ASSEMBLY OF THE MITOCHONDRIAL MEMBRANE SYSTEM: NUCLEAR SUPPRESSION OF A CYTOCHROME b IN YEAST MITOCHONDRIAL DNA

GLORIA CORUZZI AND ALEXANDER TZAGOLOF" Department of Biological Sciences, Columbia University, New York, N. Y. 10027 Manuscript received December 3, 1979 Revised copy received May 5,1980

ABSTRACT In a previous study, a mitochondrial expressing a specific enzymatic deficiency in co-enzyme QH,-cytochrome c reductase was described (TZAGO- LOFF, FOURYand AEAI 1976). Analysis of the mitochondrially translated pro- teins revealed the absence in the mutant of the mitochondrial product corres- ponding to cytochrome b and the presence of a new low molecular weight product. The premature chain-termination mutant was used to obtain sup- pressor with wild-type properties. One such revertant strain was analyzed genetically and biochemically. The revertant was determined to have a second mutation in a nuclear that is capable of partially suppressing the original mitochondrial cytochrome b mutation. Genetic data indicate that the nuclear mutation is recessive and is probably in a gene coding far a involved in the mitochondrial machinery.

YEAST mitochondrial DNA (mtDNA) is known to code for cytochrome b, a well-characterized carrier of co-enzyme QH,-cytochrome c reductase. In a previous study, a mutant of Saccharomyces oerevisiae was shown to have a genetic lesion in the cytochrome b gene (TZAGOLOFF,FOURY and AKAI1976). The mutation (M9-228) was mapped in the cob2 locus and was shown to cause the loss of a wild-type mitochondrial translation product corresponding to the cytochrome b apoprotein. The mutant was of interest because of the appearance of a new low molecular weight mitochondrial product. The presence of a prematurely terminated polypeptide in the cytochrome b-deficient mutant suggested that the mutation might be of the nonsense type. In the present study, an attempt was made to isolate a suppressor of the M9-228 mutation. Although one might expect that revertants of M9-228 would include mitochondrial tRNA suppressor , this was not borne out by genetic analyses. All of the revertants studied fell into two groups. Mutants in the first group behaved as back mutations or intragenic suppressors. These were not fur- ther examined. The second group of revertants were determined to have nuclear mutations. One such revertant (M9-22-28C/R3) has been established to have a recessive mutation in a nuclear gene (suml) that suppresses the mitochondrial mutation. Genetic and biochemical evidence indicate that the suppressor mutation acts at the level of mitochondrial translation.

Genetics 95: 891-903 August, 1980. 892 G. CORUZZI AND A. TZAGOLOFF

MATERIALS AND METHODS

Yeast strains and growth media: The mitochondrial and nuclear genotypes of the strains of Saccharomyces cereuisiae used in this study are listed in Table 1. The following media were used: YPD containing 1% yeast extract, 2% peptone and 2% glucose (unless otherwise indicated) ; YPEG containing 1% yeast extract, 2% peptone, 2% ethanol and 3% glycerol; D containing 1% yeast extract, 2% peptone, 0.1% glucose, 2% ethanol and 3% glycerol; WO containing 0.67% yeast nitrogen base and 2% glucose (unless otherwise indicated); and KAc containing 0.1% yeast extract, 1% potassium acetate and 0.05% glucose. Isolation of revr=?rtants:Revertants of a M9-228-28C were obtained in the following two ways: (1) An overnight culture of the mutant grown in liquid YPD was mutagenized with 2% EMS as previously described (CORUZZI,TREMBATH and TZAGOLOFF1979) ; approximately 2 x 107 cells (assuming 90-95% killing) were spread on solid YPEG and the plates were incubated at 30" until well-defined revertant colonies appeared (5 to 7 days). (2) Alternatively, the overnight culture was spread on solid YPD (2 x lo7 cells); a filter disc soaked with either saturated MnC1, or EMS was placed in the center of the plate. The plate was incubated for 1 to 2 days at 30" and replicated on YPEG. Revertant colonies were picked after 5 to 7 days at 30" and further purified. Induction of po mutants: Cells were grown to stationary phase in liquid YPD and treated with ethidium bromide (5 pg/ml), as described by DEUTSCHet al. (1974). The cells were washed with sterile water and spread for single colonies on D plates. Following 3 to 4 days incubation at 30",

TABLE 1 Mitochondrial and nuclear genotypes of S. cerevisias strains

Genotypes Name Nuclear Mitochondrial Mit locus D273-10B/Al a: met p+ w+ D273-10B/AI PO a: met po WO CB11 a adel p+ wf cB11po a adel po wo a M9-228-28C a adel pfwf mit- cob2 M9-228-6D a met pf of mit- cob2 a M9-228-28C/R3 a adel sum1 p+wf mit cob2 a M9-228-28C/R3po a adel suml po 00 a M9-228-28C/R3-A1 OL arg sumf pf w+ mil- cob2 (Y M9-228-28C/R3-Alpo a: arg suml po 00 M9-94/A1 a: met p+w+ mit oxil a M9-94-4B a adel pfwf mit oxil M9-3/A3 a: met p+w+ mit oxi2 a M9-3-6C a adel p+w+ mit oxi2 M3-9 a met pfwf mit oxi3 a M3-9-2B a adel pfwf mit oxi3 M74O/A1 (Y trp pfwf mit cob1 a M7-4.0-5B a adel p+w+ mit cob1 M17-162 Q: met p+ wf mit- cob2 a M17-162-4D a adel p+of mit- cob2 M19-229 OL met pfof mit- phol a M19-229 a adel pfwf mit phol M339-45 a: met pfwf mir pho2 a M339-45 a adel pfwf mit- pho2

suml refers to the nuclear suppressor mutation described in this paper. CYTOCHROME b SUPPRESSOR 893 two small colonies were picked. Each clone was remutagenized with ethidium bromide. The loss of mtDNA after the 2 cycles of mutagenesis was verified in several clones by the absence of markers in all the known mit loci ( SLONIMSKIand TZAGOLOFF1976). Mitotic segregatiom Haploid strains of opposite mating type carrying different auxotrophic mutations were mixed in liquid YPD (10% glucose) medium and incubated at 30" for 6 hr. The cells were washed with sterile water and transferred to liquid WO (10% glucose) medium for prototrophic selection of the diploid progeny. After 24 hr at 30" the diploid cells were plated for single colonies on WO plates. The colonies formed after 2 days were replicated onto YPEG and scored for growth. Tetrad analysis: The haploid strains were mixed manually on solid YPD modia. The diploid strains formed after 6 hr of incubation were replicated onto KAc medium to induce sporulation. Asci were usually observed 4 days after incubation of the sporulation plates at 25" and were dissected by standard micromanipulation techniques ( MORTIMERand HAWTHORNE1969). The spore progeny were checked for growth on glycerol, and for mating type and auxotrophy. Enzyme assays: Mitochondria were prepared according to the small-scale procedure of NEEDLEMANand TWGOLOFF(1975) and assayed for NADH-cytochrome c reductase, cytochrome c oxidase and oligomycin-sensitive ATPase activities (TZAGOLOFF,AKAI and NEEDLEMAN1975a). Analysis of mitochondrial products: Conditions for the growth of cells and the in vivo labeling of mitochondrial products with [35S] methionine in the presence of cycloheximide have been described (TZAGOLOFF,AKAI and NEEDLEMAN1975b). Mitochondria prepared according to the small-scale method of NEEDLEMANand TZAGOLOFF(1975) were solubilized in 0.05 M dithiothrei- tol, 0.05 M sodium carbonate, 12% sucrose, 2% sodium dodecyl sulfate and 0.04% bromphenol blue. Electrophoresis of total mitochondrial was performed in the presence of SDS on 7.5 to 15% polyacrylamide slab gels, as described by CHUAand BENNOUN(1975). The radio- actively labelled mitochondrial products were visualized by autoradiography of the dried slab gels.

RESULTS Genetic properties of revertants of M9-228: Revertants induced by MnC1, and EMS were crossed to a wild-type haploid strain in yeast. The diploid strains derived from each cross were tested for the presence of mitotic segregants with the original glycerol-negative . This test should discriminate between back mutations, intragenic suppressor mutations and extragenic suppressor mu- tations (Table 2). Of the 104 MnC1,-induced revertants crossed to the wild type, 76 produced only wild-type segregants (Table 3).Therefore, the revertants were classified as either back mutations, intragenic supressors or nuclear dominant sup- pressors. Since the number of segregants scored was only 100 to 300 colonies, it was not possible to distinguish between back mutations and intragenic suppressors ([SUM]). No attempts were made to distinguish between back mutations, intragenic suppressors and nuclear dominant suppressor mutations (SUM). The remaining 28 revertants obtained from the MnC1, mutagenesis and the 12 revertants induced by EMS all behaved as extragenic suppressors; in the cross to the wild-type strain, both glycerol positive (growth on glycerol) and glycerol negative (absmce of growth on glycerol) segregants were observed (Table 3). Based on the mitotic segregation test, this second group of revertants could have either an extragenic mitochondrial suppressor mutation ([SUM]) or a nuclear recessive suppressor mutation (sum). The two could be distinguished by a cross to a po tester (Table 2). In the cross to the po tester, only glycerol-negative segre- gants were observed for all 28 MnClz-induced revertants and the 12 EMS- 894 G. CORUZZI AND A. TZAGOLOFF TABLE 2 Tests to distinguish mitochondrial and nuclear suppressor mutations

Genotypes Phenotype of Type Input output segregants Mitochondrial back mutation Cross to p+ : [mit+] x [mit+] [mitf] glycerol + Cross to' po: [pol x Cmit+l [mit+] glycerol+ Mitochondrial extragenic suppressor [mitf] glycerol+ Cross to p+ : [mit+] x [mit-SUMl [mit+ SUM] glycerol+ [mit SUM] glycerol+ [mit-] I glycerol- Cross to po: [pol x [mitSUM] [mit- SUM] glycerol + Nuclear dominant suppressor Cross to p+: [mit+] x [mit]SUM [mit+] SUM/+ glycerol+ [mit- ] SUM/+ glycerol+ Cross to po: [pol x [mit]SUM [mif- ] SUM/+ glycerol+

Nuclear recessive suppressor Cross top+: [mit+] x [mit]sum [mit+] sum/+ glycerol+ [mit ] sum/+ glycerol- Cross to po: [pol x [mit-] sum [mit- ] sum/+ glycerol-

The genetic crosses are designed to distinguish between the various types of revertants. The symbols used to indicate the genotypes are as follows: Mitochondrial alleles are bracketed, nuclear alleles are without brackets; dominant suppressors are denoted by SUM and recessive suppressors are denoted by sum. The wild-type allele of the suppressor mutation is indicated by f.In the haploid state, the omission of a nuclear genotype indicates that it is wild type.

TABLE 3 Mitotic segregation in EMS- and MnCl,-induced revertants

Number of revertants Total number of displaying mitotic Mutagen EVertilllts segregation of mutant phenotype EMS 12 I2 MnCl, 104 28

a M9-228-28C was mutagenized with EMS or MnCI,, as described in MATERIALS AND METHODS. The 116 revertants obtained from both mutagenesis treatments were mated to the wild-type haploid D273-10B/Al. The diploid progeny that came from the crosses were spread on WO for single colonies and scored for glycerol growth. Of the 116 revertants tested, 40 (12 from the EMS and 28 from the MnCI, mutagenesis) showed both glycerol-positive and -negative segre- gants, as expected for recessive nuclear suppressors. The remaining 76 revertants produced only glycerol positive diploid progeny in the cross, as expected for mitochondrial suppressors. CYTOCHROME b SUPPRESSOR 895 induced revertants. One of the revertants obtained from the EMS treatment, a M9-228-28C/R3 (subsequently referred to as R3), was chosen for a more detailed genetic and biochemical analysis. Mitotic and meiotic segregation of the suppressor mutation: The input geno- types in a cross of a nuclear recessive suppressor to a wild-type strain (Table 2) predict that one half of the mitotic segregants should be glycerol negative and have the same phenotype as the original mit- mutant. This was confirmed by the results of the crosses shown in Table 4. The R3 revertant was mated to the wild-type respiratory competent haploid strain D273-1 OB/Al under the condi- tions of glucose repression to minimize the selective growth of the glycerol-posi- tive segregants. Of the 222 segregants scored, 95, or 43%, were glycerol negative. Furthermore, enzyme assays on 1 8 randomly picked glycerol-negative segregants indicated a single deficiency in co-enzyme QHz-cytochromec reductase, a pheno- type identical to that of the original mutant, a M9-228-28C. In order to perform a reciprocal cross, an a mating-type haploid strain with the mit- and suppressor mutation (sum2) was obtained. This strain, designated a R3/A1 was constructed by crossing R3 to D273--10B/AI and inducing sporu- lation shortly after mating had occurred. One of the glycerol-positive spore pro- geny in a tetrad that had segregated 2:2 was ascertained to carry both the sun1 and mit- alleles. Since the strain was prototrophic, it was mutagenized with EMS, and an arginine auxotroph (a R3/A1) was isolated. When ,aR3/A1 was crossed to the wild-type CB11,55% of the mitotic segregants were scored as gly- cerol negative (Table 4). In agreement with the results of the previous cross, randomly picked glycerol-negative segregants exhibited a single deficiency in co-enzyme QHz-cytochromec reductase. The mode of inheritance of the mit- and suppressor mutations was also exam- ined by tetrad analysis. When the original mit- mutant, U M9-228-28C, was mated to D273-10B/A1, all the meiotic spore progeny showed non-Mendelian 4:O or 0:4 segregation patterns, confirming the mitochondrial nature of the cyto- chrome b mutation. Most of the tetrads obtained from the cross of R3 to D273-

TABLE 4 Mitotic segregation of the cytochrome b deficient phenotype in crosses of R3 and a R3/AI to a wild-type strain

Enzymatic phenotype NADH Total diploid % Glycerol Cytochrome cytochrome Cross colonies scored neeative ATPase oxidase c reductase D273-10B/AI X R3 222 43.2 + + - CBI1 x a R3/A1 208 54.8 + + -

R3 and a R3/A1 were mated to wild-type haploid strains of opposite mating type in liquid YPD media (10% glucose). After 6 hr, the cells were transferred to liquid WO media (10% glucose) and incubated at 30" for 24 hr. The rototrophically selected diploid progeny were spread for single colonies on WO. After two jays, the colonies were replicated onto YPEG media. Randomly picked glycerol-negative diploids (17 in the cross with R3 and 28 in the cross with a R3/A1) were grown in liquid media. Mitochondria were prepared and assayed for respiratory and ATPase activities (TZAGOLOFF,AKAI and NEEDLEMAN1975a). 896 G. CORUZZI AND A. TZAGOLOFF TABLE 5 Tetrad analysis of diploid progeny issued from crosses of R3 to D27310B/AI

Number of tetrads with segregation ratios Cross Total tetrads 4:O 2:2 04 3:l 1:3 D273-10B/AI x R3 68 44 11 4 1 8 D273-10B/Al x R3 po 3 3 0 0 0 0 D273-10B/AI X a M9-228-28C 13 10 0 3 0 0

The two haploid strains were mixed manually on solid YPD media and mated for 6 hr. Sporu- lation was induced on KAc medium. Dissected asci were scored for growth on glycerol. The standard convention is used. 4:O indicates that all four spores grew on glycerol and 0:4 indicates that all four spores were glycerol negative.

10B/A1 segregated 4: 0 or 2: 2, although other ratios (0:4,3: 1 and 1: 3) were also found (Table 5).From the input genotypes in this cross, only 4: 0 and 2: 2 segre- gation ratios should be observed if the R3 revertant has a nuclear recessive sup- pressor mutation (Table 6). The postulated genotype of glycerol-positive spores from the 2:2 tetrads (Table 4) was confirmed by a mitotic segregation test. The glycerol positive meiotic spore progenies from several 2:2 tetrads were crossed to the wild-type D273-10B/A1 or CBI 1. Glycerol negative mitotic segregants were specifically deficient in co-enzyme QHz-cytochrome c reductase activity, indicating a 2:2 segregation of the suppressor mutation during meiosis. The occur- rence of ratios other than 4:O and 2:2 was probably due to incomplete segrega- tion of the mit mutation, since a short mating time was used in order to allow diploids with a suml/f nuclear genotype and a [mit]mitochondrial genotype to sporulate. The third analysis was done on a cross involving a pa derivative of R3 and D273-10B/Al. In this cross, all tetrads dissected were found to segregate 4:O. This would be expected if segregants with the genotype [mitf]sum1 were glycerol positive (Table 6). Other genetic tests for the recessive nature of the suppressor mutation in R3: A series of genetic tests was designed to confirm further the nuclear recessive nature of the suppressor mutation. As shown in Table 7, if sum2 is a nuclear

TABLE 6 Genotypes and of meiotic spore progeny obtained from crosses of R3 and R3p0 to a wild type strain

~ ~~ Input Genotypes of Predicted genotypes Tetrad Cross genatypes diploids of spore progeny phenotypes D273-10B/AI [mit+J [mit+]suml/+ 2 [mitflsuml : 2 [mit+] 4:O x R3 [mit lsuml [mit- ]sumi/+ 2 [mit- ]sum1 : 2 [mit ] 2:2 D273-1 OB/AI [mit+] X [mit+]suml/f 2 [mit+]suml : 2 [mit+J 4:O R3p0 [polsum1

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t .-gg% m -0)u8 6 .- y1 .5 g e, 0 a33 a zv5 ?? g@ roo $2 E.2 J F4m'wae pkc XSKC *s M 0 XXLXX .gag vI -"aaZ%M m8.2 24QQ am e45 U " & b&& g8ax83 -sgga a.2 ,mm ddd>alal -%$o 00 mt.ZZZZZ Cho'] nbauSa 289 b 82 LE 898 G. CORUZZI AND A. TZAGOLOFF gene, each cross indicated should produce diploids with a [mit] suml/+ genotype. Therefore, assuming that sum1 is recessive, all the crosses will result in glycerol-negative segregants with a co-enzyme QHz-cytochrome c reductase deficient phenotype. This prediction was validated by the results of the crosses, strongly implying that sum1 acts in a recessive manner and is unable to suppress the M9-228 mutation in the presence of the wild-type allele. Crosses homozygous for suml: Although the results of the various crosses described so far indicate sum1 to be a nuclear recessive gene, they do not exclude the possibility that, for some reason, suppression by sum1 is expressed only in a haploid nuclear background. Therefore, the phenotype of diploids homozygous for sum1 was examined. Diploids with a [mit-] suml/suml genotype were con- structed by reciprocal crosses of R3 and a R3/A1 and their po derivatives (Table 8). In each cross, the diploids should have the same complement of nuclear and should be homozygous for the suppressor mutation. The results of this experi- ment showed that all four crosses produced a high proportion of glycerol-positive diploids. The glycerol-negative segregants observed in the crosses were deter- mined to be p- mutants with pleiotropic respiratory deficiencies. The high pro- portion of p- mutants (approximately 50%) indicates that the suml/suml nuclear background causes an instability in mtDNA. Mitochondrial products in a M9-228-28C and R3: The mitochondrial products of the mit- mutant a M9-228-28C and the revertant R3 were characterized by in vivo labeling of the two strains with ["%I methionine in the presence of cyclo- heximide. The labeled products were analyzed by polyacrylamide gel electro- phoresis of total mitochondrial proteins depolymerized in sodium dodecyl sul- fate. The results of this experiment (Figure 1) show that the mit mutant is defi- cient in a single product corresponding to apocytochrome b. In agreement with previous data (TZAGOLOFF,FOURY and AKAI1976), this mutant has a new mito- chondrial product with a lower apparent molecular weight.

TABLE 8 Homozygous crosses of R3 and a R3/AI

Grawth of diploid segregants Genotype on glyceml Cross Input output Predicted Observed R3 X a R3/A1 [mit-]sum1 x [mit-]sum1 [mit]sumi/sumi ++ R3po x sy R3/A1 [polsum1 x [mii-]sum1 [mit]suml/suml + + R3 x a R3/Alpo [mit-]sum1x [polsud [mit]suml/sumi ,+ +

~ ~~~~ Haploid strains were mated in liquid YPD (10% glucose) and transferred to liquid WO media (.IO% glucose). Following 24 hr of prototrophic selection, the cells were spread on WO for single colonies. After 2 days growth at 30°, colonies were replicated onto YPEG media. The frequency of glycerol-positive segregants was about 50% in all cases. Glycerol-negative segre- gants (10 from each cross) were picked and their mitochondria assayed for ATPase, cytochrome oxidase and NADH-cytochrome c reductase activities (TZAGOLOFF,AKAIand NEEDLEMANi975a). All the glycerol-negative segregants assayed were found to be pleiotropically deficient in the three enzymes. CYTOCHROME b SUPPRESSOR 899 AB C

5

FIGURE1.-Mitochondrial products of D273-10B/A1, a MS228-28C and R3 were labeled with [*WJ methionine in the presence of cycloheximide as described previously (T"xo, AILUand NEEDLEMAN1975b). Mitochondria were isolated and depolymerized in 2% sodium dodecyl sulfate), 0.05 M dithiothreitol, 0.05 M sodium carbonate, 12% sucrose and 0.04% bromo- phenol blue. The samples were applied to polyacrylamide gels (7.5 to 15% polyacrylamide gradient containing 0.4 M Tris-C1 (pH 9.18) and 0.1% sodium dodecyl sulfate (CHUAand BENNOUN1975). Following electrophoresis, the gels were dried and the mitochondrial products visualized by autoradiography. Equivalent amounts of protein were added to each slot. Lane A, D273-10B/Al [mit+];lane B, a M9-228-28C [mir];lane C, a M9-228-28C/R3 [m't-]sud. The following products have been identified: (1) var 1, (2) subunit 1 of cytochrome oxidase, (3) subunit 2 of cytochrome oxidase, (4) cytochrome b, (5) new product, (6) subunit 9 of the ATPase. The identification of the bands is based on previously published results with similar gel systems (DOUGLASand Bmw1976; CAE~RALet al. 1978; CLAISSEet al. 1978).

Two significant differences are observed in the protein pattern of R3. First is a decrease of radioactivity in all the mitochondrial products. Second, while the normal apocytochrome b band is restored in part, the revertant still contains a significant amount of the prematurely terminated polypeptide. These results suggest that sum1 is only partially effective in suppressing the mit mutation. In addition, the suppressor lowers the overall protein synthetic activity of the mitochondria. 900 G. CORUZZI AND A. TZAGOLOFF

DISCUSSION The respiratory deficient mutant of Saccharomyces cerevisiae, a M9-228-28C, has previously been shown to have a mitochondrial mutation in the structural gene for cytochrome b (TZAGOLOFF,FOURY and AKAI 1976). Evidence for this conclusion included: (1) genetic linkage of the M9-228 allele to the cob2 locus of mtDNA, (2) a specific enzymatic deficiency in co-enzyme QH,-cytochrome c reductase, (3) absence of spectral cytochrome b, and (4) substitution of apo- cytochrome b by a new low molecular weight mitochondrial translation product. Recently, mutants with a similar phenotype have been described by CLAISSEet al. ( 1978), ALEXANDERet al. ( 1979) and HAIDet ai. (1979). The presence of a new mitochondrial product in a M9-228-28C suggested that the mutation caused premature chain termination due to a new termination codon generated either by a frameshift or base substitution. Therefore, the mutant was of potential usefulness in studying suppressor mutants of mitochondrial DNA. In this study, 116 revertants of a M9-228-28C were isolated and characterized genetically. The revertants were induced with either EMS or MnCl,, an effective mutagen of mtDNA (PUTRAMENT,BARANOWSKA and PRAZMO1973; TZAGOLOFF, AKAIand NEEDLEMAN1975a). The initial indicated that the revertants fell into two groups. Most of the MnCI,-induced revertants were determined to have either back rnu- tations or dominant nuclear suppressors. Some of the MnCl, and all of the EMS- induced revertants, on the other hand, had genetic properties conforming to nu- clear recessive mutants. Interestingly, none of the revertants behaved as mito- chondrial tRNA suppressors. This does not exclude the possibility that the M9- 228 mutation is of the nonsense type, since the absence of isoaccepting mitochon- drial tRNAs for many amino acids in yeast (WESOLOWSKIand FUKUHARA1979) might be expected to reduce the probability of tRNA suppressor mutations. The group of revertants preliminarily characterized as nuclear suppressor mu- tants appeared to be the most interesting class, and one representative strain (R3) was chosen for more detailed genetic and biochemical analysis. The presence in R3 of a recessive nuclear suppressor mutation was established by the following evidence: (1) Crosses of R3 to a respiratory competent wild-type strain resulted in - 50% glycerol-negative segregants with the original cytochrome b deficiency. This indicated that the suppressor muiation is extragenic. (2) A cross of R3 to a wild-type haploid strain produced tetrads in which the suppressor mutation segre- gated 2:2. The suppressor mutation was also established to be nuclear from the results of a cross to a po strain. In such a cross, only glycerol-negative segregants were found. This phenotype agreed with the nuclear recessive classification of the suppressor mutation. (3) The recessive nature of the suppressor mutation was further substantiated by the phenotypes of strains homozygous and heterozygous for the suppressor. The cytochrome b deficiency was suppressed in homozygous, but not in heterozygous, strains. Since the genetic evidence indicated that the cytochrome b mutation in R3 is suppressed by a nuclear gene product, it was of interest to examine the mitochon- CYTOCHROME b SUPPRESSOR 901 drial products formed in the suppressor strain. Analytical gel electrophoresis of the mitochondrial products in R3 showed an overall depression of mitochondrial protein synthesis and only a partial restoration of the wild-type apocytochrome b. Based on the intensity of the radioactive mitochondrial products in R3, the suppression is estimated to be at most only 50%. Nonsense suppression at the level of translation has been demonstrated in bac- terial systems (GORINIand BECKWITH1966; GAREN1968; ROSSETand GORINI 1969; PHILLIPS1971; REEVESand ROTH1971) as well as in yeast (HAWTHORNE and LEUPOLD1974; SMIRNOVet al. 1978). Informational suppressors act at some step in the translation of the mutant mRNA, allowing the synthesis of a functional polypeptide. In yeast, the most carefully studied suppressors have been found to be tRNAs (SHERMAN,ONO and STEWART1979). The mutant tRNA is capable of reading the termination codon as an amino acid codon during translation. Such suppressor mutations have been shown to act in a dominant or semidominant fashion (HAWTHORNEand LEUPOLD 1974). Mutations in ribosomal proteins can also suppress nonsense mutations. The mu- tated ribosomal protein allows nonsense mutations to be recognized as amino acid codons by altering the stringency of codon-anticodon pairing. Compared to tRNA suppressors, ribosomal protein mutants suppress a wider range of nonsense co- dons in bacteria (ROSSETand GORINI1969). In yeast, “omnipotent suppressor” mutations shown to act recessively are thought to have altered ribosomes (HAW- THORNE and LEUPOLD1974; GERLACH1976), Ribosomal suppressor mutations in bacteria have been shown to increase resistance to streptomycin (ROSSETand GORINI1969). A third type of nonsense suppression at the level of translation has been de- scribed in yeast (SMIRNOVet al. 1974; 1976; 1978). In this case, the suppressor mutation partially or completely inactivates a protein involved in polypeptide chain termination. Such mutations have been found to be recessive and tempera- ture sensitive (SMIRNOVet al. 1974; 1976; 1978). The nuclear recessive suppressor described here is novel in that it acts on the translation of a mitochondrial messenger. Since there is no evidence for the im- portation of nuclear tRNAs into mitochondria (BONITZet al. 1980), it is unlikely that suppression of the M9-228 allele is due to a cytoplasmic suppressor tRNA acting on mitochondrial translation. The recessive nature of the suppressor in R3 suggests that the mutation is in a structural or enzymatic component of the mitochondrial protein synthesizing system. It is known that most, if not all, of the mitochondrial ribosomal proteins and other protein factors required for translation are encoded by nuclear genes (SCHATZand MASON1974). A mutation in a ribosomal protein could act reces- sively if the wild-type protein is more efficiently used during ribosome assembly. A mutation in a termination factor can also be recessive if it causes a partial loss of function and is favorably competed against by the wild-type factor. The presence of both the normal and prematurely terminated apocytochrome b in R3 also tends to support suppression at the level of translation. The fact that the re- 902 G. CORUZZI AND A. TZAGOLOFF vertant still exhibits some of the low molecular weight product indicates that the read-through is not completely efficient.

This research was supported by Grant HL22174 from the Public Health Service.

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