Current Genetics
Current Genetics 1, 97-102 (1980)
©
by Springer-Verlag 1980
Sporulation in Mitochondrial OXI3 Mutants of Saccharomyces cerevisiae A C o r r e l a t i o n w i t h t h e G e n e t i c Map A. Hartig and M. Breitenbach Institut for Allgemeine Biochemie and Ludwig Boltzmann-Forschungsstelle for Bioehemie, W~ihringerStrasse 38, A-1090 Wien, Austria
Summary. By use o f a set o f mitochondrial oxi3 mutants ( m i t - , defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level o f respiration (below 1% o f the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation o f these results with the genetic map o f the 0 X I 3 region reveals that one group o f mutants, mapping in the central part of the OXI3 region, is capable o f sporulation.
functional cytochrome oxidase. In pulse labelling experiments subunit I o f cytochrome oxidase is usually not detected, while the other mitochondrially encoded polypeptides are present in their normal amounts. Most mutations involved in our study block sporulation completely, whereas some still exhibit sporulation, although at reduced rates. The latter constitute a distinct cluster of mutational sites on the fine structure map of the OXI3 region as published b y Schweyen et al. (1977).
Key words: Sporulation - R e s p i r a t i o n - OXI3 gene cluster - Mitochondrial genome.
Materials and Methods
Strains. The strains used in the present study are summarized Introduction It has been known since the early days o f spore research, that yeast sporulation depends on oxygen (Hansen, 1902). This was confirmed in more detail later (Miller et al., 1957) b u t the exact amount o f oxygen required for sporulation and the exact nature of the metabolic reaction(s) wherein oxygen is involved remain to be discovered. Ephrussi and Hottinguer, (1951) showed that respiratory deficient yeast cells, obtained b y treatment with acriflavine (rho- petites), are unable to sporulate. It was generally believed that this is due to the lack o f chemical energy (ATP production b y respiration) in these cells. We will present data on the sporutation in m i t mutants which - in contrast to r h o - mutants - are defective in one or the other mitochondrial function only (Slonimski and Tzagoloff, 1976). For this study we have chosen a series o f mutations which map in the 0X13 region o f mitDNA 1. These oxi3 mutants lack
Offprint requests to: M. Breitenbach 1 mitDNA = mitochondrial DNA
in Table 1.
Media. The following mixtures were used as liquid media or with 2% agar. All ingredients were from Difco (Detroit, U.S.A.). YPD: 2% Glucose, 2% Bacto-peptone, 1% yeast extract. SD: 0.67% yeast nitrogen base without amino acids, 2% glucose. YPG: 3% glycerol, 2% bacto-peptone, 1% yeast extract. Sporulation medium 1: 1% potassium acetate; sporulation medium 2: 1% potassium aeetate, 0.1% yeast extract, 0.05% glucose.
Tetrad Analysis was performed with a mieromanipulator built by Dr. C. Bieglmayer of our laboratory. Asei were suspended in glusulase (ENDO Laboratories, Garden City, N.Y, USA), diluted 1:40 with sterile water for 30 rain, streaked onto agarose and dissected with a microneedle. Oxygen Measurement. A YSI oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH, USA Type: 53) was used. All measurements were performed at exactly 30 °C in a volume of 3 ml. In order to increase the sensitivity of the assay a high cell density (5 x 107 cells/ml) was used. The oxygen uptake per 106 cells and per 1 rain was calculated based on a concentration of 243 nmoles/ml of 02 in pure water at saturation with air at 30 °C (Landolt-BSrnstein, 1962). The electrode was calibrated with pure air-saturated water at 30 °C before each measurement. The detection limit of our method was determined by serially diluting fully respiratory adapted cells of the diploid V266 and measuring at each dilution. The data were O172-8083/80/0001/0097/$ 01.20
98
A. Hartig and M. Breitenbach: Sporulation in Mitochondrial OXI3 Mutants
Table 1 Strain
777-3A
Genotype Nuclear
Mitochondrial
a, adel, opl
rho +, mit +
Origin
Remarks
derived from 777-3A
oxi3-M6331 a
~, adel, opl
rho +, m i t -
Munich collection
KL 14-4A
a, his 1, trp 2
rho °
Munich collection
F 101- lb
a, leu 2
rho °
our laboratory
F 102-2 b
a, leu2
rho °
our laboratory
V266 c
a his1 +
trp2 +
+ adel
+ opl
rho~ mit ÷
our laboratory
777-3A x KL14-4A rho ° used as a wild type control
V266Crho °
a hisl a +
trp2 +
+ adel
+ opl
rho °
our laboratory
V266 treated with EtBr d, used as a rho ° control
V266 -M6331e
a hisl +
trp2 +
+ adel
+ opl
rho~ m i t -
our laboratory
oxi3-M6 3 31 x KL14-4A rho °
a oxi3-M6331 is one of 25 haploid oxi3 m i t - strains which were used for the present study. All strains are named in a similar way by a four digit number. b strains F101-1 and F102-2 were obtained from crosses of D273-10B with E-28/1 (D273-10B is identical w i t h A T C C 2 4 6 5 7 , E28/1 is a gift of the organizers of the Cold Spring Harbor yeast genetics course). R h o - -induction was performed by growing the strains in the presence of 100 ppm EtBr. The absence of mitDNA was shown by DAPI e fluorescence microscopy (Williamson and Fennell, 1975). c The rho ° mating partners in the crosses were chosen because they are non-suppressive. As they carry no mitochondrial genetic information, pure mitochondrial genotypes can be expected in the primary diploids. V266 rho ° was obtained by EtBr treatment as described above. A set of 25 diploid oxi3 m i t - mutants were tested for their sporulation properties. A detailed list of them is given in Table 2. d EtBr = Ethidium bromide e DAPI = 4',6-diamidino, 2-phenyl indole
treated according to the literature (Currie, 1969) and the absolute detection limit was determined to be 1 nmole/min. At the cell density used, this corresponds to about 1% of the respiration of the wild type, or 6 pmoles/min x 106 cells. Catalase A Determination was carried out on polyacrylamide gels as described by Ruis and coworkers (Susani et al., 1976). Determination o f Genotypes. The nuclear genotypes of the haploids obtained from the sporulation of diploid m i t - strains were determined in the usual way by plating on selective media and checking the mating type with appropriate tester strains. The mitochondrial genotypes of the above mentioned haploids were tested by plating on YPG and by crossing with appropriate rho ° tester strains. The mitochondrial genotypes of single colony isolates of the series of diploid oxi3 m # - strains had to be determined because r h o - production usually occured. R h o - and m i t - colonies were distinguished by three criteria: a) colony size, b) ability to revert at a low frequency, and c) ability to respire at a very low residual level (see results).
Results G r o w t h o n YPG, M i t o c h o n d r i a l G e n o t y p e o f t h e Diploids. The results o b t a i n e d w i t h 27 m i t - diploids series
o f o x i 3 m u t a n t s are summarized in Table 2. I n c l u d e d are t w o controls: Strain V 2 6 6 and V 2 6 6 r h o °. The diploids were cloned on solid SD and single c o l o n y isolates checked for their m i t - or r h o - genotypes. The r h o - frequencies were b e t w e e n 1 and 50%. The reversion frequencies o f the m i t - single c o l o n y isolates were b e l o w 0.0002% after t w o days g r o w t h in YPD. As the revertants can a c c u m u l a t e even o n YPD, fresh single c o l o n y isolates were used for every n e w e x p e r i m e n t . S o m e o f t h e diploids were leaky as indicated in Table 2, i.e. t h e y exhibit very slow g r o w t h o n liquid or solid YPG. S p o r u l a t i o n ability: The diploid strains were tested on solid sporulation m e d i a 1 and 2. U n d e r these conditions isogenic wild t y p e ceils sporulated to a b o u t 20%. V 2 6 6 r h o ° never showed a single ascus in several t h o u sand cells. S o m e o f t h e n o n - l e a k y o x i 3 diploids s h o w e d a l o w ( 5 - 1 5 % ) b u t reproducible yield o f asci o n sporulat i o n m e d i u m 2, but n o t o n m e d i u m 1. F o r these strains, sporulation ability correlated well w i t h the d e l e t i o n map: The m u t a n t s located in the three central positions o f the m a p are able to sporulate, the m u t a n t s m a p p e d to the right and t o the left o f t h e m are unable to sporulate (Fig. 1). As sporulation m e d i u m 2 contains b o t h l o w
99
A. Hartig and M. Breitenbach: Sporulation in Mitochondfial OXI3 Mutants Table 2 Diploid strain
V266 rho + V266 rho ° V266-M4071 V266-M4191 V266-M6351 V266-M7351 V266-M2131 V266-M2111 V266-M 1702 V266-M2031 V266-M4051 V266-M4021 V266-M6331 V266-M6201 V266-M4061 V266-M3792 V266-M6321 V266-M6431 V266-M3831 V266-M3861 V266-M4121 V266-M3781 V266-M6111 V266-M6291 V266-M6121 V266-M4031 V266-M6011
a b c d
Leakiness (growth on YPG)
wild type + + + + + +
Oxygen uptake a pmoles/min x 106 cells
% ofwildtype
469 5 10,6
Current Genetics 1, 97-102 (1980)
©
by Springer-Verlag 1980
Sporulation in Mitochondrial OXI3 Mutants of Saccharomyces cerevisiae A C o r r e l a t i o n w i t h t h e G e n e t i c Map A. Hartig and M. Breitenbach Institut for Allgemeine Biochemie and Ludwig Boltzmann-Forschungsstelle for Bioehemie, W~ihringerStrasse 38, A-1090 Wien, Austria
Summary. By use o f a set o f mitochondrial oxi3 mutants ( m i t - , defective in cytochrome oxidase) we have shown that sporulation is possible at a very low level o f respiration (below 1% o f the wild type respiration). A specific role for oxygen in biosynthesis during sporulation is suggested. Correlation o f these results with the genetic map o f the 0 X I 3 region reveals that one group o f mutants, mapping in the central part of the OXI3 region, is capable o f sporulation.
functional cytochrome oxidase. In pulse labelling experiments subunit I o f cytochrome oxidase is usually not detected, while the other mitochondrially encoded polypeptides are present in their normal amounts. Most mutations involved in our study block sporulation completely, whereas some still exhibit sporulation, although at reduced rates. The latter constitute a distinct cluster of mutational sites on the fine structure map of the OXI3 region as published b y Schweyen et al. (1977).
Key words: Sporulation - R e s p i r a t i o n - OXI3 gene cluster - Mitochondrial genome.
Materials and Methods
Strains. The strains used in the present study are summarized Introduction It has been known since the early days o f spore research, that yeast sporulation depends on oxygen (Hansen, 1902). This was confirmed in more detail later (Miller et al., 1957) b u t the exact amount o f oxygen required for sporulation and the exact nature of the metabolic reaction(s) wherein oxygen is involved remain to be discovered. Ephrussi and Hottinguer, (1951) showed that respiratory deficient yeast cells, obtained b y treatment with acriflavine (rho- petites), are unable to sporulate. It was generally believed that this is due to the lack o f chemical energy (ATP production b y respiration) in these cells. We will present data on the sporutation in m i t mutants which - in contrast to r h o - mutants - are defective in one or the other mitochondrial function only (Slonimski and Tzagoloff, 1976). For this study we have chosen a series o f mutations which map in the 0X13 region o f mitDNA 1. These oxi3 mutants lack
Offprint requests to: M. Breitenbach 1 mitDNA = mitochondrial DNA
in Table 1.
Media. The following mixtures were used as liquid media or with 2% agar. All ingredients were from Difco (Detroit, U.S.A.). YPD: 2% Glucose, 2% Bacto-peptone, 1% yeast extract. SD: 0.67% yeast nitrogen base without amino acids, 2% glucose. YPG: 3% glycerol, 2% bacto-peptone, 1% yeast extract. Sporulation medium 1: 1% potassium acetate; sporulation medium 2: 1% potassium aeetate, 0.1% yeast extract, 0.05% glucose.
Tetrad Analysis was performed with a mieromanipulator built by Dr. C. Bieglmayer of our laboratory. Asei were suspended in glusulase (ENDO Laboratories, Garden City, N.Y, USA), diluted 1:40 with sterile water for 30 rain, streaked onto agarose and dissected with a microneedle. Oxygen Measurement. A YSI oxygen electrode (Yellow Springs Instrument Co., Yellow Springs, OH, USA Type: 53) was used. All measurements were performed at exactly 30 °C in a volume of 3 ml. In order to increase the sensitivity of the assay a high cell density (5 x 107 cells/ml) was used. The oxygen uptake per 106 cells and per 1 rain was calculated based on a concentration of 243 nmoles/ml of 02 in pure water at saturation with air at 30 °C (Landolt-BSrnstein, 1962). The electrode was calibrated with pure air-saturated water at 30 °C before each measurement. The detection limit of our method was determined by serially diluting fully respiratory adapted cells of the diploid V266 and measuring at each dilution. The data were O172-8083/80/0001/0097/$ 01.20
98
A. Hartig and M. Breitenbach: Sporulation in Mitochondrial OXI3 Mutants
Table 1 Strain
777-3A
Genotype Nuclear
Mitochondrial
a, adel, opl
rho +, mit +
Origin
Remarks
derived from 777-3A
oxi3-M6331 a
~, adel, opl
rho +, m i t -
Munich collection
KL 14-4A
a, his 1, trp 2
rho °
Munich collection
F 101- lb
a, leu 2
rho °
our laboratory
F 102-2 b
a, leu2
rho °
our laboratory
V266 c
a his1 +
trp2 +
+ adel
+ opl
rho~ mit ÷
our laboratory
777-3A x KL14-4A rho ° used as a wild type control
V266Crho °
a hisl a +
trp2 +
+ adel
+ opl
rho °
our laboratory
V266 treated with EtBr d, used as a rho ° control
V266 -M6331e
a hisl +
trp2 +
+ adel
+ opl
rho~ m i t -
our laboratory
oxi3-M6 3 31 x KL14-4A rho °
a oxi3-M6331 is one of 25 haploid oxi3 m i t - strains which were used for the present study. All strains are named in a similar way by a four digit number. b strains F101-1 and F102-2 were obtained from crosses of D273-10B with E-28/1 (D273-10B is identical w i t h A T C C 2 4 6 5 7 , E28/1 is a gift of the organizers of the Cold Spring Harbor yeast genetics course). R h o - -induction was performed by growing the strains in the presence of 100 ppm EtBr. The absence of mitDNA was shown by DAPI e fluorescence microscopy (Williamson and Fennell, 1975). c The rho ° mating partners in the crosses were chosen because they are non-suppressive. As they carry no mitochondrial genetic information, pure mitochondrial genotypes can be expected in the primary diploids. V266 rho ° was obtained by EtBr treatment as described above. A set of 25 diploid oxi3 m i t - mutants were tested for their sporulation properties. A detailed list of them is given in Table 2. d EtBr = Ethidium bromide e DAPI = 4',6-diamidino, 2-phenyl indole
treated according to the literature (Currie, 1969) and the absolute detection limit was determined to be 1 nmole/min. At the cell density used, this corresponds to about 1% of the respiration of the wild type, or 6 pmoles/min x 106 cells. Catalase A Determination was carried out on polyacrylamide gels as described by Ruis and coworkers (Susani et al., 1976). Determination o f Genotypes. The nuclear genotypes of the haploids obtained from the sporulation of diploid m i t - strains were determined in the usual way by plating on selective media and checking the mating type with appropriate tester strains. The mitochondrial genotypes of the above mentioned haploids were tested by plating on YPG and by crossing with appropriate rho ° tester strains. The mitochondrial genotypes of single colony isolates of the series of diploid oxi3 m # - strains had to be determined because r h o - production usually occured. R h o - and m i t - colonies were distinguished by three criteria: a) colony size, b) ability to revert at a low frequency, and c) ability to respire at a very low residual level (see results).
Results G r o w t h o n YPG, M i t o c h o n d r i a l G e n o t y p e o f t h e Diploids. The results o b t a i n e d w i t h 27 m i t - diploids series
o f o x i 3 m u t a n t s are summarized in Table 2. I n c l u d e d are t w o controls: Strain V 2 6 6 and V 2 6 6 r h o °. The diploids were cloned on solid SD and single c o l o n y isolates checked for their m i t - or r h o - genotypes. The r h o - frequencies were b e t w e e n 1 and 50%. The reversion frequencies o f the m i t - single c o l o n y isolates were b e l o w 0.0002% after t w o days g r o w t h in YPD. As the revertants can a c c u m u l a t e even o n YPD, fresh single c o l o n y isolates were used for every n e w e x p e r i m e n t . S o m e o f t h e diploids were leaky as indicated in Table 2, i.e. t h e y exhibit very slow g r o w t h o n liquid or solid YPG. S p o r u l a t i o n ability: The diploid strains were tested on solid sporulation m e d i a 1 and 2. U n d e r these conditions isogenic wild t y p e ceils sporulated to a b o u t 20%. V 2 6 6 r h o ° never showed a single ascus in several t h o u sand cells. S o m e o f t h e n o n - l e a k y o x i 3 diploids s h o w e d a l o w ( 5 - 1 5 % ) b u t reproducible yield o f asci o n sporulat i o n m e d i u m 2, but n o t o n m e d i u m 1. F o r these strains, sporulation ability correlated well w i t h the d e l e t i o n map: The m u t a n t s located in the three central positions o f the m a p are able to sporulate, the m u t a n t s m a p p e d to the right and t o the left o f t h e m are unable to sporulate (Fig. 1). As sporulation m e d i u m 2 contains b o t h l o w
99
A. Hartig and M. Breitenbach: Sporulation in Mitochondfial OXI3 Mutants Table 2 Diploid strain
V266 rho + V266 rho ° V266-M4071 V266-M4191 V266-M6351 V266-M7351 V266-M2131 V266-M2111 V266-M 1702 V266-M2031 V266-M4051 V266-M4021 V266-M6331 V266-M6201 V266-M4061 V266-M3792 V266-M6321 V266-M6431 V266-M3831 V266-M3861 V266-M4121 V266-M3781 V266-M6111 V266-M6291 V266-M6121 V266-M4031 V266-M6011
a b c d
Leakiness (growth on YPG)
wild type + + + + + +
Oxygen uptake a pmoles/min x 106 cells
% ofwildtype
469 5 10,6
