protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

Biochimie, 69 (1987) 517 - 529 © Soci~t~ de Chimie biologique/Elsevier, Paris

517

Research article

Protein synthesis in mitochondria from yeast strains carrying nam and mira suppressor genes

Wlodzimierz ZAGORSKI 1, Miroslaw KOZLOWSKI 1, Maria MIESZCZAK 1, Athanase SPYRIDAKIS 2, Maurice CLAISSE 2 and Piotr P. SLONIMSKI 2

1 Institute o f Biochemistry and Biophysics, Polish Academy o f Sciences, Rakowiecka 36 str., 02-532 Warsaw, Poland, and 2Centre de Gdndtique Moldculaire du CNRS, Laboratoire Associ6 ?t I'Universit6 Pierre-et-Marie-Curie, F-91190 Gif-sur- Yvette, France

(Received 12-2-1987, accepted 4-5-1987)

Summary - Yeast mitochondria isolated from two different wild type strains (gal + and gal-), whether grown on galactose or glucose, synthesize all mitochondrial polypeptides with similar efficiencies and in proportions approximating those detected in vivo. Mitochondria isolated from mit- mutants synthesize in vitro a mutant pattern of mitochondrial proteins, indistinguishable from the in vivo products. The mutant pattern is restored to the wild type one in mitochondria isolated from pseudorevertant strains carrying an additional nuclear (nam3-1 and R705) or mitochondrial (mim3-1) informational suppressor gene. Sup- pression is expressed in isolated nitochondria without the obligatory presence of cytosol at the level of both respiratory control and specific polypeptide synthesis.

Translation in isolated mitochondria is sensitive to paromomycin. The antibiotic differentiates between translation in mitochondria from wild type strains and that in nam-type gene carrying strains. This strongly suggests that ham-type _m__utation~ affect the m_itoribosome, enhancing ambiguity of translation, t h~ allowing for the pseudoreversion of mit- phenotypes.

mitochondriai translation I ribosomal suppression I nam, mira suppressor genes I translational ambiguity / paromomycin

R~sum~ - Synth~se prot~ique dans les mitochondries de levure portant les g~nes suppresseurs, ham et mira. Les mitochondries provenant de deux diffdrentes souches sauvages de levure (gal + et gal-) sont capables de synth~tiser les protdines mitochondriales dans les m~mes proportions que les cellules intactes. Parmi les produits de traduction dirig6s par les mitochondries isol6es des mutants mi t - , on remarque la disparition des polypeptides cod6s par les gimes spdcifiquement touch,spar les mutations exoniques et intro- niques. Les polypeptides en question sont de nouveau synthdtisds dans les mitochondries provenant des souches pseudordvertantes, dans lesquelles en plus des mutations mi t - , on a introduit les gbnes suppresseurs localisds sur le ~6nome nucl6aire (nam3-1 et R705) ou sur le g~nome mitochondrial (mim3-i).

La synthbse des pi'ot6ines mitochondriales est sensible ?t la paromomycine. L "antibiotique agit diff6rem- ment sur la traduction dans les mitochondries sauvages ou isol6es de souches poss6dant les g~nes suppresseurs de type nam. Ceci suggbre que les mutations nam touchent les mitoribosomes, en augmentant l'ambigu~'td ribosomale et facilitant la pseudor6version.

traduction mitochondriale / ~uppression ribosomale I g~nes suppresseurs, nam, nfim / ambigui'td de traduction I

paromomycine

Abbreviations: EDTA: ethylenediaminetetraacetic acid; TCA: trichloroacetic acid; PAGE: polyacwlamide gel electrophoresis; SDS: sodium dodecyl sulfate.

518 W. Zag6rski et al.

Introduction

The mitochondrial translational system differs from orthodox cytoplasmic systems in many ways. First- ly, in mitochondria, the meaning of several codons is at variance with the universal code (for review, see [1]). Secondly, in this system, recognition of all 61 codons is assured by a more restricted number of types of adaptor molecule [2].

This particular mode of polypeptide formation is performed by a peculiar ribosomal machinery. Despite its superficial similarity to the bacterial ribosome, the mitochondrial ribosome has a different structure. For example, in the case of the yeast mitoribosome [3], the small subunit (38S) and the large subunit (54S) contain 33 and 37 proteins, respectively. These numbers are somewhat higher than in typical prokaryotic ribosomes [4], but lower than in yeast cytoplasmic ribosomes [3,5]. Moreover , the large subuni t s of yeast mitoribosomes do not contain 5S rRNA, an obligatory component of prokaryotic as well as eukaryotic cytoplasmic ribosomes [6]. These features direct attention towards the processes of polypeptide formation in mitochondria. It may be expected that such studies will lead to a better in- sight into the general mechanisms of deciphering the code by ribosomal machinery.

The yeast mitochondrial translational system of- fers a particular opportunity for such studies, owing to the l~rge collection of well-defined yeast nuclear and mitochondrial mutations influencing polypeptide formation in the organelles. Of particular interest for studying the function of mitoribosomes are general gene non-specific suppressor mutations each allowing for in vivo pseudoreversion of several different m i t - phenotypes. Three classes of such general suppressor genes have been well characterized to date. The first, called m i m 3 (for mi tochon- dria-mitochondria interaction), described by K_ruszewska and ~ondmski [7, 8], maps in the region of mitochondrial DNA coding for 15S rRNA. The second, called ham3 (for nuclear ac- comodation of mitochondria), characterized by the same authors, maps in the nuclear genome. The third class, called sery R described by Zol/adek et al., [9] also maps in nuclear DNA, but is reported to be non-allelic.to the original ham3-1 mutation (M. Boguta, T. Zol/adek and A. Putrament, submitted for publication).

The spectra of m i t - mutations suppressed by all three suppressor genes are rather large and almost superimposable. Most of the suppressed mutations

are premature ochre terminators introduced into exons as well as introns of different mitochondrial genes. In addition, a few missense mutations in mitochondrial DNA, [7-9] can also be suppressed. Genetic data lead to the hypothesis that the suppressor mutations are expressed by changing the structure of the mitoribosome. The hypothesis in- cludes the assumption that such a change is followed by enhanced ambiguity of translation which allows the reading-through of premature terminators, and the suppression of f rame-shif t or missense mutations by the modified ribosome.

Both elements of the hypothesis seem to be amenable to direct experimental verification. In this paper, we characterize protein synthesis in mi tochondr ia isolated f rom S a c c h a r o m y c e s cerevisiae strains carrying different suppressors of m i t - mutations. In the next paper, we will concen- trate on the structure of mitoribosomes isolated from such strains.

Materials and Methods

Strains and growth conditions S. cerevisiae strains ATI2 (wild), AT12/V25 (oxil- mu- tant) and a strain carrying the nuclear suppressor gene R705, ATI2/V25-R705, (characterized by Zoladek et al., [9]) were grown in medium I [10] containing per liter: 10 g of galactose, 3 g of yeast extract (Difco), 1 g of KH2PO 4, 0.5 g of NaCI, 0.8 g of (NH4)2SO4, 0.7 g of MgSO4-7H20, 0.4 g of CaCI 2, 8/.tl of Normapur FeCI 3 ; n c, '~h~,t ; ,-~n [ , , * / - - I ")"7 , ~ . / , , , . , 3 ~ ~ r ' L ~ l "q - - f~ | . ~ l . . a . : . . . . . . .

sterilized separately. Medium sterilized in 400 ml por- tions, in 1 l Erlenmeyer flasks was supplemented with 2.5 mg of penicillin G/1. Stock inocula (200 ml) were prepared from agar slopes and after 24 h of growth in a shaker at 28°C were stored in a refrigerator (for up to 2 months). Media were inoculated with 5-10 ml of stock inoculum and grown as described above. Different preparations of mitochondria isolated from consecutive cultures, inocuiated from the same stock, retained their mutant pattern. At the time of harvesting the ODzc~°m of the wild type culture was 1.7-2.0 and the yield of cell paste was 2-2.5 g. This allows for the isolation of 6-10 mg of mitochondria, ~Jhich is enough to perform up to 100 incorporations.

gal- strains CK01, rho ~ , wild type strain, nuclear genotype a, karl - I , leu 1, can r, cytoductant from JC8/55x 777-3A/6; CK247 (called below M304i): rho +, box3-M3041, mutant in cyt. b maturase b i 2, same nuclear genotype as CK01; CK247/B291 (called below mira3-1): rho +, raira3-1, box3-M3041, spontaneous mitochondrial suppressor, same nuclear genotype a~ CK01; CK311 (called below M2101): rho +, box3-

Protein synthesis in yeast mitochondria 519

M2101, mutant in cyt. b maturase b i 2, same nuclear g~notype as CK01 ; CK311/B145 (called below ham3-1): rho +, box3-M2101, nuclear genotype a, ham3-1, karl-i, leu 1, can r spontaneous nuclear suppressor; CK235: rho +, box8 missense mutant (M2011) in the 3rd exon of the cob-box gene (J. Lazowska, unpublished), same nuclear genotype as CK01; CK271 : rho +, box6 ochre mutant (M4953) in the 6th exon of the cob-box gene, same nuclear genotype as CK01, characterized by Kruszewska and Slonimski [7] were grown in medium II containing per liter: 5 g of glucose, 3 g of yeast extract and the same salts as in medium I. Cultures were grown as above. Under these conditions, glucose repression was without importance for incorporation experiments and the yield of cell paste and mitochondria from wild type CK01 strain was the same as that from the wild type AT12 strain grown on galactose. Translational activities of mitochondria isolated from both wild type strains grown on either carbon source were also similar.

mit- mutants were grown under the same conditions as the corresponding wild type strains, but the yield of cell paste was about half. Therefore, for preparations of mitochondria, 700 ml cultures of mit- mutants were necessary.

Isolation of mitochondria Isolation was based on the method given by McKee and Poyton [10], with several minor modifications. Sphero- plasts were formed at 37°C in 1.35 M sorbitol, 0.1 M EDTANa 2, pH 7.4, and 10 mM fl-mercaptoethanol. Cell wall lysis was initiated by the addition of zymolyase 100000 (0.6 mg/1 g of initial cell paste) and continued until 80-90°70 of the cells were converted into spheroplasts. Spheroplast formation was monitored as described by McKee and Poyton [10]; usually it lasted 40-50 min. No spheroplast formation was observed

as described by McKee and Poyton [10], were harvested, resuspended in 0.6 M mannitol, 0.1 mM EDTANa 2, pH 7.4, (at 0.4 g of initial cell paste/ml) and broken by fif- teen hard strokes in an Elvehjem-Potter homogenizer, equipped with a tightly fitting teflon pestle. The homo- genate was centrifuged at 1900 x g for 5 rain, the pellet was rehomogenized and recentrifuged under the same conditions. Rehomogenization doubled the yield of mitochondria. Supernatants were pooled and mitochondria were purified by several cycles of differential centrifugation [10] and the final preparation was washed twice by centrifugation through 0.6 M mannitol. Isolated mitochondria were resuspended in mannitol solution at a concentration of 12-14 mg/ml and stored on ice.

Ribosome-free high speed supernatant was prepared from the post-mitochondrial supernatant according to Poyton and Kavanagh [11].

In vitro mitochondriai protein synthesis The standard incorporation mixture (50/zl) contained: 10 t~l of 2.4 M sorbitol; 2.5/zl of 3.0 M KCI; 1/.el of 750 mM K2HPO4/KH2PO 4, pH 7.2; 1.3/zl of a 19 L- amino acid mixture (without methionine); 4 mM each except ty=osine which was at 0.4 mM; 2 .ul of bovine

serum albumin (at a concentration of 75 mg/ml, Sigma); 1 /~l of cycloheximide (solution 2.5 mg/ml); 1 /zi of 200 mM ATP (pH adjusted with NaOH to 7.0); I gl of 25 mM GTP; 1 btl of 250 mM phosphoenolpyruvate (pH adjusted with NaOH to 7.0); 0.2/zl of phosphoenolpyruvate kinase (from a stock solution containing 10mg/ml); 1/~l of 250 mM ~t-ketoglutarate (pH adjusted to 7.0 with NaOH) and 40 pmol of pSSlmethionine (1080 Ci/mmol, Amersham). The volume was adjusted to 40/zl with water, incorporation was initiated by the addition of 1-10/zl of mitochondrial preparation. The final volume was prepared with an appropriate amount of 0.6 M mannitol. Incorporation mixtures (without mitochondria) may be prepared before the experiment and are stable at -60°C for weeks.

Mixtures were incubated in 1.5 ml Eppendorf tubes in a vigorously shaking bath, at 28°C, for 40 rain. The total incorporation of lab:led methionine into hot-TCA- precipitable products was tested by standard methods [12]. For electrophoresis, the samples were prepared in a manner slightly different from that described by McKee and Poyton [10]. At the end of the incorporation period, the mixtures were diluted with one vol of 0.6 M sorbitol-20 mM L-methionine, incubated for 10 rain at 28°C and pelleted by centrifugation at 12000 rpm for 4 rain in an Eppendorf centrifuge. The supernatant was discarded, the easily visible pellet was washed by centrifugation under the same conditions with 0.6 M sorbitol-I mM EDTANa 2, pH 7.4. We found that under these conditions all incorporated radioactivity sedimented with mitochondria. The final pellets were dissolved in dissociation buffer and the products were analyzed by SDS-PAGE as described previously [13l. For each lane less than 20/zg of protein were applied; after drying, the gels were subjected to autoradiography by standard methods for 2-8 days. For electron

. . . . : . . . . . . . . . . . l k , ~ i ~ . . . i . I t | | l ~ , | ~ l , , U l . / ~ t , | l I ~ l | | l l L C O F l [ 1 /V~,~ ~ 1 , ~ | | ~ v~JUL qIJJl. L l I G I | U L ~ . ~ I A [ I . J J A [ -

dria by repeated centrifugations in 0.3 M KCI. Mitochon- dria were negatively stained with 2°70 phosphotungstic acid and kindly photographed with a Philips 301 electron microscope by Mr. C. Grandchamp, at the Centre de G~n~tique Mol~culaire.

Results and Discussion

O p t i m u m condit ions f o r the synthesis o f mitochondrial proteins

Mitochondriel protein synthesis was fully dependent upon the presence of Mg 2+ ions (Fig. 1A). At op t imum concentra t ion , incorpora t ion was stimulated over 40 times, as compared with incorporation without MgSO 4. Mitochondria from wild type strains retained their activity when stored on ice for as long as 18 h. This was not the case with mitochondria isolated from mit- strains, losing in such prolonged storage up to 70°70 of their activity. The incorporation was also dependent upon


A

.l: "o 2o 10o aoo

~ ~ ~ m ~at ~ a q ~ m e t a ~ t

Fig. 1. Optimalization of [3SS]methionine incorporation into wild type yeast mitochondria. 50/zl incorporation mixtures (see Materials and Methods) were supplemented in A, B and D with 6 ~l of mitochondrial preparation (14 mg of prot.ein/ml) and iv C with the different indicated volumes of preparation. A. In- fluence of Mg "÷ ions.o-o : freshly isolated p repa ra t i on ;~ : preparation stored for 18 h on ice. B. Influence of K ÷ ions. C. Influence of mitochondria. D. Influence of ribosome-free high- speed supernatant (10 mg of protein/ml) isolated from the same ATI2 wild_ type ~ra in

K + ions, being optimal at 150 mM KCI (Fig. 1B). Incorporation almost linearly responded to the addition of the mitochondrial fraction (Fig. 1C) but was only poorly (if at all) stimulated by ribosome- free cytoplasm from the same strain (Fig. 1D). Full dependence upon the addition of mitochondria suggested that the system synthesized true mitochondrial polypeptides. This was further substantiated by demonstrating the insensitivity of incorporation to cycloheximide (in concentrations up to 100/zg/ml), an inhibitor acting on cytoplasmic ribosomes. Moreover, the system was fully sensitive to erythromycin (1 mg/ml), which is known to arrest mitochondrial protein synthesis. SDS- PAGE analysis showed that indeed the preparation synthesizes in vitro true mitochondrial proteins similar in relative amounts to those observed for mitochondrial peptides synthesized in whole cells (see below).

Isolated mitochondria retain the mutant pattern of respiration correlated with the synthesis o f specific polypeptides

The intactness of freshly isolated mitochondria was verified by electron microscopy (Fig. 2A). In a partially swollen mitochondrium, the inner and outer membranes can be seen (Fig. 2B). Mitochondria swollen for a longer period break up, liberating membraneous material incrusted with a regular array of spherical projections probably representing inner membrane spheres.

Protein synthesis in isolated mitochondria depended upon the different energy generating components as expected for intact mitochondria (Table I).

The oxidizable substrate, ~-ketoglutarate, stimulated incorporation with wild type mitochondria, but not with mit- mutants (not shown). In- corporation was fully dependent upon GTP and ATP and in part on phosphoenolpyruvate. With respiratory mutants , unable to oxidize ~- ketoglutarate, the translation was driven solely by the external energy-generating system composed of ATP, phosphoenolpyruvate and phosphoenolpyruvate kinase (data not shown).

Under the conditions of incorporation, mitochondria are still intact and actively respiring. This was shown by analyzing the influence of ADP on protein synthesis. It is well known that oxygen con- sumption in intact mitochondria active in oxidative phosphorylation is regulated by the supply of ADP. • l l ~ , l~,,Ot,gl¢O O l l ~ W I . l l¢lLI. I . l ¢ lL l l i~Ag lL l~ lP l l I l l W I I U L~lJ1~ y l ~ d ~ L

mitcchondria incubated with ~t- ketoglutarate and with no external source of energy is also under full control of ADP (Fig. 3A,B). The initial velocity of translation with ADP and ~t-ketoglutarate as the substrate, approaches that observed in the presence of the external-energy generating system. With a prolonged time of incubation, mitochondria become progressively inactivated, but even after 20 rain the preparation retained partial respiratory control. This direct coupling of amino acid incorporation to respiratory control is strong evidence for the functional integrity of isolated mitochondria. It is clear that wild type mitochondria in the incorporation system promoted oxidative phosphorylation, utilizing endogenously generated ATP as a source of energy for translation. Mutant mitochondria, with a faulty respiratory chain, were unable to oxidize ct-ketoglutarate, and translation was fully dependent upon the addition of an external energy- generating system. Respiratory control in mutant mitochondria was absent (Fig. 3C, D).

Similar experiments were also performed with


A

11, ~ i ~ ~ "•

!

Fig. 2. Electron photomicrographs of mitochondria isolated from the wild type (ATI2) strain. A. Fresh preparation. B. Partially swollen mitochonddum. Arrow indicates the region where outer and inner membranes are clearly visible. C. Partially disrupted mitochondrium. Arrow points to the membraneous material, liberated from organelle, incrustated with a regular array of spherical projections (inner membrane spheres). Calibration bar: 0.5 ~M.

Table I. Protein synthesis in wild type, CKOI mitochondria.

Incorporation Hot-TCA-precipitable [35S]methionine Activity mixture (cpm/25/zl) after incubation for (070)

0 min 35 min Net

Complete 43 872 497 586 447 714 100 - ~-ketoglutarate 34 730 311938 277 209 62 - PEP 32 004 176 924 144 920 32 - ATP 46 230 91489 45 259 10 - GTP 41 584 62062 20478 5


10- A A •

0

__ _ I O N N .

\ c °

,c-~-d i - ,'-o " e'o

Fig. 3. Respiratory control at the level of mitochondrial protein synthesis. ~ . Mitochondria from the wild type ATI2 strain were incubated (e-e) in the full incorporation system or with ~-ketoglutarate as the sole energy source (a--a) (o--o). B. Mitochondria from the wild type CK01 strain (o-e) (o--o) as in A. C. Mitochondria from mit- maturase mutant M2101 (~a.-A) incubated in the complete system and with =-ketoglutarate as the sole energy system (-m--t-) ; mitochondria from the pseudorevertant, mutant M2101 carrying the nam3-1 suppressor gone strain 03145/1) incubated in full system (o-e) and with =-ketogiutarate as the sole source of energy (o-o). D. Mitochon- dria from box8 exon mit - mutation M2011 carrying strain (CK235) incubated in the complete system (e-e) and with =- ketoglutarate as the sole energy system (o-o); mitochondria from exon B 6 mit- mutation carrying strain CK271 incubated in the complete system (z~-~x) and with =-ketoglutarate as the sole energy system ( - ~ l - ) . Arrows indicate the time at which I tzl of 200 raM ADP was added to the systems incubated without an external energy generating system.

5 10 15

V a r l . . ~ . . . = - .

cox,- t i ! t cyt n-.. . e e e l l • • ,,

cox i ATPase.w, ~ ~ • I I . ~ - • N

e

t"

64

Fig. 4. SDS-PAGE analysis of mitochondrial polypeptides synthesized in vitro. Mitochondria were isolated from the following strains: lanes 1-3: ATI2 wild type; lane 5: CK01 wild type; lane 6:V25 ox i l - mutant; lane 7-9 : double mutant carrying the V25 mutation and the nuclear suppressor gone R705; lane 11 : M3041 cyt b maturase mutant; lane 12: double mutant carrying the M3041 mutation and the mim3 (B291) mitochondrial suppressor gene; lane 14: M2101 cyt b maturase mutant; lane 15: double mutant carrying the M2101 mutation and the nam3-1 nuclear suppressor gene (clone B145); lane 18:M2011 cyt b missense mutant; lane !6:M4953 c~ b chain termination mu; rant; lanes 4, 10, 13 and !7: standard in vivo synthesized mitochondrial proteins (wild type strain KM91 *). In lanes 2 and 3, incorporation occurred in the presence of 30 and 60 ttg, respectively, of ribosome-free high-speed supernatant from the AT12 wild type strain. Four times more radioactivity was applied in lane 8 than in lane 7. In lane 9, incorporation took place in the presence of 60/~g of ribosome-free high-speed supernatant from the R705 gene carrying strain; the amount of radioactivity is equal to that applied in lane 8.

mitochondria isolated from pseudo wild type rever- tants of mit- mutations, carrying atl additional suppressor gone which restores respiration to whole cells. Translation in mitochondria isolated from such double mutants carrying mit- mutations and nam3 or R705 or mim3 suppressors was under full respiratory control (Fig. 3C). This directly proves that the suppressor mutations restore the active respiratory chain to mitochondria carrying missense or nonsense mutations in mitochondrial DNA.

From this data, we conclude that in vitro isolated

mitochondria retain their mutant pattern of respiration reflected in the total incorporation of labeled amino acid.

This conclusion was substantiated by direct analysis of the polypeptides synthesized in mitochondria isolated from different strains (Fig. 4). It is seen that wild type mitochondria synthesize exactly the same pattern of polypeptides in organdies which co-migrate with products formed in vivo. Judging from electrophoretic mobility, these polypeptides represent in the order of increasing

Protein synthesis in yeast mitochondt ia 523

electrophoretic mobility: varl, coxI (diffused band with apparent M r 40000), coxII (33 000), cyt. b (30000), coxIII (22000), ATPase, where varl stands for mitochondrial ribosomal polypeptide varl , coxI, II, III (for subunits I, II, III of c~ochrome oxidase), cyt b for the apocytochrome b subunit of coenzyme Q cytochrome c reductase, ATPase for suburdts of oligomycin-sensitive ATP- ase. Electrophoretic identification of m__itochondrial pol3qaeptides based upon their mobility is supported by analysis of protein synthesized in mutant mitochondria. Polypeptide coxll synthesis is defi- cient in the strain V25 carrying a V25 mutation (M. Boguta et al., submitted) identified as a premature ochre terminator codon in the middle of oxil gene coding i or this peptide [14]. The synthesis of coxII is restored in strains carrying the nuclear informational suppressor gene R705 (compare manes 6 and 7, 8 and 9 in Fig. 4).

The M3041 and M2101 mutations, located in the open reading frame of intron b i 2, prevent splic- ing of the cytochrome b premessenger RNA [7, 8]. Indeed, synthesis of the ,~yt b band is absent i- both mutants (see lanes 11 and 14, Fig. 4). The same mutations, being pleiotropic, prevent maturation of the oxi3 gene transcript, this is reflected by the lack of the oxi3 coded coxI band in laitochondria from the mutants. Once again, in a mutant carrying an additional suppressor mira3-1 or nam3-1 mutation, synthesis of both cyt b and coxI polypeptides is ~'estored (see lanes 12 and 15, Fig. 4). The cytochrome b band is displaced in products of translation in mitochondria carrying the ochre termination mutation M4953 (strain CK271) localized (J. Lazowska, personal communication) in the 6th exon of the c o b - b o x gene, but is unchanged in mitochondria carrying mutation M2011 identified (J. Lazowska, personal communication) as missense located in the same exon, (compare lanes 16, 17 and 18, Fig. 4).

Electrophoretic mobility and gener:c data unambiguously assign the peptides synthesized in vitro to full-length preducts formed under the direction of specific mitochondrial genes. Therefore, isolated mitochondria synthesize true mitochondrial polypeptides with high efficiency. This supplements the data ot McKee et aL [15], showing that not only exonic but also intronic mutations are expressed in the in vitro system. Moreover, these data are in good agreement with results presented in Fig. 3. Mutant mitochondria which do not synthesize respiratory chain peptides are unable to generate ATP from at-ketoglutarate. Suppressor mutations restore both respiratory control and synthesis of specific mitochondrial peptides. This shows that the

products synthesized in isolated mitochondria controlled by suppressor genes are fully functional.

P a r o m o m y c i n imposes on the mi tor ibosome a specif ic f u n c t i o n a l state

The restoration of respiratory capacity in mit- mutant strains carrying suppressor mutatio~g is coapled with the restoration of synthesis of polypeptides specific for the resp~.rato~ chain. As shown in Figs. 3 and 4, suppression of mitochondrial nonsense and missense mutations is expressed in i~olated mitochondria and does not requffe the presence of other cell components. This is a strong indication that the products of suppressor genes are structural components of mitochondria, directly involved in the construction of the mitochondrial translational apparatus. T.his is un- doubtedly true for mim3 class mutants, which map in the region o." ~,Jtochondrial DNA coding for 15S rRNA [7, 8]. Nuclear coded nam3-1 and R705 general reformational suppressor mutations are also believed to affect the mitoribosome. Genetic data suggest that suppressor genes code for mutated mitoribasomal protein(s) synthesized in the cytoplasm under the co,trol of the nuclear genome, transported into and active within mitochondfia ([7-9] M. Boguta et al., submitted). However, direct proof for this is still lacking. Informational suppressors may alternatively code for constituents of the translational apparatus other than the raitoribosome (e.g., enzymes modifying tRNAs, elongation or termination factors, etc.). Knowing that suppression is expresssed in isolated mitochondria, we decided to check which component(s) of the mitochondrial protein synthesizing system is (are) affected by suppressor mutations. To elucidate this, two approaches were used, one functional and the other structural. The structure of mitoribo- scmes purified from different strains was analyzed by direct methods (to be reported later).

The functional approach was based upon studying the influence of paromomycin on protein synthesis in isolated mitochondria. Paromomycin is known to enhance the ambiguity of translation in several cytoplasmic systems. The antibiotic was also shown to affect the synthesis of mitochondrial polypeptides allowing for phenotypic suppression of numerous mit - mutations [16]. It is accepted that the antibiotic interacts directly with yeast mitoribosomes. Mitochondrial resistance to paromomycin is due to a single base substitution in 15S rRNA [17]. Taking this into account, we hypothesized that the antibiotic which interferes directly with the mitoribosome function could be


-

lo moo l o o o 2o0o

p a r o m o m 0 c i n [ju01rn| ]

C

Fig. $. Paromomycin inhibition of in vitro protein synthesis in wild type mitochondria. A. Inhibition of hot-TCA-precipitable PSS]methionine incorporation in ATI2 mitochondria (o-o) and ih CK01 mitochondria (~-o). Products synthesized at different paromomycin concentrations (indicated at the top of the gels) were analyzed by SDS-PAGE. B. Incorporation into ATI2 mitochondria. C. Incorporation into CK01 mitochondria. Equal amounts of total radioactivity (24000 cpm in B and 25 000 cpm in C) were applied to each lane. S denotes standard in vivo synthesized proteins as in Fig. 4 lane 4.

of help in *.he analysis of activity or ribosomes in mitochondria carrying different mutations. With this in mind, we first tested the influence of paromomycin on translation by wild type mito- choadria (Fig. 5).

The antibiotic partially arrests the incorporation of [35S]methionine into mitochondria isolated from different wild type strains. The inhibition spans over quite a wide range of concentrations of antibiotic up to 2 mg/ml. Quite unexpectedly, the inhibition curve when presented as a semi-

logarithmic plot was distinctly sigmoidal. At low concentrations (0-1 t~g) the antibiotic does not disturb overall methionine incorporation. At concentrations of 1-100 /zg/ml, the curve falls, reaching a second plateau detected between 100 and 1000 ~g/ml. This plateau is transitory and may be overlooked because at oversaturation with the antibiotic (over 1000 b~g/ml) paromomycin completely arrests translation. Therefore, analysis of the shape of the paromomycin inhibition curve should be based on a significant collection of experimental


points. It is worth stressing that the curves detected for mitochondria from two genetically unrelated, gal + and gal- wild type strains were quite similar. This strongly confirms the reproducibility of the paromomycin effect. Knowing that paromomycin enhances the ambiguity of translation, we postulated that the steep part of the curve indicates interaction between the antibiotic and ribosomes, this was followed by the synthesis of fully non- specific polypeptides at a lower plateau. When analyzing the products of translation, we realized that this was not the case. Even at the highest concentration tested, the system still synthesized true mitochondrial polypeptides, with unchanged electrophoretic mobility. Gel analysis confirmed, however, that the overall mode of translation was disturbed by the antibiotic. Only a part of the incorporated label was recovered in full-sized products, at elevated paromomycin concentration, most [35S]methionine was found in polypeptides of various length, mostly short, migrating faster than bromophenol blue, and therefore lost under typical electrophoresis conditions. Accumulation of such polypeptides could be visualized in shorter-run gels. Formation of such TCA-precipitable peptides, heterogeneous in length, explains why in gels loaded with the same amount of total radioactivity, the label in specific polypeptides falls with increases of the antibiotic concentration.

The sigmoidal shape of the inhibition curve indicates that the reaction with the antibiotic does not follow simple first order kinetics. Several explana- tions for this fact may be advanced. Firstly, the antibiotic may interact with the system (at the level of transport or binding of paromomycin) in a cooperative manner, in which the reaction with the first molecules of paromomycin facilitates the reaction with additional molecules.

This does not satisfactorily explain the results ob- tained with different suppressor mutants (see below) where the mid-points of the inhibition curves are strongly displaced, owing to enhanced synthesis of non-specific products. Moreover, it remains unclear why inhibition is only partial, with a lower plateau established at values different for wild type strains and strains carrying nuclear coded suppressor mutations (see below). Secondly, the mitoribosome population may be composed of two classes of particles responding differently to paromomycin; unambiguous translation of full- sized products at high antibiotic concentrations reflects the action of a subclass of mitoribosomes fully resistant to paromomycin. This is incompati- ble with the genetic data showing that mitochondrial sensitivity to paromomycin is controlled by

a single base substitution in the single copy of the 15S rRNA gene [17]. Therefore, we favor the following, third interpretation. We assume that in the whole population of mitoribosomes, each par- ticle responds to the antibiotic in the same manner. Without paromomycin, fully active ribosomes unambiguously synthesize specific polypeptides. In the region of sharp decrease of the inhibition curve each ribosome interacts with the antibiotic and becomes partially inactivated. At the second, lower plateau, the whole population is saturated with paromomycin, each ribosome is partly active synthesizing quite a lot of short undefined polypeptides, but still with low probability of supporting synthesis of full-length polypeptides. This leads to the conclusion that mitoribosomes may act in two functional states 'unambiguous' and 'fully ambiguous', the latter being imposed (or revealed) on the mitoribosome by the antibiotic. Interaction with a definite range of antibiotic concentrations 'freeze- down' all mitoribosomes in the second state. The third interpretation may, in part, be compatible with the first. In fact, it is quite possible, that the onset of inhibition is generated by cooperative accumulation of the first load of the antibiotic. This would explain why inhibition is initiated only at a definite concentration of the antibiotic.

It should be stressed that the third interpretation fits well with the data on the synthesis of the coxII subunit in mitochondria carrying the V25 ochre mutation. It is well established that this mutation is subject in vivo to phenotypic suppression by paromomycin [9, 16]. Indeed, we were able to demonstrate that mitochondria supplemented with paromomycin can synthesize in vitro a polypeptide indistinguishable from subunit II of cytochrome oxidase, which, in view of the in vivo data [9, 16], must be active in respiration. In mutant mitochondria, paromomycin inhibits incorporation into TCA-precipitable products in a manner similar to that detected in the wild type strain (Fig. 6A). This should be expected, since the mitoribosomes from the oxil mutant are genetically identical to the wild type. The pattern of stimulation of coxII polypeptide synthesis confirms our interpretation of the sigmoidal shape of the inhibition curve. At low antibiotic concentrations stimulation of peptide synthesis was negligible compared to the control. Beginning with 1 /zg of paromomycin/ml, therefore, from the very onset of the overall inhibition curve, the peptide progressively accumulates. This occurs exactly in the antibiotic concentration range, where we expect a progressive rise in ambiguity. At high antibiotic concentrations, the plateau of coxII subunit synthesis is established


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Fig. 6. Paromomycin inhibition of in vitro protein synthesis in mitochondria isolated from V25 oxil- mutant. A. Inhibition of hot-TCA-precipitable pS]methionine incorporation in mutant mitochondria (o-o) and in wild type mitochondria (A-~). The triangles represent the average value for mitochondria from both CK01 and ATI2 wild type strains. B. Products synthesized by mutant mitochondria at different paromomycin concentrations (indicated at the top of the gels) were analyzed by SDS-PAGE. Equal amounts of radioactivity )90 000 cpm) were applied to each lane. S as for Fig. 5.

(Fig. 6B). As expected from the hypothesis, at the lower plateau, paromomycin-induced ribosomal ambiguity allowing for reading-through of the V25 ochre codon is accompanied by enhanced synthesis of variable length polypeptides, precipitable with TCA, but not co-migrating with authentic mitochondrial polypeptide. Therefore, the read-

through of the V25 ochre codon is optimal, whereas formation of other specific polypeptides decreases.

Paromomycin inhibits differently total protein synthesis in mitochondria isolated f rom wild type strains and those carrying nuclear suppressor mutations

The influence of paromomycin on protein synthesis was tested with mitochondria isolated from strains carrying nam3 and R705 nuclear suppressor genes. The overall shapes of the inhibition curves with both suppressor carrying strains ressembled those for wild type strains, being also sigmoidal (Fig. 7). However, the mid-points were shifted towards significantly higher paromomycin concentrations and the second plateau was established at incorporation values significantly higher than those detected for the wild type strains. This suggested that mitoribosomes from suppressor strains were simply less sensitive to paromomycin. Analysis of translation products revealed, however, that this was not the case. The nam type mutations did not introduce partial resistance to paromomycin, because at the level of specific polypeptide formation by mutant mitochondria, synthesis'of specific polypeptides diminished as of 4/zg of antibiotic/ml. At the second plateau (over 100/zg/ml) most of the label was incorporated into TCA-precipitable products of non-specific length. This is reflected in an overall rise in background radioactivity in wells loaded with the products synthesized at high j~J.LI waaA~.naaff ~Z~LJL ,..,~.~XJL~.,~,JLJLI, JL C;i, LJLI./IJ[~ I~0~,,I~, I~( U l 1 3 t d l l l L I I G 1 1 5 1 1 L

of gels, Fig. 7C, D). At the second plateau, the specific mitochondrial products represented 10°70 of total incorporation (compare lanes with 0 paromomycin and lanes with high antibiotic concentrations, Fig. 7C, D).

Therefore, mitochondria from strains carrying nuclear suppressors at elevated paromomycin concentrations incorporated 90070 of TCA-precipitable label into ill-defined, ambiguous products. This percentage is rather larger than in the wild type mitochondria. The high level of incorporation in- to non-defined polypeptides at the second paromomycin plateau seems to functionally characterize mitoribosomes from these suppressor strains. It should be stressed that similar curves are observed for two heteronuclear non-allelic suppressor mutants. Therefore, we conclude that mitoribosomes in strains carrying nuclear suppressor genes are functionally different from those in wild types. In suppressor mutants, the overall incorpora t ion of label is less sensitive to paromomycin, probably because the suppressor


A 2

0 A

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pa romornomyc in [Fg /ml ]

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e

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+++ : |+

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D

Fig. 7. Paromomycin inhibition of in vitro protein synthesis in mitochondria isolated from nam type suppressor gene carrying strains. Inhibition of hot-TCA-precipitable [35S]methionine incorporation: A. In mitochondria from a pseudorevertant strain carrying the V25 mutation and R705 nuclear suppressor (o--o) and in wild type mitochondria (z~-~). B. In mitochondria from a pseudorevertant strain carrying CK311 mutation and ham3 (clone B145/1 ) nuclear suppressor gene (o-o) and in wild type mitochondria (z~-~). Open triangles, as for Fig. 6. Products synthesized at different paromomycin concentrations (indicated at the top of the gels) were subjected to SDS-PAGE analysis. C. Products synthesized by mutant mitochondria in A. D. Products synthesized by mutant mitochondria in B. Equal amounts of radioactivh <!50000 in gel C and 50000 cpm in gel D) were applied to each lane. S as for Fig. 5.

ribosomes are intrisically able to synthesize higher amounts of ambiguous products. Thus this incorporation is only moderately inhibited by paromomycin. This shifts the mid-point of the curve of inhibition of total peptide bond synthesis by paromomycin towards higher concentrations of the antibiotic.

It should be stressed that the only fact we directly prove here is that mitoribosomes from strains

carrying nuclearly coded suppressor gene are more prone to synthesize indefinite peptides in the presence of paromomycin. This probably reflects the enhanced mitoribosomal ambiguity in such mutants. However, this ambiguity should be of a restricted type. Mitochondria from suppressor- carrying strains, without paromomycin, synthesize the same polypeptides in exactly the same proportions as mitochondria isolated from wild type


strains (Fig. 4). This suggests that neither initiation nor termination of polypeptide synthesis is disturbed by the suppressors. Mitoribosomes from strains carrying nuclear suppressors are, therefore, able to suppress premature termination codons introduced into exons or introns of mitochondrial genes, without suppressing natural termination in mitochondrial mRNA. Therefore, signals for natural termination are qualitatively different from ochre or amber premature termination codons amenable to suppression. Such differences in efficiency of suppression in other systems were assigned to the 'context effect' [18, 19]. The role of context in the action of nuclear coded mitochondrial suppressor genes was already suggested by genetic data, showing that only some (about 10%) of mit- mutations were amenable to suppression [8].

The conclusion drawn from this part of the work is that mitoribosomes from nam type mutation- carrying strains are functionally different from wild type mitoribosomes. This strongly suggests that nam type suppressor mutations alter mitoribosomes.

% ~oo

so

A

° ~ ~

1 l o

paromomycln [ j ug l m l ]

Funct ional comparison o f nuclearly a n d mitochondrial ly coded suppressors

The genetic data showed that the m i m 3 - 1 mitochondrial extragenic suppressor mutation allows for restoration of respiratory activity in several mit- mutants [7, 8]. The spectrum of suppressed mutations is almost exactly superimposable on that of mit- mutations suppressed by nam3 or

mechanism of action of mitoribosomes changed by the mira3 mutation may be similar to that of mitoribosomes affected by nam3-1 or R705 mutations. Indeed, we were able to observe that mit- mutations are suppressed in vitro in mitochondria carrying the mira3-1 mutation. The suppression was expressed without the obligatory presence of cytoplasm or nucleus. This strongly suggests that suppression results directly from an altered function of the mitoribosome. Bearing in mind the mechanism of action of the nam type mitoribosome, we speculated that the mira3-1 mutation will also show an enhanced ambiguity of translation. In fact, one can propose that the mira3 mutation, localized in 15S rRNA [7], influences the binding of ribosomal protein(s) involved in the control of ribosomal ambiguity. The affinity of the same protein(s) for the mitoribosome may be changed by the ham type mutations affecting the structure of the ribosomal protein. Therefore, nuclearly and mitochondrially coded suppressor

~ee

Fig. 8. Paromomycin inhibition of in vitro protein synthesis in mitochondria isolated from the strain carrying suppressor gene mira3. A. Inhibition of hot-TCA-precipitable [35S]methionine incorporation into mitochondria from a strain carrying the mira3-1 mutation (o-o) and in wild type mitochondria (z~--~). The triangles as for Fig. 6. B. SDS-PAGE analysis of products synthesized in A. Equal amounts of radioactivity (60 000 cpm) were a~plied to each lane. S as for Fig. 5.

mutations could hypothetically generate a similar functional state of mitoribosomes.

To test this possibility, we measured the influence of different concentrations of paromomycin on protein synthesis directed by mitochondria carrying the mira3 suppressor gene (Fig. 8).

It is clear from the data presented that the in-


hibition curve of the mutant mitochondria closely followed that observed for wild type ones, being quite different from the curve recorded with mitochondria from strains carrying ham type genes.

As in the case of wild type mitochondria, the inhibition curve is sigmoidal, with an inflection point observed at 1 #g of antibiotic and a second plateau is detected between 100 and 200 #g of paromomycin. The second plateau is established at 70% inhibition, thus at a value characteristic for wild type mitochondria. At this plateau, again as in the case of wild type strains, all mitochondrial proteins are synthesized. This result does not allow us to state that the overall level of ribosome ambiguity induced by the mim3 mutation is higher than that characteristic for wild type mitochondria.

Therefore, the paromomycin experiment shows that, at least when tested for paromomycin-induced misreading, mitoribosomes carrying the mira3 mutation differ from those modified by the ham mutations. However, in view of the striking similarity of action of both suppressors, we rather expect ribosomal suppression resulting from the nam3 type and the mim3 ribosomes to be based upon mechanistically similar principles.

Acknowledgements

This paper is dedicated to the memory of Jerzy Zag6r- ski, 'The Righteous Between the Nations'.

Authors thank Drs. G. Dujardin and B. Guiard for discussions and suggestions, Dr. J. Lazowska for a gift of mutant M2011, and M.C. Grandchamp (Centre de G6n~tique Mol6culaire du CNRS) for electron photomicrographs.

The work was supported by the Polish Academy of Sciences (project 3.13) and by grants from the CNRS (ATP Biologie Mol~culaire du G~ne), from the Ligue Na- tionale Fran~aise contre le Cancer and from the Fonda- tion de la Recherche M6dicale.

References

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protein synthesis in mitochondria from yeast strains carrying nam and mim suppressor genes

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