Molec. gen. Genet. 151, 127-136 (1977) © by Springer-Verlag 1977
Biogenesis of Mitochondria 48 Mikamycin Resistance in Saccharomyces cererisiae-A Mitochondrial Mutation Conferring Resistance to an Antimycin A-Like Contaminant in Mikamycin D.J. Groot Obbink*, T.W. SpithilL1, R.J. Maxwell, and Anthony W. Linnane Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia
Summary. Commercial preparations of mikamycin have been shown to act as both mhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds. A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Complex III. The mitochondrial [mikl-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mikl has therefore been renamed anal to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the anal determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the anal locus is part of a gene specifying a membrane component of Complex III.
Introduction Antibiotic resistance mutations have been extensively used to study and differentiate the nuclear and extrachromosomal genetic systems such as mitochondria and chloroplasts in a variety of organisms. The prime effect of ~mtibiotics such as chloramphenicol, mikamycin, erythromycin, lincomycin, spiramycin, paromomycin and neomycin, is the inhibition of protein * Current address." Microbiology Department, Royal North Shore Hospital, St. Leonards, N.S.W. 2065, Australia
synthesis in mitochondria, chloroplasts and prokaryotes. At low levels these antibiotics have no effect on eukaryotic 80S-type ribosomes. In addition, chloramphenicol, paromomycin and mikamycin at higher concentrations inhibit mitochondrial respiration (Firkin and Linnane, 1968; Dixon et al., 1971; Towers et al., 1972). Commercial mikamycin, nominally a two component mixture of mikamycin A and mikamycin B, inhibits mitochondrial protein synthesis in vitro at 1 pg/ml and mitochondrial respiration at 25-50 pg/ml (Dixon et al., 1971). This observation indicated that mikamycin may have two modes or sites of action; one at the level of the mitochondrial ribosome and the other at the mitochondrial membrane-bound respiratory system itself. An examination of this dual effect is the subject of this paper. Howell et al. (1974) previously showed that in vivo mikamycin resistance in yeast was the result of two synergistic mutations; one a nuclear mutation and the other a mitochondrial mutation. Whereas the nuclear mutation conferred resistance to mikamycin up to a level of 25 pg/ml and showed cross resistance to chloramphenicol, tetracycline and oligomycin, the mitochondrial mutation conferred resistance only to low levels of mikamycin (8 Ixg/ml) and showed no cross resistance to other antibiotics tested. The two mutations together conferred resistance to at least 800 pg/ml mikamycin; the biochemical basis of this phenomenon was not understood. Recent genetic mapping studies have shown that the mitochondrial [mikl-r] mutation maps between two oligomycin resistance determinants which are within genes involved in the determination of membrane components of mitochondrial ATPase, and close to the cybl locus which is the presumptive structural gene for cytochrome b (Trembath et al., 1976; Groot Obbink et al., 1976; Cobon et al., 1976; Nagley et al., 1976). This region of the mitochondrial genome is widely separated from the two separate regions
128
which contain the ribosomal [eryl-r] and [parl-r] mutations respectively (Sriprakash et al., 1976a; Nagley et al., 1976; Choo et al., 1976). This suggested that the effect of the [mikl-r] mutation may involve a mitochondrial membrane component, as originally proposed by Bunn et al. (1970), and not a ribosomal component. In this paper, we report that yeast cells are essentially impermeable to the A and B components of mikamycin. The [mikl-r] mutation is in fact a mutation which confers resistance to a minor antimycin A-like contaminant present in commercial mikamycin preparations and cross resistance to antimycin A. The mikI locus may be associated with a membrane component of Complex III.
D.J. Groot Obbink et al. : Mikamycin Resistance in Yeast
In vitro Mitochondrial Protein Synthesis In vitro mitochondrial protein synthesis was carried out by the method of Lamb et al. (1968) with some modifications to the composition of the incubation mixture as described below. The uptake of 14C-leucine into trichloracetic acid precipitable material was measured in 0.5 ml of incubation medium which contained 35 mM Hepes (N-2-hydroxyethylpiperaziue-N'-2-ethanesulphonic acid) buffer pH 7.4, 100 mM potassium chloride, 13 mM magnesium chloride, 60 mM sorbitol, 1.5 mM ATP, 5 mM phosphoenolpyruvate, 25gg/ml pyruvate kinase, 1 mg/ml mitochondrial protein, 0.4 gCi/ml 14C-leucine (348 mC/mmole), 60 gg/ml oligomycin and 336 gM total concentration of the 12C-amino acid mixture of Roodyn et al. (1961). Radioactivity was determined in a Phillips liquid-scintillation analyser. The protein synthetic activity was expressed as dpm 14C-leucine incorporated per mg protein per 20 min. pIs0 is defined as the concentration of a particular antibiotic required to give 50% of the maximal inhibition obtained with that antibiotic in the sensitive strain.
Materials and Methods
Ribosomal Protein Synthesis
Materials
The isolation of mitochondrial ribosomes and assay Of ribosomal poly U-directed polyphenylalanine synthesis will be described fully elsewhere (T.W. Spithill et al., in preparation). The key steps in the extraction procedure are as follows: lysis of mitochondria by addition of Triton X-100 in the presence of 0.5 M NH4CI and removal of debris by centrifugation (Plummer et al., 1973); collection Of ribosomes (as subunits) by centrifugation of the cleared lysate through a dense sucrose cushion containing 0.2% Triton X-100 and 0.15 M NH4C1. The resuspended ribosomes were used in the poly U-directed polyphenylalanine synthesis system of Modolell (1971). Activity was expressed as pmoles 14C-phenylalanine incorporated per mg ribosomal RNA per 15 rain, assuming 1 mg RNA=25A260 units.
Antibiotics were obtained from the following sources: Mikamycin (Cal): B grade, lot 902028, 90% pure, 89% mikamycin A, 11% mikamycin B. Calbiochem, Ca., U.S.A.; Mikamycin (Ka): 95% pure, 93% mikamycin A, 7% mikamycin B. Kanegafuchi Chemical Industrial Co. Ltd., Japan; Vernamycin A: batch 5A, E.R. Squibb, N.J., U.S.A. ; Antimycin A: Type III, lot 85C-0243, cycloheximide: lot 320-2640 and oligomycin, lot 75C-0136, Sigma Chemical Co., St. Louis, Mo., U.S.A. Radiochemicals were obtained from the Radiochemical Centre, Amersham, U.K. Silica gel 60HR was obtained from E. Merck A.G. Darmstadt, W. Germany. All other materials were of analytical grade.
Strains The auxotrophic haploid strains of Saccharomyees cerevisiae used in this study are characterized by the following genotypes: L4303 a his lys2 MIK-S [mikl-r] and L4175 ~ ural lys2 MIK-S [mikl-s]. MIKand mikl refer to nuclear and mitochondrial mikamycin resistance determining loci respectively.
Growth of Cells Cells were grown with forced aeration in 11 1. fermentors to a final cell density of 2-3 mg dry weight of cells/ml in media containing 1% ethanol, 1% yeast extract and a salts solution (Wallace et al., 1968). The following additional supplements were added to the medium: for strain L4303, 50 gg/ml each of histidine and lysine; for strain L4175, 50 gg/ml each of uracil and lysine.
Isolation of Mitochondria Mitochondria were isolated by the method of Cobon et al. (1974) with the modification that the mitochondria were washed three times by centrifugation rather than gradient purified.
Thin Layer Chromatography ( TLC) and Bioassay of Mikamycin Preparations Antibiotics were separated by one-dimensional TLC on silica gel 60HR plates with two different solvent systems: chloroform: methanol (9:1) and ethanol:water (3:1). Developed and dried plates were overlaid with a suspension of cells of strain L4175 in soft molten nutrient agar containing 1% yeast extract, 2% peptone, 1% ethanol and 0.5% agar (YEPE). These plates were then incubated at 28 ° C for 48 h and zones of inhibition of growth were recorded.
Assay for Antibiotic Sensitivity on YEPE Plates Cells of strains L4175 and L4303 were suspended in soft molten nutrient agar (as above) and were separately overlaid on YEPE plates containing 0.9% agar. Paper discs (1 cm diameter) containing known amounts of antibiotic were placed on the agar and the plates were incubated at 28 ° C for 48 h. Resistance to different levels of antibiotics was qualitatively determined by observing zones of inhibition of growth around the discs.
In vivo Mitochondrial Protein Synthesis
Respiration
Cycloheximide (CHX) resistant incorporation of 3H-leucine into yeast cells was carried out as described by Tzagoloff et al. (1975).
Mitochondrial respiration was measured at 28 ° C with a Clarke oxygen electrode. The reaction medium (2 ml) contained 0.6 M
D.J. Groot Obbink et al. : Mikamycin Resistance in Yeast sucrose, 10 m M K phosphate buffer p H 7.4, 1.0 m M E D T A and 0.4 mg mitochondrial protein. Either succinate (10 gmoles) or ethanol (200 gmoles) was added as substrate and State III respiration was determined by the addition of A D P (0.5 gmoles). The effect of mikamycin and antimycin A on respiration was tested as follows. Antibiotics at the concentrations indicated in the text were added to the reaction medium and pre-incubated for 10 min at 0 ° C. The reaction was initiated by the addition of substrate and incubating at 28 ° C. Where indicated, the artificial electron acceptor T M P D (N,N,N1,Nl-tetra methyl-p-phenylenediamine) was added at a final concentration of 0.35 m M .
140 •
129
Cytochrome Spectra Cytochrome spectra of isolated mitochondria were measured in a Cary model 14 double beam recording spectrophotometer at 20 ° C. The effect of mikamycin and antimycin A on the oxidation/ reduction state of the cytochromes was measured as follows. Mitochondria were suspended at a protein concentration of 5 mg/ml in the same incubation mixture (1 ml) as described for respiration and added to the test and reference cuvettes. Mitochondrial cytochromes in the test cuvette were reduced following the addition of succinate (5 ~tmoles) and A D P (0.25 gmoles). The antibiotics at
140]
A.
120
120~,~
100
~.u
B.
• toot >
T80, u
8o
Biogenesis of Mitochondria 48 Mikamycin Resistance in Saccharomyces cererisiae-A Mitochondrial Mutation Conferring Resistance to an Antimycin A-Like Contaminant in Mikamycin D.J. Groot Obbink*, T.W. SpithilL1, R.J. Maxwell, and Anthony W. Linnane Department of Biochemistry, Monash University, Clayton, Victoria, 3168, Australia
Summary. Commercial preparations of mikamycin have been shown to act as both mhibitors of mitochondrial protein synthesis and respiration. These preparations are shown to consist of two major streptogramin components (mikamycin A and mikamycin B) and a number of minor components. The major streptogramin components which inhibit mitochondrial protein synthesis in vitro are without effect in vivo due to whole cell impermeability to these compounds. A minor antimycin A-like component is the active compound in mikamycin preparations which inhibits growth of yeast cells on ethanol. The site of this inhibition is at the level of respiratory Complex III. The mitochondrial [mikl-r] mutation confers resistance to this minor growth inhibitory component and cross resistance to antimycin A. For clarity the designation mikl has therefore been renamed anal to denote the mitochondrial determinant conferring resistance to antimycin A. Genetic and physical mapping studies localise the anal determinant in the region of mitochondrial DNA specifying cytochrome b. It is proposed that the anal locus is part of a gene specifying a membrane component of Complex III.
Introduction Antibiotic resistance mutations have been extensively used to study and differentiate the nuclear and extrachromosomal genetic systems such as mitochondria and chloroplasts in a variety of organisms. The prime effect of ~mtibiotics such as chloramphenicol, mikamycin, erythromycin, lincomycin, spiramycin, paromomycin and neomycin, is the inhibition of protein * Current address." Microbiology Department, Royal North Shore Hospital, St. Leonards, N.S.W. 2065, Australia
synthesis in mitochondria, chloroplasts and prokaryotes. At low levels these antibiotics have no effect on eukaryotic 80S-type ribosomes. In addition, chloramphenicol, paromomycin and mikamycin at higher concentrations inhibit mitochondrial respiration (Firkin and Linnane, 1968; Dixon et al., 1971; Towers et al., 1972). Commercial mikamycin, nominally a two component mixture of mikamycin A and mikamycin B, inhibits mitochondrial protein synthesis in vitro at 1 pg/ml and mitochondrial respiration at 25-50 pg/ml (Dixon et al., 1971). This observation indicated that mikamycin may have two modes or sites of action; one at the level of the mitochondrial ribosome and the other at the mitochondrial membrane-bound respiratory system itself. An examination of this dual effect is the subject of this paper. Howell et al. (1974) previously showed that in vivo mikamycin resistance in yeast was the result of two synergistic mutations; one a nuclear mutation and the other a mitochondrial mutation. Whereas the nuclear mutation conferred resistance to mikamycin up to a level of 25 pg/ml and showed cross resistance to chloramphenicol, tetracycline and oligomycin, the mitochondrial mutation conferred resistance only to low levels of mikamycin (8 Ixg/ml) and showed no cross resistance to other antibiotics tested. The two mutations together conferred resistance to at least 800 pg/ml mikamycin; the biochemical basis of this phenomenon was not understood. Recent genetic mapping studies have shown that the mitochondrial [mikl-r] mutation maps between two oligomycin resistance determinants which are within genes involved in the determination of membrane components of mitochondrial ATPase, and close to the cybl locus which is the presumptive structural gene for cytochrome b (Trembath et al., 1976; Groot Obbink et al., 1976; Cobon et al., 1976; Nagley et al., 1976). This region of the mitochondrial genome is widely separated from the two separate regions
128
which contain the ribosomal [eryl-r] and [parl-r] mutations respectively (Sriprakash et al., 1976a; Nagley et al., 1976; Choo et al., 1976). This suggested that the effect of the [mikl-r] mutation may involve a mitochondrial membrane component, as originally proposed by Bunn et al. (1970), and not a ribosomal component. In this paper, we report that yeast cells are essentially impermeable to the A and B components of mikamycin. The [mikl-r] mutation is in fact a mutation which confers resistance to a minor antimycin A-like contaminant present in commercial mikamycin preparations and cross resistance to antimycin A. The mikI locus may be associated with a membrane component of Complex III.
D.J. Groot Obbink et al. : Mikamycin Resistance in Yeast
In vitro Mitochondrial Protein Synthesis In vitro mitochondrial protein synthesis was carried out by the method of Lamb et al. (1968) with some modifications to the composition of the incubation mixture as described below. The uptake of 14C-leucine into trichloracetic acid precipitable material was measured in 0.5 ml of incubation medium which contained 35 mM Hepes (N-2-hydroxyethylpiperaziue-N'-2-ethanesulphonic acid) buffer pH 7.4, 100 mM potassium chloride, 13 mM magnesium chloride, 60 mM sorbitol, 1.5 mM ATP, 5 mM phosphoenolpyruvate, 25gg/ml pyruvate kinase, 1 mg/ml mitochondrial protein, 0.4 gCi/ml 14C-leucine (348 mC/mmole), 60 gg/ml oligomycin and 336 gM total concentration of the 12C-amino acid mixture of Roodyn et al. (1961). Radioactivity was determined in a Phillips liquid-scintillation analyser. The protein synthetic activity was expressed as dpm 14C-leucine incorporated per mg protein per 20 min. pIs0 is defined as the concentration of a particular antibiotic required to give 50% of the maximal inhibition obtained with that antibiotic in the sensitive strain.
Materials and Methods
Ribosomal Protein Synthesis
Materials
The isolation of mitochondrial ribosomes and assay Of ribosomal poly U-directed polyphenylalanine synthesis will be described fully elsewhere (T.W. Spithill et al., in preparation). The key steps in the extraction procedure are as follows: lysis of mitochondria by addition of Triton X-100 in the presence of 0.5 M NH4CI and removal of debris by centrifugation (Plummer et al., 1973); collection Of ribosomes (as subunits) by centrifugation of the cleared lysate through a dense sucrose cushion containing 0.2% Triton X-100 and 0.15 M NH4C1. The resuspended ribosomes were used in the poly U-directed polyphenylalanine synthesis system of Modolell (1971). Activity was expressed as pmoles 14C-phenylalanine incorporated per mg ribosomal RNA per 15 rain, assuming 1 mg RNA=25A260 units.
Antibiotics were obtained from the following sources: Mikamycin (Cal): B grade, lot 902028, 90% pure, 89% mikamycin A, 11% mikamycin B. Calbiochem, Ca., U.S.A.; Mikamycin (Ka): 95% pure, 93% mikamycin A, 7% mikamycin B. Kanegafuchi Chemical Industrial Co. Ltd., Japan; Vernamycin A: batch 5A, E.R. Squibb, N.J., U.S.A. ; Antimycin A: Type III, lot 85C-0243, cycloheximide: lot 320-2640 and oligomycin, lot 75C-0136, Sigma Chemical Co., St. Louis, Mo., U.S.A. Radiochemicals were obtained from the Radiochemical Centre, Amersham, U.K. Silica gel 60HR was obtained from E. Merck A.G. Darmstadt, W. Germany. All other materials were of analytical grade.
Strains The auxotrophic haploid strains of Saccharomyees cerevisiae used in this study are characterized by the following genotypes: L4303 a his lys2 MIK-S [mikl-r] and L4175 ~ ural lys2 MIK-S [mikl-s]. MIKand mikl refer to nuclear and mitochondrial mikamycin resistance determining loci respectively.
Growth of Cells Cells were grown with forced aeration in 11 1. fermentors to a final cell density of 2-3 mg dry weight of cells/ml in media containing 1% ethanol, 1% yeast extract and a salts solution (Wallace et al., 1968). The following additional supplements were added to the medium: for strain L4303, 50 gg/ml each of histidine and lysine; for strain L4175, 50 gg/ml each of uracil and lysine.
Isolation of Mitochondria Mitochondria were isolated by the method of Cobon et al. (1974) with the modification that the mitochondria were washed three times by centrifugation rather than gradient purified.
Thin Layer Chromatography ( TLC) and Bioassay of Mikamycin Preparations Antibiotics were separated by one-dimensional TLC on silica gel 60HR plates with two different solvent systems: chloroform: methanol (9:1) and ethanol:water (3:1). Developed and dried plates were overlaid with a suspension of cells of strain L4175 in soft molten nutrient agar containing 1% yeast extract, 2% peptone, 1% ethanol and 0.5% agar (YEPE). These plates were then incubated at 28 ° C for 48 h and zones of inhibition of growth were recorded.
Assay for Antibiotic Sensitivity on YEPE Plates Cells of strains L4175 and L4303 were suspended in soft molten nutrient agar (as above) and were separately overlaid on YEPE plates containing 0.9% agar. Paper discs (1 cm diameter) containing known amounts of antibiotic were placed on the agar and the plates were incubated at 28 ° C for 48 h. Resistance to different levels of antibiotics was qualitatively determined by observing zones of inhibition of growth around the discs.
In vivo Mitochondrial Protein Synthesis
Respiration
Cycloheximide (CHX) resistant incorporation of 3H-leucine into yeast cells was carried out as described by Tzagoloff et al. (1975).
Mitochondrial respiration was measured at 28 ° C with a Clarke oxygen electrode. The reaction medium (2 ml) contained 0.6 M
D.J. Groot Obbink et al. : Mikamycin Resistance in Yeast sucrose, 10 m M K phosphate buffer p H 7.4, 1.0 m M E D T A and 0.4 mg mitochondrial protein. Either succinate (10 gmoles) or ethanol (200 gmoles) was added as substrate and State III respiration was determined by the addition of A D P (0.5 gmoles). The effect of mikamycin and antimycin A on respiration was tested as follows. Antibiotics at the concentrations indicated in the text were added to the reaction medium and pre-incubated for 10 min at 0 ° C. The reaction was initiated by the addition of substrate and incubating at 28 ° C. Where indicated, the artificial electron acceptor T M P D (N,N,N1,Nl-tetra methyl-p-phenylenediamine) was added at a final concentration of 0.35 m M .
140 •
129
Cytochrome Spectra Cytochrome spectra of isolated mitochondria were measured in a Cary model 14 double beam recording spectrophotometer at 20 ° C. The effect of mikamycin and antimycin A on the oxidation/ reduction state of the cytochromes was measured as follows. Mitochondria were suspended at a protein concentration of 5 mg/ml in the same incubation mixture (1 ml) as described for respiration and added to the test and reference cuvettes. Mitochondrial cytochromes in the test cuvette were reduced following the addition of succinate (5 ~tmoles) and A D P (0.25 gmoles). The antibiotics at
140]
A.
120
120~,~
100
~.u
B.
• toot >
T80, u
8o
