Proc. Natl. Acad. Sci. USAVol. 73, No. 11, pp. 3947-3951, November 1976Biochemistry

Regulation of mitochondrial protein synthesis by cytoplasmicproteins

(protein synthesis in vitro/cytochrome c oxidase/mitochondrial biogenesis/yeast)

ROBERT 0. POYTON AND JEANNE KAVANAGHDepartment of Microbiology, University of Connecticut Health Center, Farmington, Conn. 06032

Communicated by George E. Palade, September 1, 1976

ABSTRACT Isolated yeast mitochondria, which synthesizeidentifiable polypeptides identical to those made in vivo, havebeen used in an in vitro system to study cytoplasmic control ofmitochondrial protein synthesis. It has been found that proteinsynthesis in isolated mitochondria is dependent on an endoge-nous pool of cytoplasmically synthesized proteins present withinmitochondria at the time of isolation, that protein synthesisceases apparently when this pool of proteins is depleted, andthat a cytoplasmic extract can restore protein synthesis in de-pleted mitochondria. By use of depleted mitochondria to assayfor stimulatory factors it has been found that the bulk of thestimulatory activity in the cytoplasm is of a protein nature andresides predominantly in the postpolysomal supernatant. Atleast one cytoplasmic stimulatory protein appears to exert aspecific effect on the synthesis of subunits I-II of cytochromec oxidase (ferrocytochrome c:oxygen oxidoreductase; EC1.9.3.1).

It is now clear that the biogenesis of a functional mitochondrionresults from the collaborative effort of two physically separatedand functionally distinct protein synthetic systems, mito-chondrial and cytoplasmic (1, 2). While the extramitochondrial,cytoplasmic protein synthetic system contributes the majorityof both soluble and membrane-bound mitochondrial proteins,the role of the mitochondrial protein synthetic system appearsto be limited to the production of 10-15 polypeptides that arehydrophobic and reside in the inner mitochondrial membrane(2-4). Recent studies have revealed that some, and possibly all,of these mitochondrial translation products are subunits ofenzyme complexes that also contain subunit polypeptides thatare translated on cytoplasmic ribosomes. Thus far, three (I-III)of the seven subunits of cytochrome c oxidase (EC 1.9.3.1;ferrocytochrome c:oxygen oxidoreductase), two to four of theten subunits of rutamycin-sensitive ATPase (EC 3.6.1.3; ATPphosphohydrolase), and one subunit of coenzyme QH2-cyto-chrome c reductase have been identified as mitochondrialtranslation products (4-10). This duality of origin for the sub-units of some important respiratory enzymes suggests thecoordination of mitochondrial and cytoplasmic protein synthesisat the level of individual enzymes.The degree to which mitochondrial and cytoplasmic protein

synthesis are coupled has been assessed in viwo by suppressingprotein synthesis on one translation system and determiningthe fate of the translation products of the other. These studieshave demonstrated that mitochondrial translation products arenot required for the synthesis of mitochondrial proteins of cy-toplasmic origin. For instance, in the absence of mitochondrialprotein synthesis the cytoplasmically made subunits of cyto-chrome c oxidase and oligomycin-sensitive ATPase accumulatein nearly normal amounts either in the cytoplasm or in themitochondrion (11-14).

In contrast, studies on the effect of cytoplasmic proteinsynthesis on mitochondrial protein synthesis have revealed thatsome cytoplasmically made proteins appear to exert a positive

effect on the synthesis of mitochondrial translation products(1, 2). As yet, the identity of these stimulatory proteins has notbeen established. They may be proteins that act nonspecificallyto stimulate the synthesis of all mitochondrial translationproducts, as would be expected for those proteins which arerequired for the mitochondrial protein-synthetic machineryitself (e.g., initiation or elongation factors). On the other hand,they may be proteins that stimulate specific mitochondrialtranslation products (or sets of translation products, such as thosewhich compose the mitochondrially made portion of innermembrane enzyme complexes). Proteins that fall into thiscategory may be cytoplasmically made subunits or specific"organizer proteins" (13) required for the synthesis and as-sembly of each inner membrane complex.

Since it is unlikely that the identity of these proteins can beestablished from studies in vivo alone we have recently devel-oped an in vitro system, using isolated mitochondria fromSaccharomyces cerevisiae, which offers a promising alternative(15). In work described in this paper we have been able todemonstrate (i) that the synthesis of total mitochondrial proteinand subunits I, II, and III of cytochrome c oxidase cease whena pool of proteins made in the cytoplasm, and present in themitochondrion at the time of isolation, is depleted; and (ii) thatmitochondrial protein synthesis resumes when a cytoplasmicextract is added. We have used this as an assay system for cy-toplasmic factors that affect mitochondrial protein synthesisin general and the synthesis of cytochrome c oxidase in par-ticular.

MATERIALS AND METHODSGrowth of Cells and Preparation of Mitochondria. S.

cerevisiae strain D273-1OB (ATCC 24657) was grown in amedium (16) containing per liter: 10 g of galactose, 3 g of yeastextract, 1 g of KH2PO4, 0.8 g of (NH4)2SO4, 0.7 g of MgSO4-7H20, 0.5 g of NaCI, 0.4 g of anhydrous CaCl2, and 5 mg ofFeCI3-6H20. Mitochondria were isolated from early tomidexponential phase cells as described (15), but with the fol-lowing modifications: cells were washed four times with steriledistilled water prior to resuspension in 1.35 M sorbitol-0.05 Mcitrate-phosphate buffer; spheroplasts were formed at 280; andspheroplasts were incubated for 45 min at 280 after glusulasetreatment. Subsequent steps were performed at 40. Once ob-tained, spheroplasts were lysed by osmotic shock in 0.6 Mmannitol-1 mM Na2EDTA (pH 6.7) and subjected to differ-ential centrifugation. After removal of cell debris by centrifu-gation for 5 min at 2000 X g, the supernatant was fractionatedby centrifugation for 10 min at 12,000 X g into a cytoplasmicextract (supernatant) and mitocbondrial fraction (pellet). Themitochondrial fraction was resuspended in 0.6 M mannitol (pH7.0), washed twice by centrifugation (10 min at 24,000 X g),

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and suspended in 0.6 M mannitol (pH 7.0) at 5 mg of proteinper ml.

Preparation of Cytoplasmic Extract Fractions. Cytoplasmicfractions were generally prepared from the same cells used forthe preparation of mitochondria. The cytoplasmic extractprepared as described above was fractionated by differentialcentrifugation to give a "S-100" supernatant (30 min at 100,000X g) and a postpolysomal supernatant (60 min at 300,000 X g).Where indicated, the "S-100" supernatant (5-10 mg of proteinper ml) was dialyzed for 6 hr against 10 mM potassium phos-phate (pH 7.0) at 40, treated with ribonuclease A (40 Mug/ml)for 30 min at 370, treated with trypsin (100 ,ug/ml) for 30 minat 370 and then with lima bean trypsin inhibitor (50 ,ug/ml),or boiled for 10 min.

Immunotitration of Supernatants. An "S-100" supernatantwas obtained from cells grown for six generations in the growthmedium described above containing 1 uCi/ml of a mixture of"4C-labeled amino acids (reconstituted algal hydrolysate, NewEngland Nuclear). It was adjusted to 0.14 M NaCl, 1% (wt/vol)sodium cholate, 10mM potassium phosphate (pH 7.0), and 40mg/ml of protein, incubated for 4 hr at 40, and centrifuged for30 min at 150,000 X g. Aliquots (500 ,g) of the centrifugedsupernatant were first titrated with increasing amounts of an-tiserum to holocytochrome c oxidase, or with antisera mono-specific for subunit IV, VI or subunits V + VII (14) in order todetermine the optimal ratio of antiserum to antigen. Themixtures were incubated for 12-16 hr at 40 and the immu-noprecipitates collected as described (13). The titrated super-natants used to stimulate protein synthesis were then preparedby incubating larger aliquots (2 mg of protein) of the "S-100"

Time (min)

FIG. 1. Effect of preincubating cells in cycloheximide on thesynthesis in vitro of total protein (A) and cytochrome c oxidase pro-tein (B). Midexponential phase cells were incubated with 100 ,g/mlof cycloheximide for the times indicated prior to the isolation of mi-tochondria. Mitochondria were isolated as described in Materials andMethods except that all buffers contained 50 yg/ml of cycloheximide.Values for cytochrome c oxidase piitein represent plateau values fromimmunotitration curves (15) and have been corrected for nonspecificprecipitation obtained with control serum (less than 9%).

supernatant with the optimal ratio of antiserum for 16 hr at 40and removing the immunoprecipitate by centrifugation. Theresultant titrated extracts were then dialyzed for 6 hr against10 mM potassium phosphate (pH 7.0). This procedure was ef-fective in removing more than 95% of all crossreacting material,as shown by subjecting the titrated extract to a second successiveimmunoprecipitation.

Protein Synthesis In Vitro. Isolated mitochondria (0.5 mgof protein per ml) were incubated in a "protein-synthesizingmixture" (15) modified to contain 2 mM ATP, 15 ;tg/ml ofpyruvate kinase (400 units/mg), 100 Mg/ml of cycloheximide,0.03 Mmol/ml of L-leucine, and 0.1 ,mol/ml of all other aminoacids. The mixture was aerated by gentle shaking. Proteinsynthesis was determined as the incorporation of L-[4,5-3H]leucine (0.02 mCi/ml; 40-60 Ci/mmol) into trichloroaceticacid-precipitable protein. Aliquots (0.2 ml) were withdrawnat the times indicated and added to an equal volume of cold10% trichloroacetic acid-10 mM L-leucine. After 10 min theprecipitates were heated at 900 for 5 min, cooled, filtered ontoWhatman (GF/C) filter discs, and rinsed first with 5% tri-chloroacetic acid-5 mM L-leucine and then with ethanol:ether(2:1). Samples were dried and radioactivity was determined.The synthesis of cytochrome c oxidase protein was followed

by measuring the radioactivity precipitated from solubilizedmitochondrial membranes (13) by antisera prepared againstholocytochrome c oxidase (14). Mitochondria (2-3 mg of pro-tein) were collected at the times indicated and chased for 10min with 5 mM unlabeled leucine prior to solubilization forimmunotitration.

Miscellaneous Procedures. Published procedures werefollowed for the purification of renatured subunits IV-VII ofcytochrome c oxidase (16), for polyacrylamide gel electro-phoresis (3), and for the determination of radioactivity inpolyacrylamide gel slices (13).

RESULTSMitochondrial protein synthesis in vitro ceases whenan endogenous pool of cytoplasmically made proteinsis depletedSeveral lines of evidence (see introduction) have indicated adependency of mitochondrial protein synthesis on the con-comitant synthesis and/or presence of cytoplasmically syn-thesized proteins. Since we have demonstrated that isolatedyeast mitochondria are capable of synthesizing polypeptidesfor up to 30 min (15), it must be assumed that the cytoplasmi-cally made proteins necessary for mitochondrial protein syn-thesis are present in the mitochondrion at the time of isolation.To examine the relevance of preexisting pools of cytoplasmi-cally made proteins within isolated mitochondria to proteinsynthesis in vitro we have attempted to deplete these pools bypreincubating cells in cycloheximide for varying lengths of timebefore harvesting them for the isolation of mitochondria. Asseen in Fig. 1A the extent of incorporation of [3H]leucine intototal (acid-precipitable) protein decreases as the time ofpreincubation in cycloheximide increases. The same declinein synthesis is observed when the incorporation into cytochromec oxidase polypeptides (subunits I, II, and III) is followed (Fig.1B). These results are fully consistent with the idea that isolatedmitochondria contain a pool of cytoplasmically made proteins(17) that are required for mitochondrial protein synthesis.However, they do not rule out the possibility that preincubationin cycloheximide may render isolated mitochondria incapableof protein synthesis by altering mitochondrial membrane in-tegrity or preventing the transport of essential metabolites.

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Proc. Natl. Acad. Sci. USA 73 (1976) 3949

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E ,0

E

Q [/

, -- 08mg Protein

. ---- Q4mg Protein

"' ------O2mg Protein

.- - Buffer

0u 4u 2u 4u 60Time (min)

FIG. 2. Stimulation of mitochondrial protein synthesis by an"S-100" supernatant. Isolated mitochondria (0.5 mg of protein perml) were incubated in a protein-synthesizing mixture for 45 min,whereupon a one-tenth volume of postmitochondrial supernatant,containing the amount of protein indicated, or 10 mM potassiumphosphate (pH 7.0) buffer was added. Aliquots containing 0.1 mg ofmitochondrial protein were taken at the times indicated and processedas described in Materials and Methods.

Stimulation of mitochondrial protein synthesis by acytoplasmic extract

A more direct demonstration of the importance of cytoplasmicfactors became possible when we observed that an "S-100"supernatant could restore protein synthesis in mitochondria thathad stopped synthesizing proteins (Fig. 2). Since the extent ofstimulation is proportional to the amount of supernatant added,it seems likely that at least some of the stimulatory factors havea stoichiometric rather than catalytic role in mitochondrialprotein synthesis.To achieve a crude characterization of the stimulatory factors

we subjected the "S-100" supernatant to trypsin, ribonuclease,heat, and dialysis prior to its addition to mitochondria. As seen

from Table 1, the stimulatory activity is trypsin- and heat-sensitive, nondialyzable, ribonuclease-resistant, and resides inthe postpolysomal supernatant. When taken together, theseproperties indicate that the bulk of the stimulatory activity isproteinaceous. The fact that the postpolysomal supernatantcontains more total activity than the "S-100" supernatant fromwhich it was derived suggests that there may be inhibitory aswell as stimulatory factors present in the "S-100" superna-tant.

Specificity of stimulationSince most, and probably all, of the proteins required for themitochondrial protein synthetic machinery (e.g., elongationand initiation factors, mitochondrial ribosomal protein.) in yeastare coded by nuclear genes and translated extramitochondriallyon cytoplasmic ribosomes (1), it can be anticipated that at leastsome of the stimulatory activity present in the cytoplasm is dueto these proteins. Are these proteins, which would stimulate thesynthesis of all mitochondrial translation products, the onlystimulatory proteins present in the cytoplasm, or are there alsoproteins that exert a stimulatory effect on the synthesis of spe-cific translation products? To study this we have analyzed the.effect of the cytoplasmically made subunits (IV-VII) of cyto-chrome c oxidase on the synthesis of the mitochondrially madesubunits (I-III). Two approaches were possible. In the first, the"S-100" supernatant was replaced by a mixture of purifiedcytoplasmic subunits (IV-VII), then the degree of stimulationof total protein (trichloroacetic acid-precipitable) and cyto-chrome c oxidase (immunoprecipitable) synthesis was deter-mined. In the second, an "S-100" supernatant was titrated withan antiserum to holocytochrome c oxidase, in order to removeall crossreacting material, and the loss of stimulatory activitywas determined. The results from the first approach provednegative since purified subunits failed to stimulate synthesisof either total protein or cytochrome c oxidase. However, thesecond approach was more informative. Although a supernatanttitrated with antiserum to holocytochrome c oxidase still re-tained the ability to stimulate total mitochondrial proteinsynthesis (Table 2), it had lost the ability to stimulate the syn-thesis of subunits I, II, and III of cytochrome c oxidase (Fig. 3).Similar results were obtained with "S-100" supernatants titratedwith antisera monospecific for two cytoplasmically madesubunits, IV and VI. Supernatants treated with antisera againstthe other two cytoplasmically made subunits, V + VII, failedto give any crossreacting material, and hence were not used inthese studies. It should be noted that inhibition due to thepresence of serum proteins in the titrated extracts can be ruledout since the untitrated extract contained an amount of controlserum from uninjected rabbits equivalent to that present in thetitrated extracts.

In order to determine the nature of the stimulatory materialremoved by immunotitration, immunoprecipitates from eachtitrated supernatant were solubilized in sodium dodecyl sulfate

Table 1. Properties of stimulatory factors present in an "S-100" supernatant

Specific stimulatoryactivity (cpm/mg of % Total

mitochondrial protein per stimulatoryCell fraction Treatment mg of cell fraction protein) activity

"S-100" supernatant None 19,214 100RNase 17,891 93Trypsin 3,036 16Heat 1,216 6Dialysis 40,517 101

Postpolysomal supernatant None 47,588 12810 mM potassium phosphate(pH 7.0) None 0 0

Mitochondria (0.5 mg/ml) were incubated in 8 ml of protein-synthesizing mixture with L-[4,5-3H]leucine at 300 for 45 min. The protein-synthesizing mixture was then partitioned into 1-ml aliquots and 0.1 ml (0.5 mg) of the indicated cell fraction was added. Stimulatory activitywas determined after an additional 30 min of incubation (at which time incorporation into protein for each addition had essentially reachedplateau) and has been corrected by subtracting those counts present at 45 min prior to the addition of the cell fractions. Specific stimulatoryactivity has been normalized to mg of cell fraction protein. Details of treating each cell fraction are given in Materials and Methods. The un-treated postmitochondrial supernatant is taken as 100%7 for computation of% total stimulatory activity.

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Table 2. Removal of stimulatory activity forcytochrome c oxidase by immunotitration

Stimulation (cpm/mg ofmitochondrial protein)of incorporation into:

Total CytochromeCell fraction protein c oxidase

"S-100" supernatant 24,150 2,510"S-10.0" supernatanttitrated with:Antiserum againstholocytochromeoxidase 20,690 160

Antiserum againstsubunit IV 21,750 250

Antiserum againstsubunit VI 19,960 300

Stimulatory activity for incorporation of L-[4,5-3H]leucine intototal protein and cytochrome c oxidase was determined asdescribed in the legend of Table 1 and in Materials and Methods.The "S-100" supernatant (40 mg of protein/ml), prepared fromcells grown in a mixture of 14C-labeled amino acids, was dividedinto four aliquots. Three aliquots were treated with the indicatedantiserum (14) to remove all crossreacting material, as describedin Materials and Methods. An equivalent amount of control serumwas added to the remaining "untreated" aliquot, which was thenprocessed exactly like the three aliquots treated with antiserum.

and analyzed on sodium dodecyl sulfate-polyacrylamide gels.Interestingly, the radioactivity profiles from all three immu-noprecipitates were essentially identical. Each exhibited a singlemajor peak of radioactivity at 55,000 daltons and no peaks ofradioactivity in the 5000-15,000 dalton region of the gel wherethe cytoplasmically made subunits of cytochrome c oxidasewould be expected to migrate. Upon eluting the 55,000 daltonprotein from several gels and analyzing its immunologicalproperties in Ouchterlony double diffusion tests against subunitspecific antisera, we have found that it crossreagts with antiserato subunits IV and VI but not with antisera to subunits I, II, V,or VII.Taken together these results suggest that a cytoplasmically

made protein that is at least antigenically related to subunitsIV and VI of cytochrome c oxidase exerts a specific stimulatoryeffect on the synthesis of the mitochondrially made subunits,I-III, and raise the possibility that there may be additionalcytoplasmically synthesized proteins that control the synthesisof other mitochondrial translation products.

DISCUSSIONPrevious studies concerned with the interplay between mito-chondrial and cytoplasmic protein synthesis have been donein vivo with cells incubated in the presence of drugs that se-

lectively inhibit one or the other protein synthetic system. Al-though these studies (1, 2, 18) have convincingly demonstratedwhat appears to be a one-way control exerted by cytoplasmicproteins on mitochondrial protein synthesis, they have notpermitted an identification of the regulatory proteins, an as-

sessment of their mode of action, or a determination of thenumber of proteins involved. In order to approach thesequestions, we have used isolated yeast mitochondria in an invitro system that has already been shown to synthesize identi-fiable proteins identical to those made in vivo (15) and to besubject to some of the same physiological controls that are op-erative in vivo (19).

I0x

E

ca

0

0

3

0,

20 40 60 80 100Slice Number

FIG. 3. Sodium dodecyl sulfate-polyacrylamide gel electropho-resis of polypeptides immunoprecipitated by antiserum againstholocytochrome c oxidase from mitochondria stimulated with: (A)"S-100" supernatant and (B) titrated "S-100" supernatant. Isolatedmitochondria, suspended at 1 mg of protein per ml of protein-syn-thesizing mixture, were partitioned into three aliquots. One aliquotwas labeled with L-[4,5-3H]leucine (0.04 mCi/ml; 59 Ci/mmol) for 45min, while the other two aliquots received an equivalent amount ofunlabeled L-leucine. At 45 min the first aliquot was processed forimmunoprecipitation (15) while the other two aliquots were labeledwith L-[U-14C]leucine (25 MCi/ml; 312 mCi/mmol) in the presence ofeither an "S-100" supernatant (1 mg of protein per mg of mitochon-drial protein) or a titrated "S-100" supernatant (1 mg of protein permg of mitochondrial protein) for 30 min, and then processed for im-munoprecipitation (15). The titrated "S-100" supernatant was pre-pared by treating the "S-100" supernatant with antiserum to holo-cytochrome c oxidase so as to remove all crossreacting material, asdescribed in Materials and Methods. A portion of the 3H-labeledimmunoprecipitate was mixed with each "4C-labeled immunopreci-pitate and coelectrophoresed on sodium dodecyl sulfate-polyacryl-amide gels. The arrow marks the position of the dye front. (0) In-corporation during the initial 45-min period; (X) incorporation uponstimulation.

In this paper we have presented evidence that: (i) proteinsynthesis in isolated mitochondria is dependent on an endoge-nous pool of cytoplasmically made proteins present withinmitochondria at the time of isolation; (Hi) most likely proteinsynthesis ceases when this pool of proteins is depleted; and (iii)a cytoplasmic extract can restore protein synthesis in depletedmitochondria. Using this in vitro system to assay for cytoplasmicfactors that regulate mitochondrial protein synthesis, we havefound that the bulk of the stimulatory activity has propertiesattributable to proteins. These proteins do not appear to bemerely protein factors that are required for the mitochondrialprotein synthetic machinery (e.g., elongation or initiationfactors) and that may have been lost or inactivated during thepurification of mitochondria, since at least one of them exertsa specific effect on the synthesis of the mitochondrial subunitsof cytochrome c oxidase.

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The observation that a titrated cytoplasmic extract that haslost the ability to stimulate the synthesis of the three mito-chondrial subunits of cytochrome c oxidase still retains theability to stimulate the synthesis of other mitochondrial trans-lation products clearly indicates that there are more than one,and possibly many, cytoplasmic regulatory proteins. At themoment it is not clear if these are specific for other mitochon-drial translation products or are nonspecific factors requiredfor total mitochondrial translation. In this regard it is interestingthat specific nuclear mutants of cytochrome c oxidase, coen-zyme QH2-cytochrome c reductase, and ATPase have beendescribed recently in which some of the mitochondrial trans-lation products of each enzyme fail to be made (13, 20). Sinceat least one of these mutant phenotypes has been shown to besuppressed by nuclear suppressor mutations that affect onlytranslation on cytoplasmic ribosomes (21) it is possible thatspecific nuclear coded and cytoplasmically synthesized proteinscontrol the synthesis of each of the currently identified mito-chondrial translation products.

Evidence presented in this study provides a biochemical basisfor these genetic results and suggests that an in vitro assaysystem using isolated mitochondria will be of value in isolating,identifying, and determining the mode of action of the regu-latory proteins.

This 'work was supported by research grants from The NationalInstitutes of Health (GM 21800) and The American Heart Associa-tion.

1. Schatz, G. & Mason, T. L. (1974) "The biosynthesis of mito-chondrial proteins," Annu. Rev. Biochem. 43, 51-87.

2. Tzagoloff, A., Rubin, M. S. & Sierra, M. F. (1973) "Biosynthesisof mitochondrial enzymes," Biochim. Biophys. Acta 301, 71-104.

3. Poyton, R. 0. & Schatz, G. (1975) "Cytochrome c oxidase frombaker's yeast. III. Physical characterization of isolated subunitsand chemical evidence for two different classes of polypeptides,"J. Biol. Chem. 250,752-761.

4. Sebald, W., Machleidt, W. & Otto, J. (1973) "Products of mito-chondrial protein synthesis of Neurospora crassa. Determinationof equimolar amounts of three products in cytochrome oxidaseon the basis of amino acid analysis," Eur. J. Biochem. 38,311-324.

5. Tzagoloff, A. & Meagher, P. (1972) "Assembly of the mito-chondrial membrane system. VI. Mitochondrial synthesis of thesubunit proteins of the rutamycin-sensitive adenosine triphos-phatase," J. Biol. Chem. 247, 594-603.

6. Mason, T. L. & Schatz, G. (1973) "Cytochrome c oxidase frombaker's yeast. II. Site of translation of the protein components,"J. Biol. Chem. 248,1355-1360.

7. Rubin, M. S. & Tzagoloff, A. (1973) "Assembly of the mito-chondrial membrane systems. X. Mitochondrial synthesis of threeof the subunit proteins of yeast cytochrome oxidase," J. Biol.Chem. 248, 4275-4279.

8. Weiss, H. (1972) "Cytochrome b in Neurospora crassa mito-chondria. A membrane protein containing subunits of cyto-plasmic and mitochondrial origin," Eur. J. Biochem. 30, 469-478.

9. Katan, M. B. & Groot, G. S. P. (1975) "Isolation, partial charac-terization, and mode of biosynthesis of cytochrome bcI (complexIII) of yeast," in Electron Transfer Chains and Oxidative-Phosphorylation, eds. Quagliariello, E., Papa, S., Palmieri, F.,Slater, E. C. & Siliprandi, N. (North-Holland Publishing Co.,Amsterdam), pp. 127-132.

10. Jackl, G. & Sebald, W. (1975) "Identification of two products ofmitochondrial protein synthesis associated with mitochondrialadenosine triphosphatase from Neurospora crassa," Eur. J.Biochem. 54, 97-106.

11. Tzagoloff, A. (1969) "Assembly of the mitochondrial membranesystem II. Synthesis of the mitochondrial adenosine triphospha-tase, F1," J. Biol. Chem. 244, 5027-5033.

12. Tzagoloff, A., Akai, A. & Sierra, M. F. (1972) "Assembly of themitochondrial membrane system. VII. Synthesis and integrationof F, subunits into the rutamycin-sensitive adenosine triphos-phatase," J. Biol. Chem. 247, 6511-6516.

13. Ebner, E., Mason, T. L. & Schatz, G. (1973) "Mitochondrial as-sembly in respiratory-deficient mutants of Saccharomyces cer-evisiae. II. Effect of nuclear and extrachromosomal mutationson the formation of cytochrome c oxidase," J. Biol. Chem. 248,5369-5378.

14. Poyton, R. 0. & Schatz, G. (1975) "Cytochrome c oxidase frombaker's yeast. IV. Immunological evidence for the parllcipationof a mitochondrially synthesized subunit in enzymatic activity,"J. Biol. Chem. 250, 762-766.

15. Poyton, R. 0. & Groot, G. S. P. (1975) "Biosynthesis of polypep-tides of cytochrome c oxidase by isolated mitochondria," Proc.Natl. Acad. Sci. USA 72, 172-176.

16. Mason, T. L., Poyton, R. O., Wharton, D. C. & Schatz, G. (1973)"Cytochrome c oxidase from baker's yeast. I. Isolation andproperties," J. Biol. Chem. 248, 1346-1354.

17. Ibrahim, N. G. & Beattie, D. S. (1976) "Regulation of mito-chondrial protein synthesis at the polyribosomal level," J. Biol.Chem. 251, 108-115.

18. Beattie, D. S., Lin, L-F. H. & Stuchell, R. N. (1974) "Studies onthe control of mitochondrial protein synthesis in yeast," in TheBiogenesis of Mitochondria, eds. Kroon, A. M. & Saccone, C.(Academic Press, New York), pp. 465-475.

19. Groot, G. S. P. & Poyton, R. 0. (1975) "Oxygen control of cyto-chrome c oxidase synthesis in isolated mitochondria from Sac-charomyces cerevisiae," Nature 255, 238-240.

20. Tzagoloff, A., Akai, A. & Needleman, R. B. (1975) "Assemblyof the mitochondrial membrane system. Characterization ofnuclear mutants of Saccharomyces cerevisiae with defects inmitochondrial ATPase and respiratory enzymes," J. Biol. Chem.250,8228-8235.

21. Ono, B., Fink, G. & Schatz, G. (1975) "Mitochondrial assemblyin respiration-deficient mutants of Saccharomyces cerevisiae.IV. Effects of nuclear amber suppressors on the accumulationof a mitochondrially-made subunit of cytochrome c oxidase,"J. Biol. Chem. 250,775-782.

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