Mucidin resistance in yeast. Isolation, characterization and genetic analysis of nuclear and mitochondrial mucidin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to the antibiotic mucidin, a specific inhibitor of electron transport between cytochrome b and c, were isolated and divided into three phenotypic groups, as follows. Class 1 mutants were cross-resistant to a variety of mitochondrial inhibitors and exhibited no resistance at the mitochondrial level. Class 2 mutants were specifically resistant to mucidin exhibiting resistance also at the level of isolated mitochondria. Biochemical studies indicated that the mucidin resistance in class 2 mutants involved a modification of mucidin binding of inhibitory sites on the mitochondrial inner membrane without a significance change in the sensitivity of mitochondrial oxygen uptake to antimycin A, 2-heptyl-4-hydroxyquinoline-N-oxide, and 2,3-dimercaptopropanol. Class 3 was represented by a mutant which showed a high degree of resistance to mucidin and was cross-resistant to a variety of mitochondrial inhibitors at the cellular level but exhibited only a resistance to mucidin at the mitochondrial level. Genetic analysis of mucidin-resistant mutants revealed the presence of both nuclear and mitochondrial genes determining mucidin resistance/sensitivity in yeast. Resistance to mucidin in class 1 mutants was due to a single-gene nuclear recessive mutation (mucPR) whereas that in class 2 mutants was caused by mutations of mitochondrial genes. Resistance in class 3 mutant was determined both by single-gene nuclear and mitochondrial mutations. In the mitochondrial mutants the mucidin resistance segregated mitotically and the resistance determinant was lost upon induction of petite mutation by ethidium bromide. Allelism tests indicated that the mucidin resistance mutations fell into two genetic loci (MUC1 and MUC2) which were apparently not closely linked in the mitochondrial genome. Recombination studies showed that the two mitochondrial mucidin loci were not allelic with other mitochondrial loci RIB1, RIB2 and OLI1. An extremely high mucidin resistance at the cellular level was shown to arise from synergistic interaction of the nuclear gene mucPR and the mitochondrial mucidin-resistance gene (MR) in a cell. The results suggest that at least two mitochondrial gene products, responsible for mucidin resistance/sensitivity in yeast, take part in the formation of the cytochrome bc1 region of the mitochondrial respiratory chain.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc(1) complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8(m)). Subsequently replacing ARG8(m) with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit(-)). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Genetic damage during thymidylate starvation in Saccharomyces cerevisiae.

Summary Thymidylate starvation in a yeast mutant auxotrophic for dTMP caused cell death and the induction of mutations in the mitochondrial genome. After 24 h of starvation almost all surviving cells were respiratory deficient petites. In addition, shorter episodes of dTMP starvation induced chloramphenicol and erythromycin resistant mutants, indicating the occurrence of mitochondrial point mutations. Suboptimal concentrations of exogenous thymidylate were also found to induce petites and a decline in cell viability and the magnitude of these effects was acutely dependent upon the dTMP concentration. Cesium chloride gradient analysis of DNA from cells undergoing thymineless incubation revealed a progressive loss of mitochondrial DNA, and a decrease in the molecular weight of nuclear DNA.     

Random mutagenesis of the B'A' core domain of yeast DNA topoisomerase II and large-scale screens of mutants resistant to the anticancer drug etoposide

Mutagenic PCR method was applied to introduce point mutations to the B'A' core domain of yeast DNA topoisomerase II. Screens for mutants resistant to the anticancer drug etoposide were carried out in a yeast ts system in the presence of high concentrations of the drug or in a drug-hypersensitive genetic background. 129 mutants were obtained from a total of 47,000 transformants. Nucleotide sequencing of 40 selected mutants showed that a large number of the mutations map to regions encoding the linker that joins the ATPase domain to the B' module and the B'A' linker. Significant reduction in catalytic activity was evident for a large fraction of mutant enzymes and all mutants were also resistant to amsacrine, another topoisomerase II drug with a different chemical structure, suggesting that few of the mutations reflect simple changes of specific amino acid side chains that are directly involved in enzyme-drug interactions.     

Isolation of mutants with aberrant mitochondrial morphology from Arabidopsis thaliana

To identify genes related to plant mitochondrial morphology and dynamics, novel mutants with respect to mitochondrial morphology were isolated from an ethyl methane sulphonate (EMS)-mutated population of Arabidopsis thaliana. Mitochondria were visualized by transforming Arabidopsis with a gene for a fusion protein consisting of GFP and a mitochondria-targeting pre-sequence. From 19,000 M2 populations, 17 mutants were isolated by fluorescent microscopic observations. All mitochondria in these mutants were longer and/or larger than wild-type mitochondria. The approximate chromosomal loci of the mutations of seven mutants that grew well were determined. The mitochondrial phenotypes of six of the mutants were recessive but the mitochondrial phenotype of the seventh mutant was dominant. Chromosomal rough mapping of the seven mutants showed that the mutations occurred at four different loci. At least one of these loci was novel, i.e., it was different from loci of other known mitochondrial morphology mutants of Arabidopsis and different from loci of Arabidopsis homologues of yeast genes related to mitochondrial morphology.     

Studies on energy-linked reactions: isolation, characterisation and genetic analysis of trialkyl-tin-resistant mutants of Saccharomyces cerevisiae.

Mutants of Saccharomyces cerevisiae resistant to triethyl tin sulphate have been isolated and are cross-resistant to other trialkyl tin salts. Triethyl-tin-resistant mutants fall into two general phenotypic classes: class 1 and class 2. Class 1 mutants are cross-resistant to a variety of inhibitors and uncoupling agents which affect mitochondrial membranes (oligomycin, ossamycin, valinomycin, antimycin, erythromycin, chloramphenicol, '1799', tetrachlorotrifluoromethyl benzimidazole carbonylcyanide-m-chlorophenylhydrazone and cycloheximide). Class 2 mutants are specifically resistant to trithyl tin and the uncoupling agent "1799' [bis-(hexafluoroacetonyl)-acetone]. Triethyl tin at neutral pH values is a specific inhibitor of mitochondrial energy conservation reactions and prevents growth on oxidisable substrates such as glycerol and ethanol. Triethyl-tin-resistant mutants grow normally on glucose and ethanol in the presence of triethyl tin (10 muM). Biochemical studies indicate that the mutation involves a modification of the triethyl tin binding site on the mitochondrial inner membrane, probably the ATP-synthetase complex. Triethyl tin resistance/sensitivity in yeast is determined by cytoplasmic (mitochondrial) and nuclear genes. The mutants fall into a nuclear and a cytoplasmic (mitochondrial) class corresponding to the phenotypic cross-resistance classes 1 and 2. In the cytoplasmic mutants the triethyl tin resistance segregates mitotically and the resistance determinat is deleted by the action of ethidium bromide during petite induction. Recombination studies indicate that the triethyl tin mutations are not allelic with the other mitochondrial mutations at the loci RI, RIII and OLI. This indicates that the binding or inhibitory sites of oligomycin and triethyl tin are not identical and that the triethyl tin binding site is located on a different mitochondrial gene product to those which are involved in oligomycin binding. Interaction and cooperative effects between different binding sites on the mitochondrial inner membrane have been demonstrated in studies of the effect of the insertion of the TETr phenotype into mitochondrial oligomycin-resistant mutants and provide an experimental basis for complementation studies at the ATP-synthetase level.     

Isolation and characteristics of new mutants of Saccharomyces cerevisiae with increased spontaneous mutability]

To isolate some new genes controlling the process of spontaneous mutagenesis, a collection of 16 yeast strains with enhanced rate of spontaneous canavanine resistant mutations was obtained. Genetical analysis allowed to define that the mutator phenotype of these strains is due to a single nuclear mutation. Such mutations were called hsm (high spontaneous mutagenesis). Recombinational test showed that 5 mutants under study carried 5 nonallelic mutations. It was revealed that the mutation hsm3-1 is a nonspecific mutator elevating the rate of both spontaneous canavanine resistant mutations and the frequency of reversions in mutations lys1-1 and his1-7. Genetical analysis revealed that mutation hsm3-1 is recessive. The study of cross sensitivity of mutator strains to physical and chemical mutagens demonstrated that 12 of 16 hsm mutants were resistant to the lethal action of UV, gamma rays and methylmethanesulfonate, and 4 mutants were only sensitive to these factors. Possible nature of hsm mutations is discussed.     

Transport of the yeast ATP synthase beta-subunit into mitochondria. Effects of amino acid substitutions on targeting.

We have isolated the yeast ATP2 gene encoding the beta-subunit of mitochondrial ATP synthase and determined its nucleotide sequence. A fusion between the N-terminal 15 amino acid residues of beta-subunit and the mouse cytosolic protein dihydrofolate reductase (DHFR) was transcribed and translated in vitro and found to be transported into isolated yeast mitochondria. A fusion with the first 35 amino acid residues of beta-subunit attached to DHFR was not only transported but also proteolytically processed by a mitochondrial protease. Amino acid substitutions were introduced into the N-terminal presequence of the beta-subunit by bisulphite mutagenesis of the corresponding DNA. The effects of these mutations on mitochondrial targeting were assessed by transport experiments in vitro using DHFR fusion proteins. All of the mutants, harbourin from one to six amino acid substitutions in the first 14 residues of the presequence, were transported into mitochondria, though at least one of them (I8) was transported and proteolytically processed at a much reduced rate. The I8 mutant beta-subunit also exhibited poor transport and processing in vivo, and expression of this mutant polypeptide failed to complement the glycerol- phenotype of a yeast ATP2 mutant. More remarkably, the expression of I8 beta-subunit induced a more general growth defect in yeast, possibly due to interference with the transport of other, essential, mitochondrial proteins.     

Chloroplast genes in Chlamydomonas affecting organelle ribosomes. Genetic and biochemical analysis of analysis of antibiotic-resistant mutants at several gene loci.

Six chloroplast gene mutants of Chlamydomonas reinhardtii resistant to spectinomycin, erythromycin, or streptomycin have been assessed for antibiotic resistance of their chloroplast ribosomes. Four of these mutations clearly confer high levels of antibiotic resistance on the chloroplast ribosomes both in vivo. Although one mutant resistant to streptomycin and one resistant to spectinomycin have chloroplast ribosomes as sensitive to antibiotics as those of wild type in vivo, these mutations can be shown to alter the wildtype sensitivity of chloroplast ribosomes in polynucleotide-directed amino acid incorporation in vitro. Genetic analysis of these six chloroplast mutants and three similar mutants (Sager, 1972), two of which have been shown to affect chloroplast ribosomes (Mets and Bogorad, 1972; Schlanger and Sager, 1974), indicates that in Chlamydomonas at least three chloroplast gene loci can affect streptomycin resistance of chloroplast ribosomes and that two can affect erythromycin resistance. The three spectinomycin-resistant mutants examined appear to be alleles at a single chloroplast gene locus, but may represent mutations at two different sites within the same gene. Unlike wild type, the streptomycin and spectinomycin resistant mutants which have chloroplast ribosomes sensitive to antibiotics in vivo, grow well in the presence of antibiotic by respiring exogenously supplied acetate as a carbon source, and have normal levels of cytochrome oxidase activity and cyanide-sensitive respiration. We conclude that mitochondrial protein synthesis in these mutants is resistant to these antibiotics, whereas in wild type it is sensitive. To explain the behavior of these two chloroplast gene mutants as well as other one-step mutants which are resistant both photosynthetically and when respiring acetate in the dark, we have postulated that a mutation in a single chloroplast gene may result in alteration of both chloroplast and mitochondrial ribosomes. Mitochondrial resistance would appear to be the minimal necessary condition for survival of all such mutants, and antibiotic-resistant chloroplast ribosomes would be necessary for survival only under photosynthetic conditions.     

Suppression of mitochondrially-determined resistance to chloramphenicol and paromomycin by nuclear genes in Saccharomyces cerevisiae.

Summary Phenotypic revertants of a drug resistant strain of Saccharomyces cerevisiae were induced by mutgenesis with manganese. Several of these drug sensitive mutants have been shown to result from mutations in the nuclear genome that cause phenotypic modification (suppression) of the mitochondrially-determined drug resistant genotype.Four mutants carrying a single recessive nuclear gene capable of modifying mitochondrial chloramphenicol resistance are described; these may be assigned to three complementation groups. Chloramphenicol resistant mutants mapping at five separate mitochondrial loci are described. At least two of the nuclear genes cause modification of mitochondrial chloramphenicol resistance determined by mutations at three of these loci, but the other two loci are apparently non-suppressible by these nuclear alleles. This indicates that these modifiers do not act by causing a general decrease in cellular or mitochondrial permeability to the drug.A single dominant nuclear modifier of mitochondrial paromomycin resistance has been identified. It is non-allelic to and does not interact with the genes modifying mitochondrial chloramphenicol resistance.     

Mitochondrial DNA and suppressiveness of petite mutants in Saccharomyces cerevisiae

Ethidium bromide is known to be a powerful mutagen for the induction of cytoplasmically inherited petite mutations in yeast. The effect of ethidium bromide on the degree of suppressiveness of the induced mutants as a function of exposure time is described. The mitochondrial DNA of 20 ethidium bromide-induced petite mutants has been studied to determine its absence or presence and its buoyant density. Ten mutants, in which we were not able to detect any mitochondrial DNA, were neutral petites. The 10 remaining mutants with mitochondrial DNA simultaneously showed a measurable degree of suppressiveness. It was not possible to correlate the buoyant density of the mutant mitochondrial DNA with the degree of suppressiveness.This study was supported in part by USPHS grant GM 10017. G.M. received a Fulbright Travel Grant.     

Introduction of cytochrome b mutations in Saccharomyces cerevisiae by a method that allows selection for both functional and non-functional cytochrome b proteins

We have previously used inhibitors interacting with the Qn site of the yeast cytochrome bc₁ complex to obtain yeast strains with resistance-conferring mutations in cytochrome b as a means to investigate the effects of amino acid substitutions on Qn site enzymatic activity [M.G. Ding, J.-P. di Rago, B.L. Trumpower, Investigating the Qn site of the cytochrome bc1 complex in Saccharomyces cerevisiae with mutants resistant to ilicicolin H, a novel Qn site inhibitor, J. Biol. Chem. 281 (2006) 36036-36043.]. Although the screening produced various interesting cytochrome b mutations, it depends on the availability of inhibitors and can only reveal a very limited number of mutations. Furthermore, mutations leading to a respiratory deficient phenotype remain undetected. We therefore devised an approach where any type of mutation can be efficiently introduced in the cytochrome b gene. In this method ARG8, a gene that is normally encoded by nuclear DNA, replaces the naturally occurring mitochondrial cytochrome b gene, resulting in ARG8 expressed from the mitochondrial genome (ARG8^m). Subsequently replacing ARG8^m with mutated versions of cytochrome b results in arginine auxotrophy. Respiratory competent cytochrome b mutants can be selected directly by virtue of their ability to restore growth on non-fermentable substrates. If the mutated cytochrome b is non-functional, the presence of the COX2 respiratory gene marker on the mitochondrial transforming plasmid enables screening for cytochrome b mutants with a stringent respiratory deficiency (mit⁻). With this system, we created eight different yeast strains containing point mutations at three different codons in cytochrome b affecting center N. In addition, we created three point mutations affecting arginine 79 in center P. This is the first time mutations have been created for three of the loci presented here, and nine of the resulting mutants have never been described before.     

Single-amino-acid substitutions within the signal sequence of yeast prepro-alpha-factor affect membrane translocation.

We used a genetic approach to identify point mutations in the signal sequence of a secreted eucaryotic protein, yeast alpha-factor. Signal sequence mutants were obtained by selecting for cells that partially mistargeted into mitochondria a fusion protein consisting of the alpha-factor signal sequence fused to the mature portion of an imported mitochondrial protein (Cox IV). The mutations resulted in replacement of a residue in the hydrophobic core of the signal sequence with either a hydrophilic amino acid or a proline. After reassembly into an intact alpha-factor gene, the substitutions were found to decrease up to 50-fold the rate of translocation of prepro-alpha-factor across microsomal membranes in vitro. Two of three mutants tested produced lower steady-state levels of alpha-factor in intact yeast cells, although the magnitude of the effect was less than that in the cell-free system.     

Sequence analysis of mitochondrial DNA in a mouse cell line resistant to chloramphenicol and oligomycin.

A mouse L-cell line, designated 111-OB3, is described which is resistant to two drugs, chloramphenicol and oligomycin. The cells contain two types of mitochondrial DNA molecules, in roughly equal proportions, which differ in that one is cleaved by endonuclease EcoRI at a novel site within the coding sequence for subunit 6 of the mitochondrial ATPase (ATPase-6). Sequence analysis reveals that the cleavage site was created by a single transversion which predicts a replacement of valine in the wild-type ATPase-6 by glutamic acid. The replacement occurs in a hydrophobic amino acid sequence which is highly conserved in mouse, human, and bovine proteins. The position of the replacement is similar to a substitution observed in one class of yeast mutants resistant to oligomycin. Both of the mitochondrial DNA molecules in 111-OB3 also have a single nucleotide change in the gene encoding the large (16S) rRNA. These observations are consistent with the hypothesis that oligomycin resistance in mammalian cells can be cytoplasmically determined and can result from alterations in ATPase-6. The appearance of the mutation before selection in oligomycin suggests a model for the origin of mitochondrial mutations in mammalian cells.     

Mitochondrial and nuclear mutations that affect the biogenesis of the mitochondrial ribosomes of yeast. II. Biochemistry.

1. Several nuclear mutants have been isolated which showed thermo- or cryo-sensitive growth on non-fermentable media. Although the original strain carried mitochondrial drug resistance mutations (CR, ER, OR and PR), the resistance to one or several drugs was suppressed in these mutants. Two of them showed a much reduced amount of the mitochondrial small ribosomal subunit (37S) and of the corresponding 16S ribosomal RNA. Two dimensional electrophoretic analysis did not reveal any change in the position of any of the mitochondrial ribosomal proteins. However one of the mitochondrial ribosomal proteins. However one of the mutants showed a striking decrease in the amounts of three ribosomal proteins S3, S4 and S15. 2. Four temperature-sensitive mitochondrial mutations have been localized in the region of the gene coding for the large mitochondrial ribosomal RNA (23S). These mutants all showed a marked anomaly in the mitochondrial large ribosomal subunit (50S) and/or the corresponding 23S ribosomal RNA.     

Ribosomal recessive suppressors cause a respiratory deficiency in yeast Saccharomyces cerevisiae

Summary Recessive suppressor mutations in yeast Saccharomyces cerevisiae alter a component of the cytoplasmic ribosomes, relaxing the control of translational fidelity. As a consequence ribosomes can misread nonsense codons as amino acids (Surguchov et al. 1980a).The suppressor mutants are often respiratory deficient, being unable to grow on non-fermentable substrates. The study of the cytochrome spectra has revealed that the cytochrome b and aa₃ contents were lower in the mutants than in the parent strains. Furthermore, the suppresor mutations often cause hypersensitivity to paromomycin and neomycin on media with a non-fermentable source of carbon. Some of the suppressor mutants exhibited both erythromycin and chloramphenicol-dependent growth on media containing ethanol or glycerol as a sole carbon source.These results suggest that the mutations altering cytoplasmic ribosomes may simultaneously impair the mitochondrial translation. A coupling of cytoplasmic and mitochondrial protein synthesis in yeast cells is proposed. The existence of a common protein component participating both in mitochondrial and cytoplasmic protein synthesis apparatus is discussed.     

The definition of mitochondrial H+ ATPase assembly defects in mit- mutants of Saccharomyces cerevisiae with a monoclonal antibody to the enzyme complex as an assembly probe

mit- mutants with genetically defined mutations in the mitochondrial structural genes of the H+-ATPase membrane subunits 6, 8 and 9 were analysed to determine the H+-ATPase assembly defects that resulted as a consequence of the mutations. These include mutants which do not synthesize one of the membrane subunits and mutants which can synthesize these subunits, but in an altered form. Protein subunits which can still be assembled to the defective H+-ATPase in these mutants were determined by immunoprecipitation using a monoclonal antibody to the beta-subunit of the enzyme complex. The results suggest that the assembly pathway of the mitochondrially synthesized H+-ATPase subunits involves the sequential addition of subunits 9, 8 and 6 to a membrane-bound F1-sector. In addition to subunits of the F0- and F1-sectors, two other polypeptides (Mr = 18,000 and Mr = 25,000) are associated with the yeast H+-ATPase. These polypeptides were not observed in the immunoprecipitates obtained from mutants in which the F0-sector is not properly assembled.     

Localization in yeast mitochondrial DNA of mutations expressed in a deficiency of cytochrome oxidase and/or coenzyme QH2-cytochrome c reductase.

1. Three methods are described for the genetic analysis of yeast cytoplasmic mutants (mit- mutants) lacking cytochrome oxidase or coenzyme QH2-cytochrome c reductase. The procedures permit mutations in mitochondrial DNA to be mapped relative to each other and with respect to drug-resistant markers. The first method is based upon the finding that crosses of mit- mutants with some but not other isonuclear q- mutants lead to the restoration of respiratory functions. Thus a segment of mitochondrial DNA corresponding to a given mit- mutation or to a set of mutations can be delineated. The second method is based on the appearance of wild-type progeny in mit- X mit- crosses. The third one is based on the analysis of various recombinant classes issued from crosses between mit-, drug-sensitive and mit+, drug-resistant mutants. Representative genetic markers of the RIBI, OLII, OLI2 and PAR1 loci were used for this purpose. 2. The three methods when applied to the study of 48 mit- mutants gave coherent results. At least three distinct regions on mitochondrial DNA in which mutations cause loss of functional cytochrome oxidase have been established. A fourth region represented by closely clustered mutants lacking coenzyme QH2-cytochrome c reductase and spectrally detectable cytochrome b has also been studied. 3. The three genetic regions of cytochrome oxidase and the cytochrome b region were localized by the third method on the circular map, in spans of mitochondrial DNA defined by the drug-resistant markers. The results obtained by this method were confirmed by analysis of the crosses between selected mit- mutants and a large number of q- clones whose retained segments of mitochondrial DNA contained various combinations of drug-resistant markers. 4. All the genetic data indicate that the various regions studied are dispersed on the mitochondrial genome and in some instances regions or clusters of closely linked mutations involved in the same respiratory function (cytochrome oxidase) are separated by other regions which code for entirely different functions such as ribosomal RNA.     

Studies on energy-linked reactions: isolation and properties of mitochondrial venturicidin-resistant mutants of Saccharomyces cerevisiae.

Venturicidin is a specific inhibitor of aerobic growth of yeast and has no effect on fermentative growth, a result which is consistent with its known mode of action on mitochondrial oxidative phosphorylation. Venturicidin-resistant mutants of Saccharomyces cerevisiae have been isolated and form two general classes: class 1, nuclear mutants which are resistant to a variety of mitochondrial inhibitors and uncouplers, and class 2, mitochondrial mutants of phenotype VENR OLYR and VENR TETR in vivo. VENR OLYR mutants show a high degree of resistance to venturicidin and oligomycin at the whole cell and mitochondrial ATPase level but, in contrast, no resistance at the mitochondrial level is observed with VENR TETR mutants. Venturicidin resistance/sensitivity can be correlated with two binding sites on mitochondrial ATPase, one of which is common to the oligomycin binding site and the other is common to the triethyl tin binding site. Biochemical genetic studies indicate that two mitochondrial genes specify venturicidin resistance/sensitivity and that the mitochondrial gene products are components of the mitochondrial ATPase complex.     

Functional Analysis of Yeast bcs1 Mutants Highlights the Role of Bcs1p-Specific Amino Acids in the AAA Domain

The mitochondrial protein Bcs1p is conserved from Saccharomyces cerevisiae to humans and its C-terminal region exhibits an AAA (ATPases associated with diverse cellular activities) domain. The absence of the yeast Bcs1p leads to an assembly defect of the iron-sulfur protein (ISP) subunit within the mitochondrial respiratory complex III, whereas human point mutations located all along the protein cause various pathologies. We have performed a structure-function analysis of the yeast Bcs1p by randomly generating a collection of respiratory-deficient point mutants. We showed that most mutations are in the C-terminal region of Bcs1p and have localized them on a theoretical three-dimensional model based on the structure of several AAA proteins. The mutations can be grouped into classes according to their respiratory competence and their location on the three-dimensional model. We have further characterized five mutants, each substituting an amino acid conserved in yeast and mammalian Bcs1 proteins but not in other AAA proteins. The effects on respiratory complex assembly and Bcs1p accumulation were analyzed. Intragenic and extragenic compensatory mutations able to restore complex III assembly to the mutants affecting the AAA domain were isolated. Our results bring new insights into the role of specific residues in critical regions that are also conserved in the human Bcs1p. We show that (1) residues located at the junction between the Bcs1p-specific and the AAA domains are important for the activity and stability of the protein and (2) the residue F342 is important for interactions with other partners or substrate proteins.