 24

4. Sucrose density gradient profiles of the preparation of
mitochondrial ribosomes and ribosomal subunits ............ 27

5. Incorporation of [ H]leucine into protein by isolated
bovine liver mitochondria ............................................ 34

6. Synthesis of protein by isolated bovine mitochondria ....... 36

7. Fluorograms of mitoribosomal subunits from cells labeled in
the absence of cytoplasmic protein synthesis .............. 40

8. Fluorogram of mitoribosomal subunits from cells labeled in
the absence of cytoplasmic protein synthesis showing high
molecluar weight contaminants ............................... 41

9. Fluorograms of mitoriboscmal subunits from cells labeled in
the absence of mitochondrial protein synthesis ............ 44

10. Fluorograms of 28 S mitoribosomal subunits froman cells
labeled in the absence of mitochondrial protein synthesis,
no chase period .......................................... 48

U. Fluorograms of 39 S mitoribosomal subunits franm cells
labeled in the absence of mitochondrial protein synthesis,
no chase period ........................................... 50

12. Total mitochondrial incorporation in the presence and
absence of inhibitors ..................................... 57

13. Effect of inhibitors on MDBK cells ........................ 59

14. Immune precipitation of mitoribosomal proteins from soluble
cell fractions ............................................ 61















ABBREVIATIONS


Ac

ATP

bicine

BSA

CAP

CHM

Ci

cm

CpM

DNA

dpm

EDTA

h

kg

L

M

MDBK cells


The amount of ribosomes which gives an absorbance
of one unit at 260 nm when dissolved in 1 ml of
solution and measured with a light path of 1 an
1 A^ unit of mitoribosomes = 90 pg (60 pg of
proein, 30 pg of RNA)
1 A260 unit of 55 S monosomes = 32 poles
1 A260 unit of 39 S subunits = 55 pmoles
1 A260 unit of 28 S subunits = 84 pmoles

acetate

adenosine-5' -triphosphate

N,N-bis [ 2-hydroxyethyl ]glycine

bovine serum albumin

D-threochloramphenicol

cycloheximide

Curie, 2.2 x 1012 dpm of radioactivity

centimeter

scintillation counts per minute

deoxyribonucleic acid

disintegrations per minute

[ethylenedinitrilo ] tetraacetic acid

hour

1000 x a force of one gravity

liter

molar, moles/liter

Madin-Darby bovine kidney cells


vii








Abbreviations-continued


mitoribosomare

ml

mM

MM

ml

imRNA

mtDNA

PAGE

pwole

PMS

rDNA

RNA

rpnm

rRNA

S

SDS

sec

TCA

Tris

tRNA

URF

P
ID
2D
2D


mitochondrial ribosome

milliliter

millimolar

mitochondrial matrix fraction of cells

millimole

messenger ribonucleic acid

mitochondrial deoxyribonucleic acid

polyacrylamide gel electrophoresis

piccamole

post-mitochondrial supernatant cell fraction

DNA which codes for ribosomal R1

ribonucleic acid

revolutions per minute

ribosamal ribonucleic acid

Svedberg unit

sodium dodecyl sulfate

second

trichloroacetic acid

tris-hydroxymethylamincmethane

transfer ribonucleic acid

unidentified reading frame

micro, x 106

one-dimensional

two-dimensional


viii















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



THE BIOSYNTHESIS OF MITOCHONDRIAL
RIBOSOMAL PROTEINS

By

GRETCHEN LYN SCHIEBER

December 1983

Chairman: Thomas W. O'Brien, Ph.D.
Major Department: Biochemistry and Molecular Biology

This research has addressed questions on the biosynthesis of the

proteins of the mammalian mitochondrial ribosome (miitoriboscme). It

has focused on the site of synthesis of the mitoribosomal proteins;

that is, are any of the proteins of the mammalian mitoribosome

products of mitochondrial protein synthesis? It is already known

that the mitoribosomes of primitive eukaryotes, yeast and

Neurospora, do contain a protein which is a product of

mitochondrial protein synthesis. However, because these systems are

quite different from the mammalian systems, the biosynthetic site of

the proteins might differ.

Two approaches were used to address this problem. In the first,

mitochondria isolated from bovine liver were allowed to synthesize

proteins in an in vitro system and the products were comigrated in

two-dimensional gel electrophoresis with the mitoriboscoal proteins.









The mitochondrial protein synthetic products, visualized by

fluorography, showed several spots of varying intensity. These were

compared to the mitoribosomal proteins, visualized by Coomassie blue

stain. Only one protein, L44, was comigratory with one of the

radioactive spots, but some spots were positioned near mitoribosomal

proteins (S13, S20, and L20). Therefore, these mitoribosomal

proteins are indicated as possible products of mitochondrial protein

synthesis.

In the second approach, bovine (MDBK) cells grown in culture

were labeled with [3 Sjmethionine in the presence and absence of

inhibitors for mitochondrial and cytoplasmic protein synthesis. The

labeling in the absence of cytoplasmic protein synthesis (with the

cells inhibited by cycloheximide) produced a "blank" fluorogram,

indicating that there is no mitochondrial product. This was further

substantiated by cell labeling studies in the absence of

mitochondrial protein synthesis showing that the mitoriboscmal

proteins, including those proteins indicated by experiments in

isolated mitochondria (see above), incorporated [3 S]nethionine in

the presence of chloramphenicol. Finally, it was demonstrated that

mitoribosomal proteins can be both translated and assembled into

complete mitoribosomes in the absence of mitochondrial protein

synthesis. These results indicate that all of the proteins of the

mammalian mitoribosome are products of cytoplasmic protein synthesis,

and that the function of the mitochondrially produced mitoribosomal

protein in lower eukaryotes must be performed by a cytoplasmically

produced protein in mammals.













INTRODUCTION


'Background



It is now firmly established that the mitochondrion is a

"semi-autonomous" organelle, simplistically thought of as some sort

of bacteria-like structure which has evolved to exist within, but

somewhat independent of, the eukaryotic cell. While mitochondria

contain their own DNA and a complete protein synthesis system,

including ribosomes, tRNA, rRNA, and all of the accessory proteins,

they synthesize only a handful of the several hundred proteins

necessary for proper mitochondrial function. The vast majority of

mitochondrial proteins are synthesized by the nucleo-cytoplasmic

protein synthesis system; coded for by genes located on nuclear DNA,

translated on cytoplasmic (80 S) ribosomes, and specifically imported

into mitochondria [1,2].


The Nucleo-Cytoplasmic Protein Synthetic Compartment and
Mitochondrial Import

Early studies on the cytoplasmic synthesis of mitochondrial

proteins were hampered by the inability to produce clean cell

fractions and by the practice of studying mitochondrial proteins as a

general class instead of studying a specific protein. In the early








and mid 1970's, Butow's group published a series of papers [3-6]

based on electronmicroscopy which indicated that cytoplasmic

ribosomes were specifically and functionally attached to the outside

of mitochondria, implying that mitochondrial proteins might be

vectorially discharged directly into the the mitochondrial matrix.

About the same time, several groups published papers indicating that

mitochondrial proteins were synthesized initially on ribosomes bound

to the endoplasmic reticulum [7-9] and directed in a manner similar

to secretary proteins. Most of these studies relied on antibodies

raised against either a specific mitochondrial protein or a general

extract of mitochondria to follow the position of newly synthesized

mitochondrial proteins in cell fractions which had been produced

after labeling in intact cells. Several authors reported a

microsomal pool early in the synthetic pathway [10,11] and some

traced the kinetics of protein movement to the mitochondria [11,12].

Occasionally, an author reported synthesis of a specific

mitochondrial protein on free ribosomes [13,14]. The general model

for the synthesis of mitochondrial proteins was expected to parallel

that of secretary proteins.

The model of mitochondrial protein import began to change as

cell-free protein synthetic systems were used to produce precursor

mitochondrial proteins. Unexpectedly, precursors of mitochondrial

proteins were directly taken up by mitochondria added to the mixture

[15-25]. In several cases, this was demonstrated in the absence of

translation on 80 S cytoplasmic riboscmes, showing that import was a

post-translational event. Other workers looked at the distribution







of mRNA which coded for mitochondrial proteins and various authors

reported either that it was isolated almost completely from free

ribosomes [26,27] or that is was on both free and mitochondrially

bound ribosomes [28-30]. Thus, for virtually all proteins studied by

these techniques, recognition for import into mitochondria appears to

be mediated by the precursor protein (without any known covalent,

post-translational modifications) and the intact mitochondrion.

Occasionally, those proteins with an N-temnninal "transit peptide"

(see below) may adventitiously begin to import during translation,

producing mitochondrially-bound cytoplasmic ribosomes, although there

are no reports of obligatory co-translational import.

The structure of the precursor mitochondrial proteins has been

studied for many different proteins in many different systems (for

review see [30]). In most cases, the precursor protein is slightly

larger than the mature mitochondrial form, although the length of the

extension varies. While some authors refer to this extension as a

"signal peptide" [31], others are starting to use the term "transit

peptide" [32] both to denote that it is not endoplasmic reticulum

associated (and probably not recognized by the signal recognition

particle [33]) and because it may encode some assembly instructions,

not just organellar destination. While, in most cases, this peptide

appears to be a short N-terminal extension on each protein [34-37],

that is not always the case. In one case, it was reported by

Poyton's group that all four of the cytoplasmically synthesized

proteins of the F1-ATPase were synthesized as a single polypeptide,

which was imported in one step [38,39]. However, reports from








several other groups [30,40-42] have shown that these four proteins

are translated in independent events. There are also reports of

uncleaved transit peptides. For example, the newly-synthesized form

of the y-subunit of F1-ATPase in yeast has the same SDS-PAGE

mobility as the form isolated frame the mature complex [41]. Finally,

there is at least one report of what appears to be an internal

transit sequence, in cytochrome c. Newly synthesized cytochrcme c

differs from the mature polypeptide only in the replacement of the

N-terminal methionine with an acetyl group. The uptake of newly

synthesized cytochrome c is competitively inhibited by apo-cytochrcnme

c [43-45] and by an internal polypeptide (trypsin fragment 66-104)

[46]. The uptake is not affected by the N-terminal fragment (either

fragment 1-65 or 1-80) or by holo-cytochrome c.



The Mitochondrial Gename

Early characterization of mitochondrial DNA showed that it was

circular and varied in size franom about 16,000 bases in mammals to

over 100,000 bases in higher plants [47]. Other reports noted that

it was replicated in an asynchronous manner [48] and was transcribed

completely [49], with the various RNAs then processed out of the

longer transcirpt. Various researchers tried to map the products of

mitochondrial DNA transcription by hybridization, eventually

positioning the ribosomal RNAs and several tRNs [50]. Early efforts

to identify the products encoded by mitochondrial DNN relied on

analysis by one-dimensional SDS-PAGE which revealed 10-12 proteins







[51-54]. When two-dimensional PAGE was used, the patterns became

more complex; usually around 25 proteins were visualized [55-58].

All of these studies were eclipsed in 1981 by the publication of

the complete sequence of a human placental mitochondrial DNA [59].

The structure was completely analyzed by comparing it to the known

information about the sequences of mitochondrially produced proteins

and mitochondrial rRNA and tRNA [60-64]. The mitochondrial DNA

contains thirteen open reading frames and the coding sequences for

mitochondrial rRNA and for 21 tRNAs. The other major region of the

DNA is the "D-loop" which contains the origin for replication. This

organization has been preserved in the mitochondrial DNA of all of

the mammalian species sequenced, including mouse [65], cow [66], and

rat [67]. Indeed, a partial sequence in Xenopus [68] indicates that

this general organization may be conserved among all of the

vertebrates.

One of the most striking features of the mammalian mitochondrial

genome is its extraordinarily high density of coding information.

There are virtually no "unused" bases. The tRNAs serve to punctuate

the sequences for mRNA and rRNA [6-9,70], often without a single

intervening nucleotide between the 3' terminus of the tRNA and the 5'

initiation codon of the message. The mRNA sequences apparently lack

any untranslated regions, either leader sequences or introns [71].

Indeed, some of the messages only acquire a termination codon when

the cleaved portion of the transcript is polyadenylated.

Five of the reading frames of mitochondrial DNA have been assigned

to known products of mitochondrial protein synthesis; the other eight









are unassigned reading frames (URFs). The assigned reading frames

include the genes for subunit 6 of the FIATPase, cytochrome b, and

cytochrome oxidase subunits I, II, and III. Despite some early

reports that subunit 9 of the F1ATPase was mitochondrially

synthesized in rats [72,73], this protein is not coded for on

mitochondrial DNA; thus, mammals, like Neurospora, must synthesize

this protein cytoplasmically [74,75] (although it is made

mitochondrially in yeast). The reading frames of the mammalian

(bovine [66]) mitochondrial gename are summarized in table 1. These

reading frames are considered significant because they are conserved

in all of the mammalian mitochondrial genomes analyzed to date and

because long-lived, polyadenylated cognate RNAs for each of these

reading frames have been isolated from mitochondria. The assigned

reading frames are exactly comparable to the known sequences of their

product proteins. The amino acid sequences of the products of the

unassigned reading frames can be predicted and this information is

currently being exploited to determine if, as expected, these

polypeptides are being translated on mitoribosomes in living systems.

To date, antibodies produced against artificially synthesized

oligopeptides have been used to inmunoprecipitate the products of the

ATPase 6 gene [76] as well as URFs A6L, 1, and 3 [76,77].

Additionally, the predicted proteolytic fingerprint pattern was used

to demonstrate the in vivo synthesis of URFs 3 and 6 [78]. No

function has yet been assigned to a product of any unidentified

reading frame.








TABLE 1


READING FRAMES OF MAMMALIAN MITOCHONDRIAL DNA


The significant reading frames of a manummalian (bovine) mitochondrial genome are determined by homology
with other mammalian species (human and mouse) and by the presence of corresponding polyadenlylated RNAs.
All data are from Anderson et al. [66].


Size (in
Name nucleotides)


Number of
Amino Acids


Molecular Weight
(of protein)*


Number of
methionines


Proteins

ATPase subunit 6

Cytochrome b

Cytochrome oxidase I

Cytochrome oxidase II

Cytochromre oxidase III


Unidentified Reading Frames

1

2

3


1378 459t


24.8

42.5

57.0

26.0

29.9


681

1140

1542

684

784


956


1042


347


226

379

514

227

261


318*

347*

115+


35.6*

39.2*

13.0*


22*

43*

5*

37*











4L 297 98t 10.8 13
5 1821 606t 68.2t 39t
6 528 175t 19.0 lit
A6L 201 66t 7.9t 4t



* in kilodaltons

predicted








Mitochondrial Ribosomes

Despite their similar function, the cytoplasmic and

mitochondrial protein synthesis systems are completely distinct.

When compared with cytoplasmic ribosomes, mitochondrial ribosomes

generally are smaller and appear in some ways (e.g. antibiotic

susceptibility) to be more like prokaryotic ribosomes [79,80]. While

there is variability in size and physical-chemical properties among

mitoribosomes of the other eukaryotes, the mitochondrial ribosomes of

mammals all appear to be of the 55 S type [81,82]. Mammalian

mitoribosomes are slightly larger than bacterial ribosomes [83] and

their composition is much more protein rich (about 85 proteins [84]),

with their rRNA content correspondingly reduced. This change in

composition is reflected in the lower sedimentation coefficient of

mammalian mitoriboscmes, 55 S versus 70 S for bacterial ribosomes and

80 S for eukaryotic cytoplasmic ribosomes [81,82].

The proteins of the bovine mitoribosome have been characterized

by two-dimensional PAGE. Typical electropherograms are shown in

figure 1. The small (28 S, 1.1 x 106 daltons) and large (39 S,

1.65 x 106 daltons) subunits of the mitoriboscme are analyzed

separately. In figure 2 are shown the schematic diagrams of these

separations, as analyzed by Matthews et al. [84]. Proteins which are

numbered appear in most of the electropherograms. However same of

them are variable and sometimes appear weakly or not at all. The

small subunit schematic also shows fourteen lettered proteins which

appear infrequently in these electropherograms.






A B






4 *



04.




Figure 1. Electropherograms of the Proteins of the Bovine Mitochondrial Ribosome.
Proteins of the small, 28 S (A) and large, 39 S (B) subunits of bovine liver mitochondrial ribosomes
are shown. The subunits were derived froman 55 S ribosomss as described in "Materials and Methods". The
first electrophoretic dimension, from left to right in the figures, is electrophoresis in 9 M urea at pH
4.3. The second dimension, from top to bottom, is electrophoresis in 0.5% SDS and 5 M urea at pH 7.2.








A B
S-68 68
, 2 4-50 50
43
ftf -44.5 l w4 44.5
8 % -6 6W
4W IA7 7o
I1t- d P- ,1 30 12 13 4 14 30
"3sC dv 14 Z-000'3
i 15w* 15 17019
1f, 17 20 921
S200 19 q.i" 230 h 2
30
j,21.22,, 31 0 W2- 2824,
23N 24 25 26 14.4 347233 ;2 03
10 0 4b -144. 3700_ 036 qb40-14
27 29 32 1* 440
28 3 3.1 O* -38 390q*44 4
41 4243
m -12.5 460* 494 -12.5
1033 4W* o47
48 4 50
51o
520



Figure 2. Schematic diagrams of the Proteins of the Bovine Mitochondrial Ribosome.
Schematic diagrams showing the positions of bovine mitoribosomal proteins when analyzed by
two-dimensional PAGE as described in "Materials and Methods". Proteins of the small, 28 S ribosomal
subunit are shown in A and of the large, 39 S ribosomal subunit are, shown in B. Numbered proteins
appeared in the majority of electrophoretic separations analyzed. lettered proteins appeared in less
than half of the electropherograms analyzed. The schematic diagrams are taken from Matthews et al., 1982
[841.








Mitochondrial Riboscme Biosynthesis

How mitochondria synthesize and assemble their ribosomes has

been studied primarily in two primitive eukaryotic systems, yeast and

Neurospora.

In yeast, it was discovered that there is one product of

mitochondrial protein synthesis that exhibits a strain specific

molecular weight polymorphism [85,86]. This protein, named "var 1,"

varies in molecular weight from 40 to 44 kilodaltons. An early

report by Groot [87,88] demonstrated that var 1 is associated with

the small mitoribosomal subunit, but concluded that the protein could

not be a "ribosomnal protein" since yeast could be grown for several

generations in the mitochondrial specific inhibitor, chloramphenicol,

without affecting mitochondrial protein synthesis. Because

chloramphenicol does not completely inhibit mitochondrial protein

synthesis in yeast, a second study was performed by Butow's group,

using the more effective antibiotic erythromycin. In the presence of

erythromycin, it was demonstrated that mitochondrial protein

synthesis was affected and that the synthesis of the small

mitoribosomal subunit was inhibited [89, 90]. In fact, the synthesis

of var 1 is highly resistant to inhibition by chloramphenicol and

largely accounts for the leakage of yeast mitochondrial protein

synthesis in the presence of this inhibitor. Inhibiting the

synthesis of var 1 with antibiotics gives rise to small mitoribosomal

subunits which are missing some proteins including var 1 [91]. It is

now known that different gene conversions give rise to the variation

in the molecular weight of this protein [92-95] and that a sequence









homologous to var 1, as well as other mitochondrial DM sequences,

can be detected in the yeast nuclear gencme [96-98].

In Neurospora, there is also one and only one mitochondrially

produced mitoribosomal protein. This protein, originally identified

as S-4a [99], is now designated S-5 [100]. Like var 1, it is one of

the larger proteins (52,000 daltons) of the small mitoribosomal

subunit. In an early study [99], Lambowitz, Chua, and Luck

demonstrated that mitoribosomes are synthesized via a series of

precursor intermediates which contain some of the ribosomal proteins

and incompletely processed rRNA. They also showed that the synthesis

of only S-5 is inhibited by chloramphenicol and permitted in the

presence of anisomycin (a specific inhibitor of cytoplasmic protein

synthesis). In other studies, Lambowitz's group has shown that

mitoribosomal small subunits from Neurospora grown in the presence

of chloramphenicol are deficient in S-5 plus other proteins which

apparently assemble onto the particle after S-5 [101], that the

removal of an intron in the transcript for the 25S large subunit rRNA

is preceded by specific binding of some of the cytoplasmically

synthesized mitoribosomal proteins [102,103], and that mitochondrial

mutations which directly affect the production of energy also affect

the synthesis of small mitoriboscmal subunits [104].



Objectives of this Work



When this work was begun, the presence of mitochondrially

produced proteins in the mitochondrial ribosomes of yeast and









Neurospora had only recently been established. The study of

mitoribosomal proteins in those systems was aided by the ease with

which comparatively large amounts of mitoribosames could be isolated

and by the adaptability of these unicellular organisms for labeling

studies. In contrast, the proteins of mammalian (bovine)

mitoribosonmes had been characterized (unpublished at the time, [84])

but there had been no studies on the biosynthesis of these ribosomes.

The main focus of this work has been on determining which, if

any, of the mammalian (bovine) mitoribosomal proteins are

mitochondrially produced. Because of the presence of var 1 and S-5,

it seemed logical to assume that the manmalian mitoriboscme would

contain at least one protein synthesized intramitochondrially.

Indeed, since the bovine mitoribosome contained so many proteins, it

seemed feasible that there could be more than one mitochondrially

produced protein. Identifying which of the mitochondrial proteins

were mitochondrial products would allow questions concerning the need

for intramitochondrial synthesis to be addressed. Would these

proteins be involved in anchoring the mitoribosome to the membrane,

indicate i ng that they might be too hydrophobic to be imported? Would

they be closely associated with the rRNA, indicating that they might

be required for early assembly? When the mammalian mitochondrial

genome was published in 1981 [34], it added a new aspect to these

studies. Since the predicted sequence of all possible mitochondrial

products became available, if a mitoribosomally produced protein was

identified, it could be assigned to an unidentified reading frame by

sequencing only a short stretch of the primary amino acids. Finally,










the extent to which mammalian mitoribosomes were synthesized

similarly to, or differently from, the ribosomes of lower eukaryotes,

could lead to insights as to the selective constraints involved in

mitochondrial evolution.

This work has examined the site of synthesis of mitoribosromal

proteins by two techniques. The protein synthetic products of

isolated bovine mitochondria incubated in an in vitro system were

analyzed to determine if they were canomigratory by two-dimensional

PAGE analysis with the enumerated proteins of bovine mitoriboscmes.

In a more powerful technique, bovine cells in culture were treated

with specific inhibitors of cytoplasmic and mitochondrial protein

synthesis to study the contribution of each system's products to the

mitoribosomal proteins.

This work also deals briefly with another aspect of

mitoribosomal biosynthesis, the mechanism of mitoribosome assembly.

This problem includes several'separate questions of how the

cytoplasmically synthesized proteins are recognized and imported by

mitochondria, how mitoribosomal proteins are processed, and how the

proteins and rRNA are assembled into a functional particle. In a

brief study, an immune precipitation experiment was used to determine

if soluble pools of unassembled mitoribosomal proteins can be

detected in the cell cytoplasm or mitochondrial matrix fractions of

cells.
















MATERIALS AND METHODS


Materials



Solutions. Solutions cited are defined in table 2.



Reagents. [3 S]methionine, >600 Ci/mmiol, was purchased from

Ammersham. [3,4,5- H]leucine, >11UO Ci/nmmol, was purchased form New

England Nuclear. The inhibitors, chloramphenicol, erythromycin, and

cycloheximide, were obtained from Sigma, as were digitonin and ultra

pure sucrose. All reagents for the growth of cells in culture were

from Gibco. Adjuvants were obtained franom Difco. Formalin-fixed S.

aureus cells were obtained from Bethesda Research Laboratories. All

other reagents were obtained from standard laboratory suppliers.



Tissue culture cells. Madin and Darby bovine kidney (MDBK)

cells [105] were used for all experiments in tissue culture. This is

a "non-transformed," continuous cell line. The initial innoculum of

cells was kindly provided by Dr. Paul Klein. The cells were grown in

Dulbecco's modified Eagle medium with high glucose containing 10%

fetal calf serum, and exhibited an average doubling time of

approximately 20 h.









TABLE 2

SOLUTIONS CITED



Solution Formulation

A 0.25 M sucrose, 1 mM EDTA, 5 mM Tris, pH 7.5

B 0.34 M sucrose, 5 mM Tris, pH 7.5

C 100 mM KC1, 20 mM MgCl2, 10 mM triethanolamine, 5 mM B-mercaptoethanol, pH 7.5

D 200 mM KC1, 2 mM MgC12, 10 mM Tris, 5 nMM 8-mnercaptoethanol, pH 7.5

E 300 nmM KC1, 5 mM MgCl2, 10 mM Tris, 5 mM B-mercaptoethanol, pH 7.5

F 25 mM KC1, 10 mM MgCl2, 10 mM Tris, 5 mM B-mercaptoethanol, pH 7.5

G 8 M urea, 12.5 nM KC1, 5 mM MgCl2, 5 mM Tris, 2.5 nmM 6-mercaptoethanol, pH 7.5

H 0.15 M NaCI, 10 mM Tris, pH 7.5

I 9 M urea, 3 M LidC, 0.001% butylated hydroxy-toluene, pH 3

J 9 M urea, 3 M LiCI, 0.001% butylated hydroxy-toluene, 100 mM B-mercaptoethanol, pH 7

K 9 M urea, 60 mM KAc, 0.01% aminoethanethiol, pH 6.7

L 5 M urea, 2% SDS, 10 MrM sodium phosphate, pH 7.2

M 5 M urea, 2% SDS, 10 mM sodium phosphate, 100 mM 8-mercaptoethanol, pH 7.2









Experiments Using Mitochondria Isolated from Bovine Liver



Preparation of Bovine Liver Mitochondria

Mitochondria were prepared frman livers of freshly killed cows by

two procedures. In the first procedure, 2 kg of ground liver tissue

was mixed with 4 L of buffer A and the cells were lysed using a

Tekmar Super Dispax polytron homogenizer. Cell debris was removed by

centrifuging in a Beckman JA-10 rotor at 3000 rpn (1000 x g) for 10

min and then mitochondria were collected by centrifugation at 8000

rpm (7000 x g) for 10 min. The mitochondria were washed three times

by resuspension and centrifugation (7000 x g for 10 min) in buffer A;

during the second wash, the buffer was supplemented with 0.1 g/L

digitonin and the slurry (20 mg mitochondrial protein/ml) was stirred

for 15 min before centrifuging. (This procedure is standardized for

the laboratory and was performed by the laboratory staff.)

The second procedure was a modification of the first, designed

to reduce bacterial contamination in the mitochondrial preparation.

Sterile solutions and glassware were used throughout this

preparation. A small lobe of fresh liver was bathed in 0C 70%

ethanol before use. Liver tissue (100 g) removed from the center of

this lobe was minced and added to 400 ml of ice cold buffer A. A

Tekmar SDT Tissuemizer polytron homogenizer was used to lyse the

cells (> 90% cell lysis). Cell debris was removed by centrifuging in

a Beckman Type 35 rotor at 2000 rpm (300 x g) for 5 min and then

mitochondria were collected by centrifugation at 8000 rpm (5000 x g)

for 5 min. The mitochondria were washed four times by resuspension










in buffer A and centrifugation (5000 x g for 5 min); as before, the

second wash was supplemented with digitonin and the slurry was

stirred for 15 min before oentrifuging.

The mitochondria from either preparation were diluted before use

to 10 mg mitochondrial protein/ml in buffer A. In order to insure

complete dispersion of the mitochondria, this preparation was

homogenized for 5 complete strokes with a TenBroeck homogenizer. The

mitochondrial solution was oxygenated for 15-30 sec and stored sealed

under oxygen at 0C until use (within 1 h).



Conditions for Mitochondrial Incubation

Mitochondria were incubated at a concentration of 1 mg

mitochondrial protein/ml at 37C. The total incubation volume

varied from 3 to 7 ml per condition, depending on the experiment.

The complete composition of the incubation mixture is shown in table

3. Basically, it is a bicine buffered salt solution supplemented

with sucrose for osmotic stability, an ATP generating system, and all

of the common amino acids. (The amino acid used as the radiolabel

was omitted from the unlabeled amino acid pool.) Inhibitors, when

used, were added at concentrations of 200 pg/ml for chloramphenicol

and 300 pg/ml for erythromycin and cycloheximide. Mitochondria were

warmed and preincubated in the incubation mixture for 5 min before

incorporation was started by the addition of the radiolabel

([3H]leucine at 100 PCi/ml or [35S]methionine at 50 VCi/ml).

The incubations were monitored using the TCA disk assay [106].

Aliquots of 10 pl were absorbed into Whatman 3MM paper disks and









TABLE 3

IN VITRO MITOCHONDRIAL INCUBATION MIXTURE


Canmponent


KC1

MgCl2

EDTA

Sucrose

Bicine, pH 7.6

KH2PO4, pH 7.6

Amino Acid Mixturet

ADP

AMP

a-ketoglutarate


Concentration

50 mM

10 mM

2.8 mM

45 mM

45 nM

15 rM

30 ig/ml

1.5 mM

2.0 nM

10 mM


tAmino Acid Mixture. As defined in Roodyn et al., 1961 [107],
with amino acid to be used for the radiolabel emitted.









precipitated in ice cold 20% TCA. The disks were washed once at 0C

in 10% TCA, once for 20-30 min at 80C in 10% TCA, and again at 0C

in 10% TCA before being dried and counted. The hot TCA wash is used

to hydrolyze amino acids from aminoacylated tRNAs so that the data

reflect only incorporation into protein.

At the end of the incubation period (60-90 min), 1 ml aliquots

for analysis by 2D-PAGE were drawn. The mitochondria were removed

from the incubation solution by centrifugation in a Beckman Microfuge

B (9000 x g) for 5 min. The supernatant was discarded and the

pellets were stored at -70C until used.



Experiments Using Cells Grown in Tissue Culture



Conditions for Incorporation of Radiolabeled Methionine

MDBK cells to be used for labeling were grown on plastic roller

bottles (850 cm2/bottle). Generally, two bottles of cells were

used for each labeling condition (approximately 1-2 x 108 cells per

condition). At the beginning of the labeling procedures, the cells

were between 60% and 80% confluent. The medium was removed from the

cells and replaced with Dulbecco's minimal essential medium with

reduced methionine (10 pM) containing 10% dialyzed calf serum.

After the addition of antibiotics, cells were preincubated to allow

the antibiotics to inhibit protein synthesis, before the addition of

a sterile solution of [35S]methionine in media (10 uM methionine)

to a concentration of 4-8 pCi/ml.









Cycloheximide (300 pg/ml) was used to inhibit cytoplasmic

protein synthesis. The protocol is summarized graphically in figure

3, A. The antibiotic was added as a 1.5 mg/ml, freshly made solution

in the low miethionine medium, with a 15 min preincubation before the

addition of [35S]methionine. Because of the tendency of

mitochondrial protein synthesis to decrease rapidly under these

conditions [52,108], the cells were labeled for only 1-2 h. At the

end of the labeling period, the cells were washed for 15 min in

medium containing cycloheximide and 1 mM unlabeled methionine (to

remove the [35S]methionine) and then with normal medium (200 pM

methionine, no antibiotics). The cells were then grown for 2-4 h to

allow newly synthesized proteins to be incorporated into

mitochondrial ribosomes before harvesting.

Chloramphenicol (100 or 200 pg/ml) was used to inhibit

mitochondrial protein synthesis. The antibiotic was added as a 50

mg/ml solution in 95% ethanol. Since cytoplasmic protein synthesis

is not affected by chloramphenicol, these cells could be grown for

longer periods in the presence of inhibitor and [ 35S]mrethionine.

Two protocols were used (figure 3, B and C). In the first, after a

preincubation of at least 15 min with chloranmphenicol, the cells were

labeled for 8-12 h in the presence of inhibitor and

[35S]methionine. After the incubation the cells were washed in the

presence of inhibitor and of 1 mM unlabeled methionine and grown for

a 2-4 h chase period in normal medium (as above). Alternatively, the

cells were preincubated with chloramphenicol for 2-3 h (to allow for

depletion of any existing mitochondrial products). Then, the cells


























Figure 3. Schematic representation of the labeling protocols for MDBK cells.
Shown as one-dimensional graphs with the axis representing time are the protocols for labeling in
the presence of cycloheximide, A, in the presence of chloramnphenicol with a chase period, B, and in the
presence of chloramphenicol without a chase period, C. Note the change in scale of the axis between
figure A and figures B and C.







p35S]METHIONINE I
I ~CYCLOHEXIMIDE !
ILOW METHIONINE MEDIA
0 2


FZ


HOURS


r35s] METHIONINE
CHLORAMPHENICOL
LOW METHIONINE MEDIA
2 4 6


NORMAL MEDIA
4


CELL HARVEST

I


CELL HARVEST


NORMAL MEDIA
10 12


HOURS


CELL HARVEST


[35S] METHIONINE
CHLORAMPHENICOL
LOW METHIONINE MEDIA


HOURS


I I
2


I 4


I I


CELL HARVEST
I


ff


I '


*


I


- F-









were incubated for 8-12 h in the presence of inhibitor and

[35S]methionine, before they were washed in the presence of

chloramphenicol and harvested (no chase period).

Control cells were grown and labeled using conditions which

paralleled each inhibitor protocol, including mock additions of

antibiotic.



Preparation of Mitochondria from Cultured Cells

Cells were removed franom their bottles with a solution containing

0.02% EDTA and 0.05% trypsin. The cells were washed two times in

buffer A: the first wash at roanom temperature and the second at 4C.

All remaining steps in the ribosane preparation were performed at

0-4C.

The cell pellet was resuspended into 16 ml of diluted buffer B

(buffer B:water, 2:3) containing 120-180 pg/ml digitonin. The cells

were homogenized -using a polytron homogenizer (Teckmar SDT

Tissuemizer) until they were 80-99% lysed. An additional 10 ml of

the digitonin solution was used to rinse the homogenizer and this was

added to the cell lysate. Cell debris was removed by centrifugation

at 1250 x g for 5 min. The supernatant was removed and the debris

pellet was re-extracted with 15 ml of diluted buffer B (buffer

B:water, 1:4). After another centrifugation, the supernatants were

combined and mitochondria were collected by spinning at 9500 x g for

10 min. The post-mitochondrial supernatant (PMS) was stored at

-70C if it was to be used in immune precipitations (see below).

The mitochondria (pellet) were washed in 20 ml of buffer A and then









resuspended into 20 ml of buffer B. A 1.0 ml aliquot of this

suspension was centrifuged in a Beckman Microfuge B (approximately

9000 x g); the supernatant was discarded and the pellet was stored

frozen at -70C until it was prepared for electrophoresis as a

sample of intact mitochondria. The remaining 19 ml was centrifuged

at 9500 x g for 10 min and the pellet was used to prepare

mitochondrial ribosomes (see below).



Isolation of Mitochondrial Ribosomes

Mitochondrial ribosomes were isolated by two techniques. For

experiments where the MDBK cells were labeled for 8-16 h (in the

presence of chloramphenicol and the parallel control) the

mitochondria were resuspended into 1.4-1.7 ml of buffer C. The

mitochondria were lysed by the addition of 200 pl of 16% Triton

X-100. In order to improve recovery of radiolabeled mitoribosomes,

15 A260 units of unlabeled bovine 55 S mitoribosomes (prepared from

bovine liver, see above) were added in enough additional buffer C to

bring the total volume to 2.0 ml. The mitochondrial lysate was spun

for 5 min in a Beckman Microfuge B (approximately 9000 x g) in order

to remove debris. The supernatant was loaded onto 10-30% sucrose

density gradients in buffer C and centrifuged in a Beckman SW28 rotor

to isolate 55 S mitoribosomes (figure 4, A). The fractions

containing ribosomes were pooled and the ribosomes were collected by

centrifugation. (If the soluble proteins at the top of the gradient

were to be used in immune precipitations, they were pooled and stored

at -70C.) The pellets were resuspended into 1-2 ml of either





















'6 2 JOK/
0
(M
c J
4-.
o 1.5

C)

o 1.0


28S
0.5
15 25/ 35 5 5253






Fraction number



Figure 4. Sucrose density gradient profiles of the preparation of
mitochondrial ribosomes and ribosomal subunits.
Mitochondrial ribosomes were isolated from MDBK cells in the
presence of carrier bovine liver mitoribosomes by sedimentation in a
non-dissociating 10 to 30 % sucrose density gradient, A. The fractions
containing 55 S monosomes (cross-hatched) were pooled and the ribosames
were collected by centrifugation. Subunits were prepared by
resuspending the monoscmes into a dissociating buffer (buffer D or E),
and then subunits were isolated in a 10 to 30 % sucrose density
gradient of the corresponding buffer, B. Fractions containing the 28 S
and 39 S subunits (cross-hatched) were separately pooled and the
subunits were collected by centrifugation.









buffer D or E, which dissociate 55 S monosomes into 28 S and 39 S

subunits, and loaded onto 10-30% sucrose density gradient in the

corresponding buffer (figure 4, B). As before, this was centrifuged

in a Beckman SW28 rotor. The fractions containing 28 S and 39 S

mitoribosomal subunits were pooled separately and collected by

centrifugation.

The MDBK cells labeled in the presence of cycloheximide (figure

3, A) and their parallel control had a comparatively short incubation

period. The ribosomes frcm these short incubations were expected to

contain a barely detectable amount of radiolabel. Therefore, the

ribosome isolation protocol was modified in a manner that improves

recovery but produces mitoribosomal subunits containing more

contamination with non-ribosomal components. The mitochondrial

pellet was resuspended and lysed as already described excepting that

buffer E was used instead of buffer C. Thus 28 S and 39 S

mitoribosomal subunits were recovered with only one sucrose density

gradient (in buffer E, equivalent to figure 4, B). The fractions

containing 28 S and 39 S mitoriboscmal subunits were again pooled

separately and centrifuged.

The pellets of 28 S and 39 S mitoribosomal subunits were

resuspended into 60 pl of buffer D and prepared for two-dimensional

PAGE.



Assay for Inhibitor Response

MDBK cells were grown to 60-80% confluence in T150 flasks. The

media was removed from the cells and replaced with Dulbecco's minimal









essential media with reduced methionine (10 pM) containing 10%

dialyzed calf serum. After the addition of antibiotics (see figure

13), cells were preincubated for 15 min to allow the antibiotics to

inhibit protein synthesis, before the addition of a sterile solution

of [35Slmethionine in media (10 pM methionine) to a concentration

of 4-8 VCi/ml. After 1.5-2 h incubation at 37C, the cells were

washed with 1 mM methionine (in the presence of the appropriate

antibiotics) and harvested. The harvested cells were washed with

normal media and then with buffer B. The cells were collected by

centrifugation (9000 x g for 5 min) and the supernatant was

discarded. The cell pellets were sometimes stored at -70C until

they were prepared for one-dimensional electrophoresis.



Inmunologic Methods



Preparation of Antisera

Antisera were prepared in rabbits by injection of ribosomal

subunits in either a standard buffer (buffer F) or a denaturing

buffer (buffer G) designed to disperse the ribosomal protein from the

rRNA. After collecting preinmmune serum from each animal, they were

initially injected in their footpads with 30 A260 units of

ribosomal subunits (in 40-120 il buffer) mixed with an equal volume

of Freund's complete adjuvant. Collection of antisera began 4 weeks

after the injection. Animals were bled no more frequently than 20 ml

every two weeks and they were booster injected as needed with a

mixture identical to the initial preparation except that incomplete










adjuvant was used. The first booster injection was given in the

footpads and, thereafter, injections were given subcutaneously in the

back. Titer was tested in Ouchterlony diffusion tests against

extracted mitoribosamal proteins.



Preparation of Imnune Precipitates

Antigen preparations used in these experiments were a by-product

of the labeling procedures with MDBK cells. One preparation used was

the post-mitochondr'ial supernatant (PMS). This solution, the

supernatant of the lysed cells after the removal of membraneous

organelles, should include the soluble proteins of the cytoplasm.

The other preparation was the top 3 ml of the SW 28 gradient in

buffer D mixed with 3 ml of buffer H. This solution should contain

the soluble proteins of the mitochondrial matrix (MM) and probably

some extracted membrane proteins as well. In each case, the

preparations used were those prepared fram NDBK cells labeled in the

presence of chloramphenicol until cell harvest, because these

preparations were expected to contain the highest specific activity

of unassembled, cytoplasmically synthesized mitoribosomal proteins.

In order to clear any pre-existing precipitates from these solutions,

they were held at 4C for > 12 h, and then centrifuged at 20,000 x g

for 30 min before being used.

For the precipitation step, 1 ml of prepared antigen solution

was mixed with 20 pl of serum (or buffer H, see figure 14) and 1 mg

BSA (in 0.1 ml buffer H). These solutions were incubated with mixing

overnight at 4C. Then 25 mg of pre-washed, formalin-fixed









Staphlococcus aureus cells were added. After incubation with

mixing at 4C for >12 h, the cells were washed four times, twice in

0.5 ml buffer H, once in 0.75 ml buffer H + 0.5% Triton X-100, and

finally in 0.75 ml buffer H. The antibodies and bound proteins were

then eluted with 100 pl of buffer M (see below) and analyzed by

one-dimensional PAGE.



Electrophoretic Methods



Samples were prepared for two-dimensional polyacrylamide gel

electrophoresis (two-dimensional PAGE) by two extraction steps in

buffer I or buffer J. Routinely, 3.5 A260 units of ribosomal

subunits (200 pg of ribosomal protein) ware extracted in 120 P1 of

buffer. This mixture was stirred for 12-16 h at 4C and then the

solubilized protein was collected in the supernatant fraction after

centrifugation at 20,000 x g for 1 h. The pellet was then

re-extracted in an additional 80 pl of buffer for 4-6 h and

centrifuged; the supernatants were combined and dialyzed versus

buffer K before electrophoresis. This procedure was frequently

modified. Ribosomal subunits prepared from tissue culture cells,

with added carrier bovine liver mitoriboscmes, were extracted in a

standard volume (120 pl, first extraction; 80 pl, second

extraction), regardless of the actual A260 measurement (1.5-8

A 260) Whole mitochondria samples were supplemented with 3.5

A260 units of isolated mitoribosomal subunits and extracted in the
standard volume or in a doubled volum. Attempts to improve the
standard volume or in a doubled volume. Attempts to improve the









extraction from the mitochondria included the addition of 0.5% Triton

X-100 or 2 M tetramethylurea to the buffers.

Samples for analysis in one-dimensional SDS-PAGE were

solubilized in buffer L or M (25-200 pl, depending on the sample

volume). The samples were incubated at 60-70C for 1 h before

loading onto the acrylamide gel slab.

The conditions for electrophoresis have been described

previously [84]. Briefly, the first dimension was electrophoresis in

potassium acetate buffer with 9 M urea at pH 4.3, 4.6% acrylamide.

The second dimension was electrophoresis with 0.5% SDS and 5 M urea

in phosphate buffer at pH 7.2, 10% acrylamide. The conditions for

one-dimensional electrophoresis were identical to the second

dimension of the two-dimensional PAGE system. After electrophoresis,

the gels were stained with Cocmassie blue R (0.25% in 50% ethanol).

The pattern of labeled proteins was visualized by fluorography

[109-111].













RESULTS


Experiments with Isolated Bovine Mitochondria



Synthesis of Protein by Isolated Bovine Mitochondria

Mitochondria, isolated from bovine liver, were allowed to

synthesize proteins in an osmotically stabilized, buffered salt

solution containing all of the cannn amino acids, one of which was

radiolabeled, and an ATP-generating system. Synthesis of protein was

followed by assaying incorporation of radiolabeled amino acid into

TCA-precipitable material. In order to exclude incorporation into

aminoacyl tRNA, the TCA precipitates, on paper disks, were washed

once in hot (80 90C) TCA solution, in addition to washes in cold

TCA. This procedure hydrolyzes the aminoacyl bond, leaving the tRNA

precipitated, but no longer charged [107]. Figure 5 shows

incorporation of [3H]leucine by mitochondria isolated by the

standard preparation method. The mitochondria actively incorporate

the amino acid for the duration of the incubation period, 1 h.

Cycloheximide does not affect this incorporation, showing that this

incorporation of amino acids is not fram contaminating cytoplasnic

ribosomes. The slight increase in the presence of this inhibitor

probably represents sampling error. Chloramphenicol eliminates

virtually all of the incorporation, as expected.













CHM
2000- STANDARD


E





S 1000
o
C._




g yI
-CAP


0 30 60

Time (min)


Figure 5. Incorporation of [ H]leucine into protein by isolated
bovine liver mitochondria.
Bovine mitochondria, freshly isolated from bovine liver, were
incubated at a concentration of 1 mg mitochondrial protein/ml with
[ H]leucine in the incubation mixture described in table 3.
Incorporation into protein was followed by hot TCA precipitation of
10 pl aliquots. Incorporation was assayed in the absence of
inhibitors (* ) and in the presence of the inhibitors cycloheximide
( i ) and chloramphenicol ( A ).









Analysis of Radiolabeled Mitochondrial Products by Two-Dimensional
PAGE

An aliquot of the uninhibited mitochondrial incubation mixture

containing 1 mg of mitochondrial protein was prepared for 2D-PAGE.

The sample was divided in half and, to one half, 28 S mitoriboscmal

subunits were added. To the other half, 39 S mitoribosomal subunits

were added. These samples were then prepared for electrophoresis by

the standard method. After electrophoresis, the stained gels showed

the pattern of genuine mitoribosomal proteins in addition to several

prominent mitochondrial proteins. The pattern of an electropherogram

with 28 S mitoribosomal carrier is shown in figure 6, A.

Flourography was used to study the pattern of mitochondrially

synthesized proteins. As expected, both electropherograms produced

the same pattern of radiolabeled spots, shown in figure 6, B.

The pattern of radiolabeled spots from the fluorogram can be

compared to the stained pattern of genuine mitoriboscmal proteins in

order to determine if any of the radioactive

(mitochondrially-produced) proteins are comigratory with, and

possibly therefore identical to, the mitoriboscmal proteins. Figure

6, C and D show the superimposed positions of the radiolabeled

proteins with both the 28 S (figure 6, C) and the 39 S (figure 6, D)

mitoribosomal protein patterns. There is a radiolabeled spot that is

comigratory with protein L44 (protein 44 of the large, 39 S,

ribosomal subunit). In addition, there are radiolabeled spots in the

vicinity of proteins S13, S20, and L20. The pale and indistinct

nature of these proteins [84] makes it impossible to determine if

these spots are actually ccmaigratory. Thus, these four mitoribosomal










A










O .. .






C : 28S D : 39S





004t
-- zo """ r,> o




o *
0 ,? *%44
a, a a0 F
0 0 '44
o o
0 00
000
S %




Figure 6. Synthesis of protein by isolated bovine mitochondria.
Proteins synthesized by isolated bovine mitochondria in an in
vitro incubation system were analyzed in the presence of carrier
proteins from bovine mitoriboscmal subunits by two-dimensional PAGE
and fluorography. The Coomassie blue stained pattern, A, shows the
pattern of the carrier 28 S proteins and several high molecular
weight mitochondrial proteins. In the corresponding fluorogram, B,
the pattern shows several scattered spots of varying intensity. In
the corresponding schematic diagrams, the positions of the
mitochondrial protein synthetic products (cross-hatched spots) are
shown in respect to the positions of the enumerated proteins of the
28 S, C, and 39 S, D, mitoribosncmal subunits (filled circles).









proteins are identified as possible products of mitochondrial protein

synthesis. However, this result must be considered cautiously in

view of its irreproducibility (next paragraph) and of the results

obtained with cells grown in culture (see below).

Two attempts were made to repeat this result. In one, the

mitochondria labeled in the presence of cycloheximide or in the

presence of chloramphenicol (see figure 5) were prepared and

electrophoresed in a manner identical to that used for the

uninhibited sample. The fluorograms which were produced franom either

sample contained one major spot in the high molecular weight region

of the gel, which was comigratory with a non-ribosomal protein. The

fact that this protein labeled in the presence of either

chloramphenicol and cycloheximide indicates that this result may be

artefactual. In the other experiment, mitochondria prepared by the

semi-sterile method were labeled in an identical manner excepting
35
that [ S]methionine was used and erythromycin was added to same of

the samples. As before, the incorporation was followed by a

TCA-precipitation assay and showed that the mitochondria incorporated

the radiolabel throughout the incubation period (1.5 h) and that

erythromycin did not affect this incorporation (data not shown). The

presence of erythromycin demonstrated both that this mixture was not

contaminated with bacteria and also that the mitochondria prepared by

this method were intact. (Mitochondrial ribosomes are sensitive to

erythromycin but the mammalian mitochondrial membrane is not

permeable to this antibiotic [112]). Aliquots of these labeled

mitochondria ware prepared for electrophoresis by a diluted standard








extraction as well as extractions supplemented with 0.5% Triton X-100

or with 2 M tetramethylurea. In all cases, fluorograms of the

two-dimensional separations were blank. One-dimensional PAGE

analysis of the residual pellets from the extractions and of the top

of the first dimension stacker gel indicated that the radiolabeled

protein primarily failed to extract, and, to a lesser extent,

aggregated during preparation for electrophoresis.



Experiments with Cells in Culture



Mitoribosomal Proteins from Cells Labeled in the Absence of
Cytoplasmic Protein Synthesis

A continuous line of bovine cells growing in culture, MDBK

cells, was used to study the biosynthesis of mitoriboscmes in vivo.

In order to inhibit labeling of the products of cytoplasmic protein

synthesis, MDBK cells were labeled in the presence of the

cycloheximide. The protocol is shown schematically in figure 3, A.

In the presence of cycloheximide, only proteins which are products of

mitochondrial protein synthesis should incorporate radioactivity.

During the chase period, in the absence of both radioactivity and the

inhibitor, any components required for assembly of ribosomes can be

synthesized and the particles should assemble, but components made by

the nucleo-cytoplasmic system will not be radiolabeled. The

ribosomal large and small subunits were prepared from the

mitochondria of these cells directly using dissociating conditions

and a single sucrose density gradient. They were analyzed by

two-dimensional electrophoresis and fluorography.








The results are shown in figure 7 together with the results from

a parallel control (cells labeled in the absence of inhibitor). No

spots are observed on fluorograms from either large or small

ribosomal subunits labeled in the presence of cycloheximide (figure

7, A and B). Th i s is in contrast to the subunit preparations from

parallel control cells (figure 7, C and D) which show a great deal of

incorporated radioactivity. The most prominent spots in these

autoradiograms are, in fact, not ribosomal proteins, but are

contaminating mitochondrial proteins of high specific radioactivity

which are included in this preparation because the subunits were

prepared by the single gradient method. The known proteins of the

mitoriboscmre are visible as the background of paler spots.

This experiment was performed twice. In one case a few high

molecular weight proteins are visible in the fluorograms of ribosomal

subunits froman cells labeled in the presence of cycloheximide (figure

8). These spots appear to be mitochondrial contaminants which have

"leaked" through the cycloheximide inhibition. They do not appear to

be mitoribosanal proteins for two reasons. They are quite similar in

the fluorograms from both subunits, but it is known that the proteins

of the two subunits are distinct [73]. Additionally, they correspond

in position to some of the intensely labeled spots of the control

fluorograms.

Thus, conditions of the labeling period were adequate for the

incorporation of [35S]methionine into mitochondrial ribosamal

proteins, but that incorporation is completely blocked by

cycloheximide, an inhibitor of cytoplasnic protein synthesis.












A. o .


K


Figure 7. Fluorograms of mitoribosamal subunits from cells labeled
in the absence of cytoplasmic protein synthesis.
Mitochondrial ribosomal subunits froman cells labeled in the
presence of cycloheximide were analyzed by two-dimensional PAGE and
fluorography. Fluorograms of the small, A, and large, B, subunits
are shown. Ribosomal subunits from cells labeled in parallel in the
absence of inhibitors are shown in C (28 S) and D (39 S).


,. ,.r.-


' W











































Figure 8. Fluorogram of mitoribosomal subunits from cells labeled in
the absence of cytoplasmic protein synthesis showing high molecluar
weight contaminants.









Mitoribosomal Proteins from Cells Labeled in the Absence of
Mitochondrial Protein Synthesis

Chloramphenicol is an inhibitor of mitochondrial (and bacterial)

protein synthesis. The cytoplasmic ribosames of eukaryotic cells are

not affected by this inhibitor. In a converse experiment to the one

described above, MDBK cells were labeled with [35S]methionine in

the presence of chloramphenicol, allowing label to be incorporated

only into those proteins produced by the nucleo-cytoplasnic system

(figure 3, B). As before, proteins made during the chase period (by

the mitochondrial protein synthesis system) should not have any

incorporated radioactivity. Since this inhibitor has a low toxicity

to these cells, the labeling period could be much longer (8-16 h),

producing ribosomes of higher specific activity. This allowed the

ribosomes to be prepared using two sucrose density gradients; the

first to isolate 55 S monosomes, which are then collected and

dissociated into 28 S and 39 S ribosonal subunits. The subunits are

then purified in a second sucrose density gradient. As before, the

ribosomal subunit proteins were separated by two-dimensional PAGE and

analyzed by fluorography.

The results are shown in figure 9. The fluorograms of 28 S and

39 S subunits labeled in the presence of chloramphenicol (figure 9, A

and D) show the expected pattern for mitoriboscmal proteins. Indeed,

the patterns show no significant differences from the fluorograms of

ribosomal subunits from cells labeled in the absence of inhibitor

(figure 9, C and F). In the experiment illustrated, the major

difference is in the small subunit pattern, where the control

fluorogram is weaker than the one produced in the presence of the


























Figure 9. Fluorograms of mitoribosomal subunits from cells labeled in the absence of mitochondrial
protein synthesis, with chase period.
Mitochondrial ribosomal subunits from cells labeled in the presence of chloramphenicol and chased in
normal medium were analyzed by two-dimensional PAGE and fluorography. Fluorograms of the 28 S small, A,
and 39 S large, D, subunits are shown, together with corresponding schematic diagrams showing the
positions and numbers of the mitoribosomal proteins in the fluorograms, B and E. Ribosoanl subunits from
cells labeled in parallel in the absence of inhibitors are shown in C (28 S) and F (39 S). Dotted
circles indicate the position of carrier proteins in the corresponding Coomassie blue stained pattern
(see text).









A
4f4

0 6
90 10

l ie 1.5
160 017

23 .25
27, 09
*9 28 3I 430
3 33% 3









7 5 06 4
81 *-11 *146
281










~46**47
501
~31 25 '238 o.26
-:.' 28_32 035
S37se3e 44
41 42ol 0
43
-, : .46*047
500


p? F








inhibitor. This is at least partially due to reduced recovery of the

uninhibited sample, the stained pattern of the carrier proteins

serving as a gauge of recovery. Other factors may be involved (see

"Discussion"). The accompanying schematic diagrams (figure 9, B and

E) enumerate the proteins in the fluorogram shown which label in the

presence of chloramphenicol. The schematic diagrams show the

location for the more faintly labeled proteins (for example, the low

molecular weight proteins of the large subunit) which are marginally

observable in the photographic prints. (Some of these faint proteins

are more easily observed in figures 10 and 11.) This experiment was

performed three times without ever detecting any proteins which could

be mitoribosomal products, that is, a protein, the labeling of which

is blocked by chloramphenicol.

The patterns of mitoriboscmal proteins in these fluorograms are

virtually the same as the stained pattern characteristically formed

by the carrier bovine liver mitoribosomal proteins revealed by

Coomassie blue stain, differing only in a few proteins which failed

to label in both the inhibited and control ribosomes. These proteins

include S26, L13, L24, L33, L34, L39 and L40. (An additional group

of proteins, S12, S13, S25, L27, and L45, failed to incorporate

radiolabel in both the inhibited and control ribosames of most

samples, but gave an ambiguous result in at least one experiment.

That is, in one case either both the inhibited and the control

ribosomes, or only the inhibited riboscmes appeared to be labeled.)

Proteins which never showed incorporation of radiolabeled methionine,

probably do not contain that amino acid. They must, therefore, be









products of cytoplasmic protein synthesis, because with the known

genome of bovine mitochondria, it is predicted that all mitoribosamal

products contain methionine (see table 1).

In some cases, for example, proteins Sl and S4 in the experiment

shown, the radiolabeled spot was not ccmigratory with the stained

spot on the electropherogram. In figure 9, B, the area of the

stained spot is shown by the dotted line, while the spot on the

fluorogram is shown as a filled spot. This change in mobility may

indicate that the radiolabeled protein is a contaminant which is

comigratory with the mitoribosomal protein. Another possible

explanation is that the mitoriboscmnal protein of the MDBK cells is

slightly modified from that of the bovine liver carrier protein, such

that it occupies a similar, but not identical, place in the

electrophoretic separation. This labeling pattern was reproducible

for Sl, but S4 showed comigratory label and stain in all other

experiments.

A second protocol for labeling cells in the presence of

chloramphenicol was designed to test a prediction. That is, if all

of the mitoriboscmal proteins are cytoplasnically synthesized, then

it should be possible to completely synthesize and assemble

mitoribosomes in the absence of mitochondrial protein synthesis.

This protocol is shown schematically in figure 3, C. This protocol

differs froman the one already described in two respects. First, after

the chloramphenicol is added, the cells are preincubated for 2 h to

allow any pre-existing mitochondrial protein synthetic products to be








utilized. Secondly, there is no chase period; chloramphenicol is

kept in the solutions until the end of the harvesting procedure.

The results of this protocol for proteins franom 28 S subunits are

shown in figure 10. Two data sets for this experiment are

illustrated, with 28 S subunits labeled in the presence of

chloramphenicol shown in A and C, and the respective parallel

controls shown in B and D. The two experiments are illustrated to

demonstrate the variability of the results with this protocol. In

fact, this experiment was repeated three times without ever producing

a fluorogram of the quality shown in figure 9, A. However, in all

cases, the experimental pattern was predominantly comparable to that

produced from the control cells and the "flaws" were not

reproducible. For example, in the experiment illustrated in A and B,

the experimental fluorogram is less intense than its control. This

could be due to reduced overall recovery of the subunits during the

centrifugation steps, but other factors may be involved (see

"Discussion"). Additionally, protein S14, seems surprisingly weak in

the control fluorogram (indicated in figure 10, A and B). In the

experiment illustrated in C and D, the fluorograms, both inhibited

and control, are of equal intensity but show several highly labeled

contaminating proteins. These contaminants include one protein which

is apparently a mitochondrial protein synthetic product (indicated by

the large arrowhead) since it is more easily observed in the control

fluorograms. Based on its molecular weight (approximately 19,000

daltons) it may be a product of URF 6. However, several factors are

inconsistent with its being a mitoriboscmal protein: 1) it is in a





























- .*.*- ** D


Figure 10. Fluorograms of 28 S mitoriboscmnal subunits from cells
labeled in the absence of mitochondrial protein synthesis, no chase
period.
The results for 28 S mitoribosomal subunits from two experiments
with MDBK cells labeled and harvested in the presence of
chloramphenicol are shown, A and C, together with their corresponding
parallel controls, B and D. Note the variation in the two results,
largely due to contamination in the second experiment (C and D). In
A and B, a change in protein S14 is indicated. In the second
experiment, one of the contaminating proteins (large arrowhead)
appears to be a mitochondrial product since it is more easily
obsevred in the control fluorogram. Another protein (*) curiously
appears only in the presence of chloramphenicol.








region of the gel where no mitoribosomal proteins have ever been

observed, 2) it appears in both subunits (although more prominently

in the contaminated 28 S fluorograms), and 3) it appeared only in

this experiment and not in the other two. The other protein

(indicated by an asterisk), which surprisingly appears only in the

mitoribosomal subunits from cells labeled in the presence of

chloramphenicol, also appears in both subunits (see below) but only

in this experiment.

In figure 11 are shown proteins froman 39 S subunits (froman the

same experiment as the 28 S subunits shown in figure 10, C and D,

above) labeled using this protocol in the presence of chloramphenicol

(A) and their parallel control (B). As predicted, the fluorograms

look very similar whether produced with mitoriboscmes from cells

grown in the presence of chloramphenicol or from the parallel

controls. In fact, the most obvious difference in the experiment

illustrated is the presence of the protein (indicated by an asterisk)

which appeared only in these fluorograms of mitoriboscmal proteins

labeled in the presence of chloramphenicol. More importantly, the

labeling of all of the mitoriboscmal proteins was not inhibited by

chloramphenicol in any of the three trials of this experiment.

The results of all of the labeling of mitoriboscmes in the

presence of chloramphenicol are summarized in table 4. Most of the

enumerated proteins of the bovine mitoriboscme were consistently

labeled in all conditions (the 3/3/4 result). An additional group of

proteins (e.g. S2, S3, or S9) do not always appear on the

electropherograms, as judged by the Coomassie blue stain pattern of













A B


















Figure 11. Fluorograms of 39 S mitoribosonal subunits from cells
labeled in the absence of mitochondrial protein synthesis, no chase
period.
4 P






Figure ii. Fluorograms of 39 S mnitoribosomal subunits from cells
labeled in the absence of mitochondrial protein synthesis, no chase
period.
The results for 39 S mitoriboscnal subunits frman MDBK cells
labeled and harvested in the presence of chloramphenicol are shown,
A, together with the corresponding parallel control, B. Note the
presence of a protein (*) which only labeled in the presence of the
inhibitor.








the carrier, but, when present, were always labeled. As mentioned

above, some proteins were never labeled (the 0/0/0 result produced by

S26, LI3, L24, L33, L34, L39 and L40) indicating that they probably

contain no methionine. Another group of proteins (S12, S13, S25,

L27, and L45), was not labeled in most experiments, but seemed

comigratory or nearly ccmigratory with a radioactive spot in at least

one experiment. These proteins might not contain methionine, but the

results make this conclusion suspect. Importantly, when they were

labeled, these proteins labeled in the pattern expected for products

of cytoplasmic protein synthesis. Some of the proteins are difficult

to resolve in the electropherograms; for those experiments where the

identification is questionable, this is indicated in the table by the

second (decimal) position. A good example of this type of ambiguity

is protein S4. As already discussed, in the experiment shown in

figure 9, stained, carrier S4 was easily observed and there is a

radiolabeled spot which migrates at the rim of the stained spot in

both the inhibited and control conditions. However, in other

experiments, radiolabeled S4 was comigratory with its stained

carrier. Thus, it is listed as "2.1," clearly observed in two

experiments and ambiguously identified in one. Also, the cracked gel

shown in figure 10, B, made S4 impossible to identify in that control

experiment, so only three out of the four possible control conditions

are reported. Likewise, it was too pale to identify in one of the

+CAP/-chase conditions so that column is a "2" instead of a "3." In

contrast, the radiolabeled spot nearest carrier protein Sl was always

below and to the left of its expected position, as shown in












TABLE 4

SYNTHESIS OF DEFINED MITORIBOSOMAL PROTEINS
IN THE PRESENCE OF CHLORAMPHENICOL

Results are reported for each defined mitoribosomal protein (as in
ref. [84]) as the nmber of separate experiments in which the
incorporation of [ S]methionine was detected. Proteins were
identified by exact comigration of the spots revealed by fluorography
( S labeled MDBK cells) and by Coomassie blue stain (bovine liver
carrier mitoribosomal proteins). Where two numbers are given (A.B)
the first number represents detection of spots which were exactly
comigratory and the second number represents spots that were in
approximately the correct area of the gel but were not clearly
comigratory with the stained spot. "N.D." (no data), indicates the
protein failed to appear on all fluorograms and in all stained
electropherograms of that condition. The prefixes "S" and "L" refer
to the small and large mitoribosomal subunits.

Protein +CAP +CAP No
Number +chase -chase Inhibitor

Sl 0.3 0.3 0.3

52 3 2 2

53 3 1 2

S4 2.1 2 2.1

55 3 2 2.1

S6 3 3 4

S7 2.1 2.1 3.1

58 3 3 4

59 1 1 2

510 3 3 3.1

Sll 1.1 0.1 2.1

S12 1.1 0 0.1

S13 0.1 0.1 0.1


3 3


S14







Table 4 continued


Protein +CAP +CAP No
Number +chase -chase Inhibitor

S15 3 3 4

S16 3 3 4

S17 0.2 0.2 0.2

S18 2 1 2

S19 0.3 0.2 0.1

S20 1.1 0.1 2

S21 3 2.1 4

S22 1.2 3 3

S23 3 3 4

S24 N.D. N.D. N.D.

S25 1.1 0 0

S26 0 0 0

S27 2 1.1 2

S28 3 3 4

S29 3 3 4

S30 3 2.1 3

S31 3 3 4

S32 2 N.D. 2

S33 3 3 4



L1 3 3 4

L2 3 3 4

L3 3 3 4

L4 3 3 4








Table 4 continued


Protein +CAP +CAP No
Number +chase -chase Inhibitor

L5 3 3 4

L6 3 3 4

L7 3 3 4

L8 3 3 4

L9 2 3 3

L10 3 3 4

LII 3 3 4

L12 3 3 4

L13 0 0 0

L14 3 3 4

L15 3 3 4

L16 3 3 4

L17 3 3 4

L18 3 3 4

L19 3 3 4

L20 3 3 4

L21 3 3 4

L22 3 3 4

L23 3 3 4

L24 0 0 0

L25 2.1 3 3.1

L26 3 3 4

L27 0 0 0.1

L28 3 3 4








Table 4 continued


Protein +CAP +CAP No
Number +chase -chase Inhibitor

L29 2 3 4

L30 2 3 4

L31 3 3 4

L32 3 3 4

L33 0 0 0

L34 0 0 0

L35 3 3 4

L36 N.D. N.D. N.D.

L37 3 3 4

L38 3 3 4

L39 0 0 0

L40 0 0 0

L41 3 3 4

L42 3 3 4

L43 3 3 4

L44 3 3 4

L45 0 0.1 0.1

L46 3 3 4

AL47 3 3 4

L48 2 2 4

L49 N.D. 3 4

L50 2 3 3.1

L51 N.D. 3 4

L52 N.D. 3 4










figure 9, B. However, it was observed in all of the experiments

except the cracked gel (figure 10, B) and, therefore, was scored as

0.3/0.3/0.3. This type of analysis gave results consistent with a

cytoplasmic site of synthesis for each of the enumerated proteins of

the mammalian mitochondrial ribosome.



Effect of Inhibitors

The interpretation of the ribosomal pattern is determined by the

effect of the inhibitor under each condition. In order to

demonstrate that the inhibitors produce the expected results, it is

necessary to show both that mitochondrial protein synthesis continued

in the presence of cycloheximide and that mitochondrial protein

synthesis ceased completely in the presence of chloramphenicol.

Aliquots of intact mitochondria franom the labeled cells (see

above) were analyzed by one-dimensional PAGE to show mitochondrial

protein synthetic products in cells labeled in the presence of

cycloheximide. In figure 12, lane A shows mitochondria from cells

labeled in the presence of cycloheximide. The expected limited band

pattern of mitochondrial products is produced from cells labeled in

the presence of cycloheximide. Because this system (modified from

Leister and Dawid, 1974 [113], see "Methods") has not been charac-

terized, specific assignment of known mitochondrial products to the

bands is not possible. This demonstrates that cytoplasmic protein

synthesis was eliminated by the cycloheximide but that mitochondrial

incorporation continued. In a comparatively short exposure, the

mitochondria from cells labeled in the presence of chloramphenicol









A B


U
'% M
,I= M


Figure 12. Total mitochondrial incorporation in the presence and
absence of inhibitors.
A sample of mitochondria from each labeling condition was
analyzed by one-dimensional SDS-PAGE and fluorography. Mitochondria
from cells labeled in the presence of cycloheximide, A, exposed for
70 days. Mitochondria from cells labeled in the presence of
chloramphenicol, B, and their parallel control, C, exposed for 18 h.








display a band pattern identical to their parallel control (figure

12, B and C). The pattern of these control cells shows a saturating

pattern of total cellular protein synthesis. Because of the

overwhelming pattern produced by cytoplasmic protein synthesis, the

absence of mitochondrial products is not observable in lane B.

The mitochondrial protein synthetic products are only observable

with one-dimensional PAGE in the absence of cytoplasmic protein

synthesis (in cells also labeled with cycloheximide). Therefore, the

effect of chloramphenicol could not be observed in the cells prepared

for the isolation of mitochondrial ribosomes. The effect of

chloramphenicol was studied using cells grown in T150 flasks and

labeled under a variety of inhibitor conditions. After a 2 h

labeling period with [35S]nmethionine, the cells were harvested and

analyzed by one-dimensional SDS-PAGE. The results are presented in

figure 13. (Because each lane is the total SDS-extractable material

from the cells of one T150 flask, the lanes are overloaded and the

band pattern is somewhat distorted.) Cells labeled in the absence of

inhibitors or in the presence of chloramphenicol show a complex,

heavily labeled pattern (lanes A and B, respectively). In the

presence of only cycloheximide, this pattern is eliminated and a

limited band pattern, the mitochondrial protein synthetic products,

is observed (lane C). This pattern is completely eliminated by the

additional presence of chloramphenicol at either 100 (lane E) or 200

(lane D) pg/ml. At very long exposures, the one-dimensional PA3E

separations from cells labeled in the presence of both inhibitors

show a complex band pattern (lanes F and G). This pattern is









A BC D E


FG


! "


b


Figure 13. Effect of inhibitors on MDBK cells.
MDBK cells were labeled with [ Simethionine in the presence
or absence of the inhibitors cycloheximide and chloramphenicol. The
total cellular material was analyzed by one-dimensional SDS-PAGE and
fluorography. A, no inhibitor; B, 200 pg/ml chloramphenicol; C,
300 pg/ml cycloheximide; D and F, 300 pg/ml cycloheximide and
200 pg/ml chloramphenicol; E and G, 300 pg/ml cycloheximide and
100 pg/ml chloramphenicol. Fluorograms were exposed for 17 h (A
and B), 4 days (C-E), and 14 days (F and G).








similar to a very short exposure of the products of uninhibited

cells, demonstrating that the cycloheximide inhibition is not

complete. However, there is no indication of the limited band

pattern of mitochondrial protein synthetic products at either

concentration of chloramphenicol, even at this long exposure.



Precursors of Mitoriboscmal Proteins



Antibodies raised against the proteins of the large (39 S)

mitoribosomal subunit were used in an attempt to detect unassembled

ribosomal proteins in the soluble fractions of the MDBK cells. TWo

cell fractions were tested, the post-mitochondrial supernatant (PMS)

which should contain the soluble proteins of the cytoplasm, and the

soluble fraction froman the mitochondrial lysate, which should contain

the soluble proteins of the mitochondrial matrix (MM) as well as the

Triton X-100 soluble proteins of the mitochondrial membranes. Each

fraction was immunoprecipitated with two different preparations of

antiserum, as well as with the corresponding preimmune sera and with

buffered saline solution (buffer H). The imnuine precipitates were

analyzed by one-dimensional SDS-PAGE and fluorography.

The results are presented in figure 14. Because of non-specific

precipitation and trapping, there are radiolabeled proteins

observable in all of the fractions. Of interest however, are those

proteins (see arrowheads in figure 14) which appear only in the

immune sera precipitations, suggesting that they are specifically

mitoribosomal proteins. Some of these proteins were










A B


C D


EF G H


Figure 14. Immune precipitation of mitoribosomal proteins from
soluble cell fractions.
Cell fractions isolated from cells labeled with
[35S]methionine in the presence of chloramphenicol were
precipitated with antisera against the proteins of 39 S mitoriboscmal
subunits and analyzed by one-dimensional SDS-PAGE and fluorography.
A-D, post-mitochondrial supernatant fraction; E-H, mitochondrial
matrix fraction. In A and E, the antigen was analyzed directly; in
the other lanes the antigen was first treated with buffer H (B and
F), pre-imnmune serum (C and G), and inine serum (D and H), and then
with fixed S. aureus cells. The inmune precipitates were analyzed
by one-dimensional SDS-PAGE and fluorography. The arrowheads
indicate the positions of bands specifically precipitated by the
immune serum.









inunmmunoprecipitated from each of the cell fractions, indicating that

there is an unassembled pool of at least same mitoribosomal proteins

in the cytoplasm and in the mitochondria. Because digitonin was used

during the cell lysis, the internal contents of some of the

membraneous organelles (Golgi, endoplasmic reticulum, etc.) may have

been released at that step, so it cannot be positively concluded that

the cytoplasmic proteins are free of proteins from other

non-mitochondrial organelles.

Theoretically, this type of analysis should also be able to

indicate the possibility of transit peptide cleavage, that is,

proteins which are immmunoprecipitated from both the

post-mitochondrial supernatant and the mitochondrial matrix

fractions, but which appear to be of lower molecular weight when

isolated from the latter. This experiment did not indicate that type

of change. Two factors may be involved; 1) the antisera used were

raised against total 39 S ribosomal subunits and their broad

recognition spectrum did not allow a specific protein to be

distinguished so that its molecular weight in the two compartments

could be directly compared, and 2) the patterns of non-specific

precipitation are so complex that they could obscure that type of

band position shift.















DISCUSSION


The mitochondrion participates in its own biosynthesis. It

contains within it a complete protein synthetic system, including

ribosomes, mRNA, tRNA, and the necessary accessory proteins. The

intraorganellar location of the mitochondrial ribosome raises several

questions about how its components are synthesized and assembled.

Based upon what is known about the synthesis of other mitochondrial

proteins and of mitoribosomes in yeast and Neurospora, it can be

envisioned that most or all of the mitoribosomal proteins are

synthesized individually in the cytoplasn and imported into the

mitochondrion where they begin a specific, ordered assembly onto an

incompletely cleaved transcript of the complete mitochondrial rDN.

region. Specific proteins assembling onto the transcript might cause

it to assume secondary or tertiary structures which signal for

cleavage of the rRNA or for assembly of still more of the

mitoriboscmal proteins. This process of structural rearrangement and

cleavage of the rRNA and of addition of mitoribosomal proteins

continues until the riboscme is completed.

Some of the 85 proteins of the mammalian mitoribosome

undoubtedly serve in a functional capacity during protein synthesis,

but others might serve a purely structural function, either creating

a structural assembly site for rRNA or protein during mitoribosomal








synthesis, or perhaps adding necessary size to the organelle in place

of some of the rRNA seen in other classes of ribosomes. The current

data on the mitochondrially produced proteins of the yeast and

Neurospora mitoribosones indicate that they may serve as a rRNA or

protein assembly site during the synthesis of the small subunit. If

so, this leads to questions as to whether this function is obligatory

and if intramitochondrial synthesis is absolutely required for a

protein serving that function.

This work has addressed questions of the synthesis of

mitochondrial ribosomes in nammalian systems, using the bovine system

as a model. The major concentration has been on determining whether

or not any of the mitoribosomal proteins are products of

mitochondrial protein synthesis. Additionally, one experiment has

looked at the presence of soluble pools of mitoriboscmal proteins

both intra- and extramitochondrially.



Protein Synthesis in Isolated Mitochondria



One approach used was to examine the protein synthetic products

of isolated bovine mitochondria. The radiolabeled proteins

synthesized by intact mitochondria isolated from bovine liver were

analyzed by two-dimensional PAGE and compared to a pattern of

authentic bovine mitoribosomal proteins added as visible carrier.

Mitoribosomal proteins which are comigratory with radiolabeled

proteins are identified as possible mitochondrial protein synthetic

products. One radioactive pattern was produced by this technique,










and its spots included one which was ccmigratory with mitoribosomal

protein AL44 and spots which migrated in the vicinity of proteins S13,

S20 and L20.

The advantage of this technique is that it studies the

mitochondrial protein synthesis system independently of the rest of

the cell. If any cytoplasmic (80 S) ribosomes are present (as trace

contamination) they cannot incorporate the radioactive amino acids

because they lack an energy source and the necessary soluble

components (e.g. charged tRNA's). If growing bacteria are present in

the mixture, however, they can incorporate the radiolabel so care

must be taken to avoid introducing bacteria during the preparation of

the mitochondria, or inhibitors can be used which differentiate

bacterial protein synthesis.

In theory, the major limitation of this system is that it has no

specific selection criterion for mitochondrial ribosomal proteins.

This in vitro system does not process or assemble proteins into

ribosomal particles, so the analysis includes all radiolabled

products extracted from intact mitochondria. The fluorogram reveals

a pattern of several scattered spots and there is no method for

determining coincidental comigration. Furthermore, since not all of

the authentic mitoribosomal proteins are always clearly resolved

[84], some of the radiolabeled spots are in an area where comigration

cannot be clearly delineated. Additionally, the presence of so many

spots on the electropherogram indicates that not all of the spots are

simple translation products of the mtDNA reading frames. One

possibility is that some of the spots could represent modified forms









of these proteins. It is not known whether or not isolated

mitochondria process proteins exactly like their in vivo

counterparts. While there have been some reports of modifications to

mitochondrial protein synthetic products [114-116], the identified

proteins produced by mammalian mitochondria correspond to their

reading frames [59-67,117,118].

In practice, the most disconcerting aspect of this approach was

its irreproducibility. Two subsequent attempts to reproduce the

first result failed, apparently because the radiolabeled proteins

were not extracted from the mitochondria. Interestingly, the 9 M

urea extraction removes virtually all mitoribosanal proteins from the

isolated ribosomal particles. If a mitoriboscmal protein is one of

the mitochondrial products, its extraction is probably blocked by the

presence of mitochondrial membranes. In general, systems for the

electrophoretic analysis of the mitochondrial protein synthetic

products rely on detergents for solubilization. However, the

electrophoresis system used in these experiments which characterizes

the mitoribosomal proteins is not compatible with detergents, so

their use was avoided. In one case, where Triton X-100 was added to

the extraction mixture, the extraction of these proteins appears to

have been improved, but when the detergent was removed during the

first electrophoretic dimension, the proteins aggregated. These

results suggest that the original "successful" experiment, might not

be a separation of ribosamal product proteins. One possibility is

that this radioactivity was incorporated into easily extracted

proteins of contaminating bacteria. Thus, this technique identifies








mitoribosomal proteins which might be products of mitochondrial

protein synthesis, but it cannot be conclusive.



Protein Synthesis in Cultured Bovine Cells



By using specific inhibitors, mitochondrial and cytoplasmic

protein synthesis were "isolated" and studied in a complete living

system, bovine tissue culture cells (MDBK cells). By studies in the

MDBK cells it was shown that the proteins of mammalian mitoribosomes

label in a manner expected for products of cytoplasmic protein

synthesis. That is, they fail to label in the presence of

cycloheximide and do incorporate label in the presence of

chloramphenicol. Furthermore, correctly sedimenting ribosomal

subunits were synthesized completely in the presence of

chloramphenicol, and the analysis of their proteins indicated normal

assembly of mitoribosomes even in the absence of mitochondrial

protein synthesis.

This experimental system offers several advantages. Firstly,

the tissue culture cells provide a complete system for studying

mitochondrial biosynthesis. Both cytoplasmic and mitochondrial

protein synthesis can be examined. Although not included in this

study, it is of course possible to manipulate other aspects of

mitoribosame biosynthesis (e.g. rRNA synthesis or protein import), as

well.

Secondly, there is a built in selection criterion for

mitoribosomal proteins. In other systems, where one or a few








specific proteins have been studied, the analysis was performed by

solubilizing the mitochondria with detergents and then precipitating

the protein of interest with a specific antibody [119]. For the

mitoribosomal proteins, however, a complete spectrum of

characterized, specific antibodies does not exist. Therefore

selection for mitoriboscmal proteins was done by allowing the cells

sufficient time, either in the absence or presence of inhibitors, to

assemble proteins into mitoriboscmal particles. Then, the ribosomal

subunits could be prepared in sucrose gradients and their protein

composition analyzed. The analysis is aided by the use of bovine

cells, as opposed to some other species, because the electrophoretic

pattern of the enumerated mitoribosomal proteins is well

characterized [84].

Finally, bovine liver mitoribosomes are easily obtained for

protein carrier. When working with the small amounts of

mitoribosomal protein from tissue culture cells, these carrier

mitoriboscmes serve several uses. Added at the time of mitochondrial

lysis, after the riboscmes are labeled and assembled, they add needed

bulk to the material being isolated. This not only makes the

manipulations easier, but is particularly important in reducing

adsorptive and handling losses. During the purification of

ribosomes, it is the A260 measurement of the carrier which is

followed and used to judge fractions to be pooled and to judge total

recovery. Thus, by definition, mitoribosomal proteins are those

which are associated with the correct gradient positions for intact,

fully assembled mitoribosomal subunits. Precursor particles, which









if present would run at a higher position in the gradient, would not

be detected by this method. During the electrophoretic analysis, the

carrier pattern shows the correct position for the ribosomal

proteins, and exact comigration can be used to identify mitoribosomal

proteins and distinguish contamination.

In analyzing these results, several factors must be considered.

The blank fluorograms which resulted with the inhibitor

cycloheximide would also have been produced if the conditions for the

labeling period (media, length of incubation, specific activity of

radiotracer, etc.) or for recovery and analysis of ribosomnal subunits

(mitochondrial isolation, length of fluorography, etc.) were

inadequate to visualize synthesis into mitoribosomal proteins. A

parallel control experiment, however, where all such factors were the

same except that cycloheximide %s emitted, demonstrated that the

synthesis of mitoribosomal proteins was observable under these

conditions. Additionally, it is known that mitochondrial protein

synthesis is sensitive to the inhibition of cytoplasmic protein

synthesis, that during prolonged incubations in the absence of

cytoplasmic protein synthetic products, mitochondrial protein

synthesis becomes inhibited and eventually mitochondrial products are

degraded. In order to insure that the cycloheximide labeling

condition was not excessive, a sample of intact mitochondria from

these cells was examined. It showed the expected limited number of

mitochondrially synthesized protein bands in one-dimensional

SDS-PAGE, demonstrating that the products of mitochondrial protein

synthesis were translated during the incubation period and that they









were not degraded during the labeling period, but, in fact, they were

intact and could be assembled during the chase period. Additionally

the electrophoretic band pattern of proteins produced by mitochondria

from cells labeled in the presence of cycloheximide (and then chased

and used for the preparation of mitoriboscmes, figure 12, A) is

roughly similar to that of total protein synthesized by cells labeled

under the same conditions to test the inhibitor effectiveness (no

chase period, figure 13, C). However, the possibility exists that a

mitoribosomal protein might be a minor product of mitochondrial

protein synthesis which is not discernable by one-dimensional

SDS-PAGE analysis. Additionally, sequestration inside the

mitochondrial membrane might give added protection from proteolysis

to electron transport proteins, which would not necessarily be true

for a riboscmal protein. Since the effect of these factors cannot be

evaluated, the blank fluorograms are not conclusive data.

Another important factor is the effectiveness of chloramphenicol

inhibition of mitochondrial protein synthesis in these cells. In

these experiments, the analysis examines only those proteins which

assemble into properly sedimenting mitoriboscmes. Because of the

small amount of material available and the addition of carrier liver

mitoribosomes, the results would not distinguish between unaffected

incorporation and partial incorporation caused by incomplete

chloramphenicol inhibition. Figure 13 demonstrates that

chloramphenicol is very effective in MDBK cells, and reduces

mitochondrial protein synthesis below detectable levels even at 100

ig/ml. One problem with this control experiment is that









chloramphenicol inhibition could not be tested directly with the

cells used to produced the riboscmes shown in figures 9, 10, and 11,

but had to be tested in a separate set of cells grown specifically

for that purpose. However, all indications are that the

chloramphenicol inhibition in the MDBK cells was complete.

All of these experiments were performed using [3 S]methionine

as the radioactive amino acid. This analysis is limited to detection

by 35S (either methionine or cysteine) because only this isotope

can be used to synthesize amino acids of adequate specific activity

for detection by these methods. Fortunately, since the amino acid

sequence of all possible mitochondrial products can be predicted from

the known DNA sequence (see table 1), it appears that all

mitochondrial protein synthetic products contain a high proportion of

methionine residues. Additionally, the methionine residues of these

predicted polypeptides are distributed throughout the sequences,

making it unlikely that all of them would be removed by any

hypothetical processing of the transcript or of the protein. Indeed,

those proteins (e.g. L13) missing on fluorograms of mitoribosomes

from cells labeled in both the presence and absence of

chloramphenicol probably simply lack methionine residues. These

unlabeled proteins, therefore, cannot be mitochondrial products.

Finally, this analysis is based upon the ability to detect all

of the mitoribosomal proteins by a specific system of two-dimensional

PAGE. Yet a few of the mitoribosomal proteins are variable in their

appearance in this system of analysis. For example, protein L36 was

never detected on any of the electropherograms or fluorograms from









these experiments. Additionally, the possibility must be considered

that there are ribosomal proteins that are never detected in the

two-dimensional PAGE analysis. A protein would be consistently

overlooked in this analysis if it was comigratory with another

protein (one "spot" which is actually two proteins) or if it was not

solubilized by the extraction conditions. By analogy to lower

eukaryotes, these data also address the possibility that a

mitochondrially produced mitoribosonal protein might be missed in

this analysis because it was not displayed in these two-dimensional

PAGE separations. It is known that in yeast and Neurospora, the

small ribosomal subunits formed in the absence of mitochondrial

protein synthesis are defective. They migrate incorrectly in

sedimentation analysis, and they specifically lack a few proteins

which assemble after the mitochondrial product. In our bovine

system, the subunits examined were those which migrated to the

position of assembled subunits in sucrose density gradients. In the

protocol shown in figure 3, B (cells labeled in the presence of

chloramphenicol and then chased) if chloramphenicol blocks

mitoribosome synthesis at a precursor stage, then the particles made

during the labeling in the presence of chloramphenicol would have

been completed during the chase period. Proteins added during the

chase period should not be labeled, though. So the particles

recovered from the gradients would be deficiently labeled in those

proteins which were added after any mitochondrial product.

Therefore, it is important to note that the subunits produced by this










experiment (figure 9) produce the same pattern of spot intensities as

their parallel control.

However, that experiment does not address the possibility that

proteins made cytoplasmically during the labeling period, might still

be available for assembly during the chase period. This situation

could occur either for proteins which required several minutes for

import into the mitochondria, or if unassembled proteins inside the

organelle are not quickly degraded. If such proteins are retained

into the chase period, then, when the inhibition is released, a

mitochondrially synthesized protein could be produced and assembly

could continue. These scenarios are addressed by the second protocol

for labeling in the presence of chloramphenicol (no chase period,

figure 3, C). As seen in figures 10 and 11, mitoribosomes from cells

labeled and assembled in the presence of chloramphenicol show the

same patterns as their uninhibited counterparts. This indicates that

the complete absence of detectable mitochondrial protein synthesis

did not block synthesis or assembly of both subunits of the

mitoribosome. If mammalian mitoribosomes contained a protein

analogous to either S-5 in Neurospora or var 1 in yeast, this

experiment should have produced a distorted or deficient pattern.

These data do not completely rule out the possibility of

"hidden" mitoribosomal proteins, proteins which were not detected

either because they are not present in the electropherogram or

because they are not distinguished from another similarly migrating

protein. However, if such a mitoriboscmal protein does exist, it

does not affect the ability of any other protein to assemble onto the










mitoribosome, and, therefore, is functioning in some manner which

differs from the behavior of the protein of the mitoribosomes in

lower eukaryotes.

Continuously disconcerting aspects of these experiments were the

problems in producing good quality fluorograms, especially of the

small ribosomal subunit. Problems were anticipated in incorporating

sufficient radioactivity and in obtaining adequate recovery of the

subunits (see above). However, some experiments produced excellent

fluorograms of the subunits (e.g. figure 9). Several of the small

subunit fluorograms were also damaged by differing degrees of

contamination. This latter factor probably indicates that the

preparation method for obtaining mitoriboscmes franom tissue culture

cells is not optimized. However, this contamination is independent

of mitoribosome synthesis, since, when it occurred, it was present

equally in both the experimental and control fluorograms.

In general, the small ribosomal subunits produced weak

fluorograms. The two exceptions were fluorograms of subunits froman

cells labeled in the presence of chloramphenicol and then given a

chase period in normal media. These fluorograms were significantly

better than those of small subunits either frman cells labeled

continuously in the presence of chloramphenicol or from control

cells. Two factors may be involved. Firstly, it has frequently been

reported that there is a burst of mitochondrial synthetic activity in

cells being released from chloramphenicol inhibition. This increased

level of synthesis may maximize assembly of mitoriboscmes in cells

labeled by this protocol. Secondly, Lambowitz's group has noted that









especially the synthesis of the small mitoribosomal subunit in

Neurospora, more so than the other mitochondrially produced

proteins, is sensitive to a mutation which causes a defect in the

F ATPase [104]. That is, weak fluorograms produced from cells

labeled continuously in the presence of chloramphenicol may reflect a

repression of synthesis which is not directly caused by inhibition of

the synthesis of a mitoribosomal protein.

In summary, the experiments in cultured bovine cells provide the

most complete analysis of the site of synthesis of the proteins of

the mammalian mitochondrial ribosome. All of the experiments in this

system indicate that in mammals, as opposed to the lower eukaryotes,

all of the proteins of the mitoribosome are cytoplasmically

synthesized.



Pools of Unassembled Mitoribosomal Proteins



Knowing the site of synthesis of the mitoriboscomal proteins,

cytoplasm or mitochondrial matrix, answers only one of several

questions raised by mitoribosome biosynthesis. Other questions

raised include 1) Are the proteins synthesized on bound or free

ribosomes and, if bound, to what membrane? 2) In what cellular

compartment do unassembled mitoribosomal proteins exist? 3) How much

time elapses between translation in the cytoplasm and import into the

mitochondria?, 4) What is the mechanism of import into the

mitochondria?, and 5) What is the assembly pattern of mitoribosomes?

Virtually all of these questions can theoretically be addressed by










immune precipitations of mitoribosomal proteins and their precursors

in carefully prepared cellular fractions.

This type of study was not a major part of this work, but one

experiment was performed which utilized cell fractions prepared

during the labeling studies in MDBK cells. This imminune precipitation

with a broad spectrum antiserum raised against the proteins of the

large mitoriboscmnal subunit detected some unassembled mitoribosomal

proteins in both the post-mitochondrial supernatant and the

mitochondrial matrix (figure 14). Furthermore, since mitoribosomal

proteins could be detected among the soluble proteins of the

post-mitochondrial supernatant, this suggests that these proteins are

free in the cytoplasm and possibly synthesized on free polysomes.

Two problems limit the amount of information which can be

obtained from this experiment. First, the conditions used for the

precipitation were not optimized and the one-dimensional SDS-PAGE

profiles showed several contaminating proteins. In some cases, what

appear to be specific precipitation bands might just be increased

aggregation of contaminating proteins in the lattice of antibodies

and protein A. Second, the cells were lysed using a hypotonic

digitonin solution. Digitonin is a detergent which solubilizes all

cellular membranes except the inner mitochondrial memnbrane [120].

Although the amount of digitonin used was below the concentration

necessary to disrupt membranes thoroughly, it may have caused some

leakage of, for example, the contents of the endoplasmic reticulum or

the Golgi into the cytoplasm. Therefore, the "soluble proteins of









the cytoplasm" nay include proteins which were actually sequestered

during translation on bound riboscmes.

Ideally, this type of experiment is probably best performed by

using monoclonal or adsorbed polyclonal antibodies to follow the

progression of a specific protein. Also, the cell fractions should

be prepared differently. For example, inhibitors of mitochondrial

import could be used during a labeling period to prepare a fraction

for the study of the extramitochondrial form of these proteins, or

ethidium bromide could be used to inhibit the synthesis of

mitochondrial ribosomal RNN to study the imported but unassembled

proteins. The major problem with such experiments would probably be

incorporating sufficient radiolabel into the proteins, since the

inhibitors would probably necessitate a relatively short labeling

period.



Implications for Mitochondrial Evolution and Biogenesis



This project was originally designed to determine which of the

proteins of the mammalian mitochondrial ribosome was a mitochondrial

product, in hopes of assigning it to one of the unidentified

mitochondrial genes (URF's). That the manmmalian mitoribosomal

proteins are all products of the nucleo-cytoplasmic system was an

unexpected result. This reflects yet another difference between the

mitochondrial protein synthetic systems of higher and lower

eukaryotes. If indeed, the mitochondrial product protein in lower

eukaryotes functions in ribosome assembly, that function is performed










in mammal s by a protein or proteins imported from the cytoplasm. It

might be that a protein of identical function is imported. This

would argue against theories that the mitochondrially produced

proteins have retained intramitochondrial synthesis because they are

"too hydrophobic", or have some other structural feature which makes

them incapable of import. Because of the differences in the

structure of the two classes of mitoriboscmes, it is also possible

that the ribosomal domain involved is sufficiently changed in mammals

that a different assembly mechanism is used. Considering the highly

proteinaceous nature of the mammalian mitoribosome, perhaps this is

not so surprising. It appears that regions of RNA and protein in

other ribosomal classes have been replaced in the mammalian

mitoribosome with structures containing only protein. It might be

that the ribosomal region of S-5 or var 1 in lower eukaryotes is

functionally the same in mammalian mitoribosaomes but structurally

completely different.

This result has important implications for mitochondrial

evolution and biogenesis. The only other known example of a change

in mitochondrial products is the "DCCD-binding protein" of the

F ATPase, which is made mitochondrially in yeast and

cytoplasmically in Neurospora and mammals. However, this change

does not seem to affect the general structure of the mitochondrial

gene map in lower eukaryotes. In contrast, the genarme of mammals

seems very efficient. Its reduced size evolved predominantly by

eliminating intervening sequences. Although an exact number of

reading frames of the mitochondrial gencarme of lower eukaryotes is not










yet available, the number of mitochondrially produced structural

proteins seems comparable in lower eukaryotes and mammals. The high

density of information on mammalian mitochondrial DNP. suggests that

mitochondria are under selection for conservation of genetic

material, although the reason for such a selective pressure is

difficult to discern. The close homology of the mitochondrial DNN

reading frames in species as diverse as human and mouse, suggests

that the proteins encoded by these genes are required for proper

mammalian mitochondrial function. Yet, the data presented here

demonstrate that mitochondria are not obligated to make a set of

specific types of proteins, but that the protein synthetic products

are evolving in response to, or in partnership with the proteins

encoded on the nuclear gename.














REFERENCES


1. Teintze, M., Hennig, B., Schleyer, M., Schmidt, B., and Neupert,
W. (1982) Biogenesis of mitochondrial membrane proteins. In:
Membranes in Growth and Developmnent (Hoffman, J.F., Giebisch,
G. H., and Bolis, L., eds) pp. 37-47, A. R. Liss Inc., New York.

2. Schatz, G. (1979) How mitochondria import proteins from the
cytoplasm. Fed. Eur. Biochem. Soc. Letters 103, 203-211.

3. Kellems, R. E., and Butow, R. A. (1972) Cytoplasmic-type 80S
ribosomes associated with yeast mitochondria. I. Evidence for
ribosome binding sites on yeast mitochondria. J. Biol. Chem.
247, 8043-8050.

4. Kellems, R. E., Allison, V. F., and Butow, R. (1974) Cytoplasmic
type 80S ribosomes associated with yeast mitochondria. II.
Evidence for the association of cytoplasmic ribosomes with the
outer mitochondrial membrane in situ. J. Biol. Chem. 249,
3297-3303.

5. Kellems, R. E., and Butow, R. (1974) Cytoplasmic type 80S
ribosomes associated with yeast mitochondria. III. Changes in
the amount of bound ribosomes in response to changes in
metabolic state. J. Biol. Chem. 249, 3304-3310.

6. Kellems, R. E., Allison, V. F., and Butow, R. (1975) Cytoplasmic
type 80S ribosomes associated with yeast mitochondria. IV.
Attachment of ribosomes to the outer membrane of isolated
mitochondria. J. Cell Biol. 65, 1-14.

7. Godinot, C., and Lardy, H. A. (1973) Biosynthesis of glutamate
dehydrogenase in rat liver. Demonstration of its microsamal
location and hypothetical mechanisms of transfer into
mitochondria. Biochemistry 12, 2951-2060.

8. Hallermayer, G., Zimmermann, R., and Neupert, W. (1977) Kinetic
studies on the transport of cytoplasmically synthesized proteins
into the mitochondria in intact cells of Neurospora crassa.
Eur. J. Biochem. 81, 523-532.

9. Harmey, M. A., Hallermayer, G., Korb, H., and Neupert, W. (1977)
Transport of cytoplasmically synthesized proteins into the
mitochondria in a cell free system from Neurospora crassa.
Eur. J. Biochem. 81, 533-544.







10. Kadenbach, B. (1966) Synthesis of mitochondrial proteins:
Demonstration of a transfer of proteins froman microsoames into
mitochondria. Biochim. Biophys. Acta 134, 430-442.

11. Kawajiri, K., Harano, T., and Omura, T. (1977) Biogenesis of the
mitochondrial matrix enzyme, glutamate dehydrogenase, in rat
liver cells. I. Subcellular localization, biosynthesis, and
intracellular translocation of glutamate dehydrogenase. J.
Biochem. 82, 1403-1416.

12. Hallermnayer, G., and Neupert, W. (1976) Studies on the synthesis
of mitochondrial proteins in the cytoplasm and on their
transport into the mitochondrion. In: Genetics and Biogenesis
of Chloroplasts and Mitochondria (Bucher, Th., Neupert, W.,
Sebald, W., and Werner, S., eds) pp. 807-812, North Holland, New
York.

13. Robbi, M., Berthet, J., Trouet, A., and Beaufay, H. (1978) The
biosynthesis of rat liver cytochrome c. I. Subcellular
distribution of cytochrome c. Eur. J. Biochem. 84, 333-340.

14. Robbi, M., Berthet, J., Trouet, A., and Beaufay, H. (1978) The
biosynthesis of rat liver cytochrome c. II. Subcellular
distribution of newly synthesized cytochrome c. Eur. J.
Biochem. 84, 341-346.

15. Ades, I. Z. (1982) Transport of newly synthesized proteins into
mitochondria -- A review. Mol. Cell. Biol. 43, 113-127.

16. Conboy, J. G., and Rosenberg, L. E. (1981) Posttranslational
uptake and processing of in vitro synthesized ornithine
transcarbamylase precursor by isolated rat liver mitochondria.
Proc. Nat. Acad. Sci. USA 78, 3073-3077.

17. Fluckiger, J., Behra, R., Jaussi, R., and Christen, P. (1982)
Intracellular location of the precursor of mitochondrial
aspartate aminotransferase and its in vitro import into
mitochondria. Experientia 38, 740.

18. Marra, E., Passarella, S., Doonan, S., Quagliariello, E., and
Saccone, C. (1980) Protease resistance of aspartate
aminotransferase imported in mitochondria. Fed. Eur. Biochem.
Soc. Letters 122, 33-36.

19. Miralles, V., Felipo, V., Hernendez-Yago, J., and Grisolia, S.
(1983) Transport of the precursor for rat liver glutamate
dehydrogenase into mitochondria "in vitro." Biochem. Biophys.
Res. Coamn. 110, 499-503.

20. Morita, T., Miura, S., Mori, M., and Tatibana, M. (1982)
Transport of the precursor for rat liver ornithine
carbamoyltransferase into mitochondria in vitro. Eur. J.
Biochem. 122, 501-509.








21. Ries, G. Hundt, E., and Kadenbach, B. (1978)
Immunoprecipitation of a cytoplasmic precursor of rat-liver
cytochrcme oxidase. Eur. J. Biochem. 91, 179-191.

22. Sakakibara, R., Kamisaki, Y., and Wada, H. (1981) Import of a
putative precursor of rat liver mitochondrial glutamic
oxaloacetic transaminase into mitochondria. Biochem. Biophys.
Res. Ccmm. 102, 235-242.

23. Shore, G. C., Carignan, P., and Raymond, Y. (1979) In vitro
synthesis of a putative precursor to the mitochondrial enzyme,
carbamyl phosphate synthetase. J. Biol. Chem. 254,
3141-3144.

24. Zwizinski, C., Schleyer, M., and Neupert, W. (1983) Transfer of
proteins into mitochondria: Precursor to the ADP/ATP carrier
binds to receptor sites on isolated mitochondria. J. Biol.
Chem. 258, 4071-4074.

25. Maccecchini, M.-L., Rudin, Y., and Schatz, G. (1979) Transport
of proteins across the mitochondrial outer membrane. A
precursor form of the cytoplasmically made intermembrane enzyme
cytochrame c peroxidase. J. Biol. Chem. 254, 7468-7471.

26. Aziz, L. E., Chien, S.-M., Patel, H. V., and Freeman, K. B.
(1981) A putative precursor of rat liver mitochondrial malate
dehydrogenase. Fed. Eur. Biochem. Soc. Letters 133, 127-129.

27. Morita, T., Mori, M., Tatibana, M., and Cohen, P. P. (1981) Site
of synthesis and intracellular transport of the precursor of
mitochondrial ornithine carbamoyltransferase. Biochem. Biophys.
Res. Comm. 99, 623-629.

28. Northemann, W. Schmelzer, E., and Heinrich, P. C. (1981) The
size and distribution of cytochrome c oxidase subunit IV mRNA
between free and membrane-bound polyribosomes. Eur. J.
Biochem. 119, 203-208.

29. Suissa, M., and Schatz, G. (1982) Import of proteins into
mitochondria: Translatable mRNAs for imported mitochondrial
proteins are present in free as well as mitochondria-bound
cytoplasmic polyscmes. J. Biol. Chem. 257, 13048-13055.

30. Heinrich, P. C., Northemann, W., and Schmelzer, E. (1981)
Proteolytic precursor processing in the biosynthesis of
mitochondria. Acta biol. med. germ. 40, 1451-1463.

31. Kaput, J., Golfz, S., and Blobel, G. (1982) Primary structure of
a signal peptide for post-translational translocation into
mitochondria. J. Cell Biol. 95, 275a.









32. Bennett, J. (1982) Sic transit peptide. Trends in Biochem.
Sci. 7, 269.

33. Walter, P., and Blobel, G. (1981) Translocation of proteins
across the endoplasmic reticulum. II. Signal recognition protein
(SRP) mediates the selective binding to microsomal membranes of
in vitro assembled polysomes synthesizing secretary protein.
J. Cell Biol. 91, 551-556.

34. Ades, I. Z., and Harpe, K. G. (1981) Biogenesis of mitochondrial
proteins. Identification of the mature and precursor fonnrms of
the subunit of 6-aminolevulinate synthetase franom embryonic chick
liver. J. Biol. Chem. 256, 9329-9333.

35. Cote, C., Solioz, M., and Schatz, G. (1979) Biogenesis of the
cytochrome bc, complex of yeast mitochondria: A precursor form
of the cytoplasmically made subunit V. J. Biol. Chem. 254,
1437-1439.

36. Jaussi, R., Sonderegger, P., and Christen, P. (1981)
Demonstration of a precursor of mitochondrial aspartate
aminotransferase (mAATase) in cultured chicken embryo
fibroblasts (CEF). Experientia 37, 626-627.

37. Mori, M., Morita, T., Ikeda, F., Amaya, Y., Tatibana, M., and
Cohen, P. P. (1981) Synthesis, intracellular transport, and
processing of the precursors for mitochondrial ornithine
transcarbamylase and carbamoylphosphate synthetase I in isolated
hepatocytes. Proc. Nat. Acad. Sci. USA 78, 6056-6060.

38. Poyton, R. 0., and McKenmie, E. (1979) A polyprotein precursor
to all four cytoplasmically translated subunits of cytochrame c
oxidase from Saccharomyces cerevisiae. J. Biol. Chem. 254,
6763-6771.

39. Poyton, R. 0., and McKemmie, E. (1979) Post-translational
processing and transport of the polyprotein precursor to
subunits IV and VII of yeast cytochrome c oxidase. J. Biol.
Chem. 254, 6772-6780.

40. Lewin, A. S., Gregor, I., Mason, T. L., Nelson, N., and Schatz,
G. (1980) Cytoplasmically made subunits of yeast mitochondrial
F I-ATPase and cytochrome c oxidase are synthesized as
individual precursors, not as polyproteins. Proc. Nat. Acad.
Sci. USA 77, 3998-4002.

41. Maccecchini, M.-L., Rudin, Y., Blobel, G., and Schatz, G. (1979)
Import of proteins into mitochondria: Precursor form of the
extramitochondrially made F -ATPase subunits in yeast. Proc.
Nat. Acad. Sci. USA 76, 343-347.

42. Mihara, K., and Blobel, G. (1980) The four cytoplasmically made
subunits of yeast mitochondrial cytochrome c oxidase are








synthesized individually and not as a polyprotein. Proc. Nat.
Acad. Sci. USA 77, 4160-4164.

43. Hennig, B., and Neupert, W. (1982) Assembly of cytochrame c.
Apocytochrome c is bound to specific sites on mitochondria
before its conversion to holocytochrcme c. Eur. J. Biochem.
121, 203-212.

44. Korb, H., and Neupert, W. (1978) Biogenesis of cytochrome C in
Neurospora crassa: Synthesis of apocytochrame C, transfer to
mitochondria and conversion to holocytochrome C. Eur. J.
Biochem. 91, 609-620.

45. Matsuura, S., Arpin, M., Margoliash, E., Sabatini, D., and
Morimoto, T. (1979) Synthesis of cytochrame c in rat liver free
polysomes and in vitro transfer into mitochondria. J. Cell
Biol. 83, 437a.

46. Matsuura, S., Arpin, M., Hannum, C., Margoliash, E., Sabatini,
D., and Morimoto, T. (1981) In vitro synthesis and
post-translational uptake of cytochrome c into isolated
mitochondria: Role of a specific addressing signal in the
apocytochraome. Proc. Nat. Acad. Sci. USA 78, 4368-4372.

47. Borst, P. (1972) Mitochondrial nucleic acids. Ann. Rev.
Biochem. 41, 333-376.

48. Crews, S., Ojala, D., Pasakony, J., Nishiguchi, J., and Attardi,
G. (1979) Nucleotide sequence of human mitochondrial DNA
containing the precisely identified origin of replication.
Nature 277, 192-198.

49. Murphy, W. I., Attardi, B., Tu, C., and Attardi, G. (1975)
Evidence for complete symmetrical transcription in vivo of
mitochondrial DNA in HeLa cells. J. Mol. Biol. 99, 809-814.

50. Amalric, F., Merkel, C., Gelfand, R., and Attardi, G. (1978)
Fractionation of mitochondrial RNA from HeLa cells by high
resolution electrophoresis under strongly denaturing conditions.
J. Mol. Biol. 118, 1-25.

51. Costantino, P., and Attardi, G. (1975) Identification of
discrete electrophoretic components among the products of
mitochondrial protein synthesis in HeLa cells. J. Mol. Biol.
96, 291-306.

52. Yatscoff, R. W. and Freeman, K. B. (1977) Discrete
electrophoretic products of mitochondrial protein synthesis in
the Chinese hamster ovary cell line. Can J. Biochem. 55,
1064-1074.








53. Yatscoff, R. W., Freeman, K. B., and Vail, W. J. (1977) Site of
biosynthesis of mammalian cytochrome c oxidase subunits. Fed.
Ear. Biochem. Soc. Letters 81, 7-9.

54. Yatscoff, R. W., Aujame, L., Freeman, K. B., and Goldstein, S.
(1978) Interspecific variations in proteins synthsized by
mammalian mitochondria. Can. J. Biochem. 56, 939-942.

55. Bhat, N. K., Niranjan, B. G., and Avadhani, N. G. (1981) The
complexity of mitochondrial translation products in mammalian
cells. Biochem. Biophys. Res. Coammn. 103, 621-628.

56. Bhat, N. K., Niranjan, B. G., and Avadhani, N. G. (1982)
Quantitative and comparative nature of mitochondrial translation
products in manummalian cells. Biochemistry 21, 2452-2460.

57. Attardi, G. and Ching, E. (1979) Biogenesis of mitochondrial
proteins in HeLa cells. Meth. Enzymol. 56, 66-79.

58. Ching, E., and Attardi, G. (1982) High-resolution
electrophoretic fractionation and partial characterization of
the mitochondrial translation products from HeLa cells.
Biochemistry 21, 3188-3195.

59. Anderson, S., Bankier, A. T., Barrell, B. G., deBruijn, M. H.
L., Coulson, A. R., Drouin, J., Eperon, I. C., Nierlich, D. P.,
Roe, B. A., Sanger, F., Schreier, P. H., Smith, A. J. H.,
Staden, R., and Young, I. G. (1981) Sequence and organization of
the human mitochondrial genome. Nature 290, 457-465.

60. Attardi, G. (1981) Organization and expression of the mammalian
mitochondrial genome, a lesson in econanomy. Trends in Biochem.
Sci. 6, 86-89.

61. Attardi, G. (1981) Organization and expression of the mammalian
mitochondrial genome, a lesson in economy. (part 2) Trends in
Biochem. Sci. 6, 100-103.

62. Attardi, G., Cantatore, P., Ching, E., Crews, S., Gelfand, R.,
Merkel, C., Montoya, J., and Ojala, D. (1981) Organization and
expression of genetic information in human mitochondrial DNA.
In: International Cell Biology 1980-1981 (Schweiger, H. G.,
ed) pp. 225-238, Springer-Verlag, New York.

63. Borst, P. and Grivell, L. A. (1981) Small is beautiful --
Portrait of a mitochondrial genome. Nature 290, 443-444.

64. Grivell, L. A. (1983) Mitochondrial DNA. Sci. Amer. 248,
78-89.

65. Bibb, M. J., VanEtten, R. A., Wright, C. T., Walberg, M. W., and
Clayton, D. A. (1981) Sequence and gene organization of mouse
mitochondrial DNA. Cell 26, 167-180.









66. Anderson, S., deBruijn, M. H. L., Coulson, A. R., Eperon, I. C.,
Sanger, F., and Young, I. G. (1982) Complete sequence of bovine
mitochondrial DNA: Conserved features of the mammalian
mitochondrial genome. J. Mol. Biol. 156, 683-717.

67. Pepe, G., Holtrop, M., Gadaleta, G., Kroon, A. M., Cantatore,
P., Gallerani, R., DeBenedetto, C., Quagliariello, C., Sbisa,
E., and Saccone, C. (1983) Non random patterns of nucleotide
substitutions and codon strategy in the manmalian mitochondrial
genes coding for identified and unidentified reading frames.
Biochem. Int. 6, 553-563.

68. Wong, J. F. H., Ma, D.-P., Wilson, R. K., and Roe, B. A. (1983)
Nucleotide sequence analysis of Xenopus laevis mitochondrial
tRNA genes. Fed. Proc. 42, 2240.

69. Battey, J., and Clayton, D. A. (1980) The transcription map of
Human mitochondrial DNA implicates transfer RNA excision as a
major processing event. J. Biol. Chem. 255, 11599-11606.

70. Ojala, D., Montoya, J., and Attardi, G. (1981) tRNA punctuation
model of RNA processing in human mitochondria. Nature 290,
470-474.

71. Montoya, J., Ojala, D., and Attardi, G. (1981) Distinctive
features of the 5'-terminal sequences of the human mitochondrial
mRNAs. Nature 290, 465-470.

72. Dianoux, A.-C., Bof, M., and Vignais, P. V. (1978) The
dicyclohexylcarbodiimide-binding protein of rat liver
mitochondria is a product of the mitochondrial protein
synthesis. Eur. J. Biochem. 88, 69-77.

73. Kuzela, S., Luciakova, K., and Lakota, J. (1980) Amino acid
incorporation by isolated rat liver mitochondria into two
protein components of mitochondrial ATPase complex. Fed. Eur.
Biochem. Soc. Letters 114, 197-201.

74. Schmidt, B., Hennig, B., Zinrerman, R., and Neupert, W. (1983)
Biosynthetic pathway of mitochondrial ATPase subunit 9 in
Neurospora crassa. J. Cell Biol. 96, 248-255.

75. van den Boogaart, P., Samallo, J., and Asteribbe, E. (1980)
Similar genes for a mitochondrial ATPase subunit in the nuclear
and mitochondrial genome of Neurospora crassa. Nature 298,
187-189.

76. Chomyn, A., Mariottini, P., Gonzalez-Cadavid, N., Attardi, G.,
Strong, D. D., Trovato, D., Riley, M. and Doolittle, R. F.
(1983) Identification of the polypeptides encoded in the ATPase
6 gene and in the unassigned reading frames 1 and 3 human mtDNA.
Proc. Nat. Acad. Sci. USA 80, 5535-5539.









77. Manottini, P., Chcmyn, A., Attardi, G., Trovato, D., Strong, D.
D., and Doolittle, R. F. (1983) Antibodies against synthetic
peptides reveal that the unidentified reading frame A6L,
overlapping the ATPase 6 gene, is expressed in human
mitochondria. Cell 32, 1269-1277.

78. Oliver, N. A., Greenberg, B. D., and Wallace, D. C. (1983)
Assignment of a polymorphic polypeptide to the human
mitochondrial DNA unidentified reading frame 3 by a new peptide
mapping strategy. J. Biol. Chem. 258, 5834-5839.

79. O'Brien, T. W., and Matthews, D. E. (1976) Mitochondrial
ribosomes. In: Handbook of Genetics (King, R. C., ed) volume
5, pp. 535-580, Plenum Press, New York.

80. Buetow, D. E., and Wood, W. M. (1978) The mitochondrial
translation system. Subcellular Biochem. 5, 1-85.

81. O'Brien, T. W. (1971) The general occurrence of 55S ribosomes in
mammalian liver mitochondria. J. Biol. Chem. 246, 3409-3417.

82. Sacchi, A., Ferrini, U., Londei, P., Camuarano, P., and Miraldi,
N. (1977) Mitochondrial and cytoplasmic riboscmaes fram mammalian
tissues: Further characterization of ribosonal subunits and
validity of buoyant-density methods for determination of the
chemical composition and partial specific volume of
ribonucleoprotein particles. Biochem J. 168, 245-259.

83. Hamilton, M. G., and O'Brien, T. W. (1974) Ultracentrifugal
characterization of the mitochondrial ribosome and subribosomal
particles of bovine liver: Molecular size and composition.
Biochemistry 13, 5400-5403.

84. Matthews, D. E., Hessler, R. A., Denslow, N. D., Edwards, J. S.,
and O'Brien, T. W. (1982) Protein composition of the bovine
mitochondrial ribosome. J. Biol. Chem. 257, 8788-8794.

85. Douglas, M. G., and Butow, R. A. (1976) Variant forms of
mitochondrial translation products in yeast: Evidence for
location of determinants on mitochondrial DNA. Proc. Nat. Acad.
Sci. USA 73, 1083-1086.

86. Perlman, p. S., Douglas, M. G., Strausberg, R. L., and Butow, R.
A. (1977) Localization of genes for variant forms of
mitochondrial proteins on mitochondrial DNA of Saccharcmyces
cerevisiae. J. Mol. Biol. 115, 675-694.

87. Groot, G. S. P. (1974) The biosynthesis of mitochondrial
ribosomes in Saccharomyces cerevisiae. In: The Biogenesis of
Mitochondria (Kroon, A. M., and Saccone, C., eds) pp. 443-452,
Academic Press, New York.









88. Groot, G. S. P., Grivell, L. A., VanHarten-Loosbroek, N.,
Kreike, J., Moorman, A. F. M., and VanOCnmen, G. J. B. (1977) The
role of the mitochondrial genetic system in the biogenesis of
the mitochondrial inner membrane. In: Structure and Function of
Energy Transducing Membranes (vanDam, K., and vanGelder, eds)
pp. 177-186, Elsevier/North Holland, New York.

89. Terpstra, P., Zanders, E., and Butow, R. A. (1979) The
association of var 1 with the 38S mitochondrial ribosncmal
subunit in yeast. J. Biol. Chem. 254, 12653-12661.

90. Terpstra, P., and Butow, R. A. (1979) The role of var 1 in the
assembly of yeast mitochondrial ribosomes. J. Biol. Chem.
254, 12662-12669.

91. Maheshwari, K. K., Marzuki, S., and Linnane, A. W. (1982) The
formation of defective small ribosomal subunits in yeast
mitochondria on the absence of mitochondrial protein synthesis.
Biochem. Int. 4, 109-115.

92. Lopez, I. C., Farrelly, F., and Butow, R. A. (1981) Trans action
of the var 1 determinant region on yeast mitochondrial DA;
specific labelings of mitochondrial translation products in
zygotes. J. Biol. Chem. 256, 6496-6501.

93. Hudspeth, M. E. S., Ainley, W. M., Shumard, D. S., Butow, R. A.,
and Grossman, L. I. (1982) Location and structure of the var 1
gene on yeast mitochondrial DNA: Nucleotide sequence of the 40.0
allele. Cell 30, 617-626.

94. Strausberg, R. L., and Butow, R. A. (1981) Gene conversion at
the var 1 locus on yeast mitochondrial DNA. Proc. Nat. Acad.
Sci. USA 78, 494-498.

95. Stausberg, R. L., Vincent, R. D., Perlman, P. S., and Butow, R.
A. (1981) Assymetric gene conversion at inserted segments on
yeast mitochondrial DNA. Nature 276, 577-583.

96. Farrelly, F., and Butow, R. A. (1983) Rearranged mitochondrial
genes in the yeast nuclear gencrme. Nature 301, 296-301.

97. Fox, T. D. (1983) Mitochondrial genes in the nucleus. Nature
301, 371-372.

98. Reid, R. A. (1983) Can migratory mitochondrial DNA activate
oncogenes? Trends in Biochem. Sci. 8, 190-191.

99. Lambowitz, Am. M., Chua, N.-H., and Luck, D. J. (1976)
Mitochondrial ribosome assembly in Neurospora. Preparation of
mitochondrial ribosomal precursor particles, site of synthesis
of mitochondrial ribosomal proteins and studies on the poky
mutant. J. Mol. Biol. 107, 223-253.









100. Lambowitz, A. M., LaPolla, R. J., and Collins, R. A. (1979)
Mitochondrial ribosome assembly in Neurospora. Two-dimensional
gel electrophoretic analysis of mitochondrial ribosomal
proteins. J. Cell Biol. 82, 17-31.

101. LaPolla, R. J., and Lambowitz, A. M. (1977) Mitochondrial
ribosome assembly in Neurospora crassa. Chloramphenicol
inhibits the maturation of small ribosome subunits. J. Mol.
Biol. 116, 189-205.

102. LaPolla, R. J., and Lambowitz, A. M. (1979) Binding of
mitochondrial ribosomal proteins to a mitochondrial ribosomal
precursor RNA containing a 2.3-kilobase intron. J. Biol. Chem.
254, 11746-11750.

103. LaPolla, R. J., and Lambowitz, A. M. (1982) Mitochondrial
ribosome assembly in Neurospora. Structural analysis of mature
and partially assembled ribosomal subunits by equilibrium
centrifugation in CsCl gradients. J. Cell Biol. 95, 26-277.

104. Collins, R. A., Bertrand, H., LaPolla, R. J., and Lambowitz, A.
M. (1981) A novel extracellualr mutant of Neurospora with a
temperature-sensitive defect in mitochondrial protein synthesis
and mitochondrial ATPase. Molec. Gen. Genet. 181, 13-19.

105. Madin, S. H., and Darby, Jr., N. B. (1958) Established kidney
cell lines of normal adult bovine and ovine origin. Proc. Soc.
Exp. Biol. Med. 98, 574-576.

106. Mans, R. J., and Novelli, G. D. (1961) Measurement of the
incorporation of radioactive amino acids into protein by a
filter-paper disk assay. Arch. Biochem. Biophys. 94, 48-53.

107. Roodyn, D. B., Reis, P. J., and Work, T. S. (1961) Protein
synthesis in mitochondria. Requirements for the incorporation of
radioactive amino acids into mitochondrial protein. Biochem.
J. 80, 9-21.

108. Costantino, P., and Attardi, G. (1977) Metabolic properties of
the products of mitochondrial protein synthesis in HeLa cells.
J. Biol. Chem. 252, 1702-1711.

109. Laskey, R. A., and MillI,4A. D. (1975) Quantitative film
detection of H and C in polyacrylamide gels by
fluorography. Eur. J. Biochem. 56, 335-341.

110. Laskey, R. A., ad Mills.A D. (1977) Enhanced autoradiographic
detection of P and -I using intensifying screens and
hypersensitive film. Fed. Eur. Biochem. Soc. Letters 82,
314-316.









111. Chamberlain, J. P. (1979) Fluorographic detection of
radioactivity in polyacrylamide gels with the water soluble
fluor, sodium salicylate. Analyt. Biochem. 98, 132-135.

112. DeVries, H., Arendzen, A. J., and Kroon, A. M. (1973) The
interference of the macrolide antibiotics with mitochondrial
protein synthesis. Biochim. Biophys. Acta 331, 264-275.

113. Leister, D., and Dawid, I. (1974) Pysical properites and protein
constituents of cytoplasmic and mitochondrial riboscmes of
Xenopus laevis. J. Biol. Chem. 249, 5108-5118.

114. Rascati, R. J., and Parsons, P. (1979) Biosynthesis of
cytochrome c oxidase by isolated rat liver mitochondria. J.
Biol. Chem. 254, 1594-1599.

115. Sevarino, K. A., and Poyton, R. 0. (1980) Mitochondrial membrane
biogenesis: Identification of a precursor to yeast cytochrome c
oxidase subunit II, an integral polypeptide. Proc. Nat. Acad.
Sci. USA 77, 142-146.

116. van't Sant, P., Mak, J. F. C., and Kroon, A. M. (1981) Larger
precursors of mitochondrial translation products in Neurospora
crassa: Indications for a precursor of subunit 1 of cytochrome
c oxidase. Eur. J. Biochem. 121, 21-26.

117. Darley-Usmar, V. M., and Fuller, S. D. (1981) M-values of
mature subunits I and III of beef heart cytochrcme c oxidase in
relationship to nucleotide sequences of their genes. Fed. Eur.
Biochem. Soc. Letters 135, 164-166.

118. Feldman, F., and Mahler, H. R. (1974) Mitochondrial biogenesis.
Retention of terminal formylmethionine in membrane proteins and
regulation of their synthesis. J. Biol. Chem. 249,
3702-3709.

119. Hare, J. F., and Hodges, R. (1982) No unique mitochondrial
translation products in respiratory chain-linked NADH
dehydrogenase. Biochem. Biophys. Res. Ccmmn. 105, 1250-1256.

120. Schnaitman, C., Erwin, V. G., and Greenawalt, J. W. (1967) The
submitochondrial localization of monoamine oxidase: An enzymatic
marker for the outer membrane of rat liver mitochondria. J.
Cell Biol. 32, 719-735.