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ASSEMBLY of the MITOCHONDRIAL MEMBRANE SYSTEM: NUCLEAR SUPPRESSION of a CYTOCHROME B MUTATION in YEAST MITOCHONDRIAL DNA

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ASSEMBLY of the MITOCHONDRIAL MEMBRANE SYSTEM: NUCLEAR SUPPRESSION of a CYTOCHROME B MUTATION in YEAST MITOCHONDRIAL DNA ASSEMBLY OF THE MITOCHONDRIAL MEMBRANE SYSTEM: NUCLEAR SUPPRESSION OF A CYTOCHROME b MUTATION 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 mutant 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 mutants 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 gene 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 protein involved in the mitochondrial translation 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 mutations, 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 proteins 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 phenotype. 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.
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