Molec. gen. Genet. 176, 411 415 (1979) © by Springer-Verlag 1979
Sporulation of Mitochondrial Respiratory Deficient mit- Mutants of Saccharomyces cerevisiae Elke Pratje, Reinhard Schulz, Susanne Schnierer, and Georg Michaelis Universitfit Bielefeld, Fakultfit f~r Biologic, Postfach 8640, D-4800 Bielefeld 1, Federal Repubhc of Germany
Summary. The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient m i t - mutants. These mutants were isolated in an o p l strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, O X I 3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1 - cytochrome c oxidase subunit 2, O X I 2 - subunit 3, O X I 3 - subunit 1, and C O B - cytochrome b. The ability of diploid m i t - mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit + tetrads, both mit + and m i t - tetrads, or only m i t - tetrads. Mutants forming m i t - tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some m i t - mutants have retained the ability to perform meiosis and sporulation.
multaneously lack components necessary for mitochondrial protein synthesis, electron transport and oxidative phosphorylation. The availability of another class of mitochondrial respiratory deficient mutants, designated m i t - , provides an assay of mitochondrial functions required for meiosis and spore development. These m i t - mutants result from single point mutations or very small deletions in the mitochondrial genome. They have retained a functional mitochondrial protein synthesis and display specific defects either in cytochrome c oxidase, cytochrome b or in the ATPase complex (Tzagoloff et al., 1975; Slonimski and Tzagoloff, 1976). These mutants should therefore provide information regarding the role of mitochondrial protein synthesis, electron transport and specific mitochondrial gene products for meiosis and sporulation. Here we have isolated and characterized genetically and biochemically a collection of 153 m i t - mutants. All mutations representing four mitochondrial genes were analyzed for their effects on sporulation. Surprisingly, mutants were found which are capable of producing asci with four m i t - spores.
Materials and Methods Introduction
Sporulation of Saccharomyces cerevisiae is an aerobic process and seems to depend on a functional mitochondrial system (for reviews see Fowell, 1969; Haber and Halvorson, 1975; Esposito and Esposito, 1975). It is well-established that diploid, cytoplasmically inherited petite mutants are unable to sporulate. These respiratory deficient mutants, resulting from large deletions or complete loss of mitochondrial DNA, siS e n d offprint requests to" E. Pratje
Strams. The following strains of S a c c h a r o m y c e s cerevtsiae were used in this study:
777-3A: KL14~A: KL14-4A/2: GM10-2B/2: DS-22A/2:
~ a d e l o p l rho + a h i s l trp2 rho + C A P 3R2 1 0 L ~ g s P A R 4 s R 4 a hisI trp2 rho ° a lys2 rho ° c~ ade2 hts4 rho °.
The respiratory deficient m i t - mutants were isolated m strain 777 3A according to Kotylak and Slonimska (1977) The stable p e t t t e clones used for deletion mapping were derived from strain KL14-4A. The diploid strain GM50 was obtained from the cross 777-3A x KL 14~A/2.
0026-8925/79/0176/0411/$01.00
412
E. Pratje et al. : Sporulatlon of Mitochondrial Respiratory Deficient Mutants
Media. The following media were used: Y P G = I % Bacto-yeast extract, 1% Bacto-peptone, 2% glucose, solidified with 2.3% Bacto-agar; YPG10 used for growth of mit- mutants, consisted of 1% Bacto-yeast extract, 1% Bacto-peptone, 10% glucose; N3 = 1% Bacto-yeast extract, 1% Bacto-peptone, 2% glycerol, 0.1M sodium-potassium phosphate buffer pH 6.25; WO=0.67% Bacto-nitrogen base free of amino acids, 2% glucose, 50 mM sodium-potassium phosphate buffer pH 6.25, solidified with 2.3% Bacto-agar. Synthetic complete media consisted of 0.67% Bacto-yeast nitrogen base free of amino acids, 2% carbon source (lactate, glycerol, ethanol, succinate, Tween 80, or acetate), 50ram sodiumpotassium phosphate buffer pH 6.25, adenine sulfate, uracil, L-arglnine • HC1, L-histidlne • HC1, L-methionine and L-tryptophan at 20 rag/l, L-lsoleucine, L-leuclne, L-lysine.HC1 and L-tyrosine at 30 mg/1, L-phenylalanine at 50 rag/l, L-aspartac acid and L-glutamlc acid at 100 mg/1, L-valine at 150 rag/l, L-threonine at 200 mg/I and L-serine at 375 mg/1. Chloramphenicol, paromomycin or oligomycin plates were prepared by adding the drugs to autoclaved N3 agar cooled to 60°C. Final concentrations: 4 g chloramphenlcol/l, 4 g paromomycin/1, and 3 mg ohgomycin/1.
Sporulatlon. Diploid yeast strains were sporulated as described by Petersen et al. (1978). Spore formation was observed by light microscopy during an incubation period of seven days. Isolation of undisseeted tetrads and dissection of tetrads were performed usmg a de Fonbrune micromanipulator. Biochemical Analysis of Mitochondrtal Translatlon Products Mitochondrial translation products were analyzed essentially as described by Douglas and Butow (1976). Strain 777-3A and all mttmutants derived from it were crossed with the rho ° tester KL 14-4A/ 2, since a more specific labeling of mitochondrial translation products was obtained in diploid strains. Cells were grown to the late logarithmic phase and labeled in semisynthetic medium for 60 min at 28°C with 3sSO42 (200 gCi/ml) in the presence of 100gg cycloheximide/ml. Mitochondria were isolated and the proteins were separated by electrophoresis on slab gels containing 10 to 15% exponential gradients of polyacrylamide. Sodium dodecylsulfate was replaced by lithium dodecylsulfate in order to perform electrophoresls at 4°C using higher voltage. Gels treated with 2,5-diphenyloxazole to enhance the sensitivity of the procedure (Bonner and Laskey, 1974) were radioautographed with a Kodak "X-Omat" film. Oxygen Consumption. Oxygen uptake was determined polarographically at 28 ° C as described by Pratje and Michaelis (1977).
Results
Isolation and Genetic Characterization of mit- Mutants Respiratory deficient mit- mutants were isolated in strain 777-3A following the procedure of Kotylak and Slonimski (1977). This approach takes advantage of the recessive nuclear mutation opl which permits survival o f m i t - mutants and confers lethality to petite cells (Kovacova et al., 1968). MnCI2 was applied as a mutagen at a final concentration of 4 m M (Putrament et al., 1973). Colonies derived from mutagenized
cells were mated with a K L 1 4 ~ A / r h o ° tester strain. M i t - mutants could easily be identified as diploids which failed to grow on glycerol. Such colonies were found with a frequency of about 0.8%. In this study 153 purified mit- mutants, designated 777-3A/101 to 385 were isolated and examined. Complementary petite mutants were obtained from strain K L 1 4 ~ A either spontaneously or by raising the growth temperature to 37 ° C. The primary petite colonies were replica crossed with an antibiotic sensitive rho + strain and analyzed for the retention of three mitochondrial markers : chloramphenicol resistance CAP, paromomycin resistance PAR, and oligomycin resistance OLI1. For deletion mapping 56 stable petite subclones of known mitochondrial genotype were selected. The 56 petite clones were crossed with the 153 mit strains in all possible combinations and the resulting diploids were scored for growth on glycerol. Restoration of a given mit mutation depends on whether or not the mit + allele is retained by the petite tester. Figure 1 summarizes the results of deletion mapping with 27 discriminating petite testers. The 153 mit- mutations can be grouped into four different regions, OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b (Tzagoloff et al., 1975 ; Slonimski and Tzagoloff, 1976). No PHO mutant exhibiting deficiencies in oligomycin-sensitive ATPase has been found in this collection of mitisolates.
Characterization o f Mitochondrially Translated Polypeptides in mit- Mutants Mitochondrial translation products have been analyzed essentially as described by Douglas and Butow (1976). The proteins were labeled in vivo with 3sSO~ in the presence of cycloheximide. They were analyzed by electrophoresis on exponential lithium dodecylsulfate - polyacrylamide slab gels. Comparison of the protein patterns of various diploid mitmutants with wild-type GM50 showed either no alterations, an abnormal mobility of one protein, the presence of new bands not seen in the wild-type, or the specific loss of one band. Figure 2 illustrates for each of the four genetic loci one mit- mutant that lacks specifically one maj or mitochondrial translation product. The gene-polypeptide relationships are as follows: OXI3 mutant GM50/134 lacks specifically cytochrome c oxidase subunit I, OXI2 mutant GM50/385 lacks subunit III, and OXI1 mutant GM50/248 lacks subunit II, whereas cytochrome b is missing in COB mutant GM50/144. These results are consistent with recent studies on OXI3 (Slonimski
E. Pratje et al. : Sporulation of Mitochondrial Respiratory Deficient Mutants "
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Sporulation of Mitochondrial Respiratory Deficient mit- Mutants of Saccharomyces cerevisiae Elke Pratje, Reinhard Schulz, Susanne Schnierer, and Georg Michaelis Universitfit Bielefeld, Fakultfit f~r Biologic, Postfach 8640, D-4800 Bielefeld 1, Federal Repubhc of Germany
Summary. The role of mitochondrial protein synthesis, electron transport, and four specific mitochondrial gene products on sporulation were studied in respiratory deficient m i t - mutants. These mutants were isolated in an o p l strain and localized on the mitochondrial genome by petite deletion mapping. All 153 mutations studied could be assigned to the four mitochondrial regions OXI1, OXI2, O X I 3 and COB, known to affect cytochrome c oxidase and cytochrome b. The specific loss of one mitochondrially translated polypeptide was found in some mutants of each locus: OXI1 - cytochrome c oxidase subunit 2, O X I 2 - subunit 3, O X I 3 - subunit 1, and C O B - cytochrome b. The ability of diploid m i t - mutants to sporulate was systematically investigated. About one third of the mutants, representing three loci, were incapable of forming spores. All other cultures produced either respiratory competent mit + tetrads, both mit + and m i t - tetrads, or only m i t - tetrads. Mutants forming m i t - tetrads mapped in all four loci. These results demonstrate that in contrast to petite mutants some m i t - mutants have retained the ability to perform meiosis and sporulation.
multaneously lack components necessary for mitochondrial protein synthesis, electron transport and oxidative phosphorylation. The availability of another class of mitochondrial respiratory deficient mutants, designated m i t - , provides an assay of mitochondrial functions required for meiosis and spore development. These m i t - mutants result from single point mutations or very small deletions in the mitochondrial genome. They have retained a functional mitochondrial protein synthesis and display specific defects either in cytochrome c oxidase, cytochrome b or in the ATPase complex (Tzagoloff et al., 1975; Slonimski and Tzagoloff, 1976). These mutants should therefore provide information regarding the role of mitochondrial protein synthesis, electron transport and specific mitochondrial gene products for meiosis and sporulation. Here we have isolated and characterized genetically and biochemically a collection of 153 m i t - mutants. All mutations representing four mitochondrial genes were analyzed for their effects on sporulation. Surprisingly, mutants were found which are capable of producing asci with four m i t - spores.
Materials and Methods Introduction
Sporulation of Saccharomyces cerevisiae is an aerobic process and seems to depend on a functional mitochondrial system (for reviews see Fowell, 1969; Haber and Halvorson, 1975; Esposito and Esposito, 1975). It is well-established that diploid, cytoplasmically inherited petite mutants are unable to sporulate. These respiratory deficient mutants, resulting from large deletions or complete loss of mitochondrial DNA, siS e n d offprint requests to" E. Pratje
Strams. The following strains of S a c c h a r o m y c e s cerevtsiae were used in this study:
777-3A: KL14~A: KL14-4A/2: GM10-2B/2: DS-22A/2:
~ a d e l o p l rho + a h i s l trp2 rho + C A P 3R2 1 0 L ~ g s P A R 4 s R 4 a hisI trp2 rho ° a lys2 rho ° c~ ade2 hts4 rho °.
The respiratory deficient m i t - mutants were isolated m strain 777 3A according to Kotylak and Slonimska (1977) The stable p e t t t e clones used for deletion mapping were derived from strain KL14-4A. The diploid strain GM50 was obtained from the cross 777-3A x KL 14~A/2.
0026-8925/79/0176/0411/$01.00
412
E. Pratje et al. : Sporulatlon of Mitochondrial Respiratory Deficient Mutants
Media. The following media were used: Y P G = I % Bacto-yeast extract, 1% Bacto-peptone, 2% glucose, solidified with 2.3% Bacto-agar; YPG10 used for growth of mit- mutants, consisted of 1% Bacto-yeast extract, 1% Bacto-peptone, 10% glucose; N3 = 1% Bacto-yeast extract, 1% Bacto-peptone, 2% glycerol, 0.1M sodium-potassium phosphate buffer pH 6.25; WO=0.67% Bacto-nitrogen base free of amino acids, 2% glucose, 50 mM sodium-potassium phosphate buffer pH 6.25, solidified with 2.3% Bacto-agar. Synthetic complete media consisted of 0.67% Bacto-yeast nitrogen base free of amino acids, 2% carbon source (lactate, glycerol, ethanol, succinate, Tween 80, or acetate), 50ram sodiumpotassium phosphate buffer pH 6.25, adenine sulfate, uracil, L-arglnine • HC1, L-histidlne • HC1, L-methionine and L-tryptophan at 20 rag/l, L-lsoleucine, L-leuclne, L-lysine.HC1 and L-tyrosine at 30 mg/1, L-phenylalanine at 50 rag/l, L-aspartac acid and L-glutamlc acid at 100 mg/1, L-valine at 150 rag/l, L-threonine at 200 mg/I and L-serine at 375 mg/1. Chloramphenicol, paromomycin or oligomycin plates were prepared by adding the drugs to autoclaved N3 agar cooled to 60°C. Final concentrations: 4 g chloramphenlcol/l, 4 g paromomycin/1, and 3 mg ohgomycin/1.
Sporulatlon. Diploid yeast strains were sporulated as described by Petersen et al. (1978). Spore formation was observed by light microscopy during an incubation period of seven days. Isolation of undisseeted tetrads and dissection of tetrads were performed usmg a de Fonbrune micromanipulator. Biochemical Analysis of Mitochondrtal Translatlon Products Mitochondrial translation products were analyzed essentially as described by Douglas and Butow (1976). Strain 777-3A and all mttmutants derived from it were crossed with the rho ° tester KL 14-4A/ 2, since a more specific labeling of mitochondrial translation products was obtained in diploid strains. Cells were grown to the late logarithmic phase and labeled in semisynthetic medium for 60 min at 28°C with 3sSO42 (200 gCi/ml) in the presence of 100gg cycloheximide/ml. Mitochondria were isolated and the proteins were separated by electrophoresis on slab gels containing 10 to 15% exponential gradients of polyacrylamide. Sodium dodecylsulfate was replaced by lithium dodecylsulfate in order to perform electrophoresls at 4°C using higher voltage. Gels treated with 2,5-diphenyloxazole to enhance the sensitivity of the procedure (Bonner and Laskey, 1974) were radioautographed with a Kodak "X-Omat" film. Oxygen Consumption. Oxygen uptake was determined polarographically at 28 ° C as described by Pratje and Michaelis (1977).
Results
Isolation and Genetic Characterization of mit- Mutants Respiratory deficient mit- mutants were isolated in strain 777-3A following the procedure of Kotylak and Slonimski (1977). This approach takes advantage of the recessive nuclear mutation opl which permits survival o f m i t - mutants and confers lethality to petite cells (Kovacova et al., 1968). MnCI2 was applied as a mutagen at a final concentration of 4 m M (Putrament et al., 1973). Colonies derived from mutagenized
cells were mated with a K L 1 4 ~ A / r h o ° tester strain. M i t - mutants could easily be identified as diploids which failed to grow on glycerol. Such colonies were found with a frequency of about 0.8%. In this study 153 purified mit- mutants, designated 777-3A/101 to 385 were isolated and examined. Complementary petite mutants were obtained from strain K L 1 4 ~ A either spontaneously or by raising the growth temperature to 37 ° C. The primary petite colonies were replica crossed with an antibiotic sensitive rho + strain and analyzed for the retention of three mitochondrial markers : chloramphenicol resistance CAP, paromomycin resistance PAR, and oligomycin resistance OLI1. For deletion mapping 56 stable petite subclones of known mitochondrial genotype were selected. The 56 petite clones were crossed with the 153 mit strains in all possible combinations and the resulting diploids were scored for growth on glycerol. Restoration of a given mit mutation depends on whether or not the mit + allele is retained by the petite tester. Figure 1 summarizes the results of deletion mapping with 27 discriminating petite testers. The 153 mit- mutations can be grouped into four different regions, OXI1, OXI2, OXI3 and COB, known to affect cytochrome c oxidase and cytochrome b (Tzagoloff et al., 1975 ; Slonimski and Tzagoloff, 1976). No PHO mutant exhibiting deficiencies in oligomycin-sensitive ATPase has been found in this collection of mitisolates.
Characterization o f Mitochondrially Translated Polypeptides in mit- Mutants Mitochondrial translation products have been analyzed essentially as described by Douglas and Butow (1976). The proteins were labeled in vivo with 3sSO~ in the presence of cycloheximide. They were analyzed by electrophoresis on exponential lithium dodecylsulfate - polyacrylamide slab gels. Comparison of the protein patterns of various diploid mitmutants with wild-type GM50 showed either no alterations, an abnormal mobility of one protein, the presence of new bands not seen in the wild-type, or the specific loss of one band. Figure 2 illustrates for each of the four genetic loci one mit- mutant that lacks specifically one maj or mitochondrial translation product. The gene-polypeptide relationships are as follows: OXI3 mutant GM50/134 lacks specifically cytochrome c oxidase subunit I, OXI2 mutant GM50/385 lacks subunit III, and OXI1 mutant GM50/248 lacks subunit II, whereas cytochrome b is missing in COB mutant GM50/144. These results are consistent with recent studies on OXI3 (Slonimski
E. Pratje et al. : Sporulation of Mitochondrial Respiratory Deficient Mutants "
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