Portal de Periódicos da CAPES

Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae

1999; Springer Nature; Volume: 18; Issue: 1 Linguagem: Inglês

10.1093/emboj/18.1.58

1460-2075

Jie Lai‐Zhang,

RNA modifications and cancer

Article4 January 1999free access Epistatic interactions of deletion mutants in the genes encoding the F1-ATPase in yeast Saccharomyces cerevisiae Jie Lai-Zhang Jie Lai-Zhang Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author Yan Xiao Yan Xiao Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author David M. Mueller Corresponding Author David M. Mueller Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author Jie Lai-Zhang Jie Lai-Zhang Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author Yan Xiao Yan Xiao Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author David M. Mueller Corresponding Author David M. Mueller Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA Search for more papers by this author Author Information Jie Lai-Zhang1, Yan Xiao1 and David M. Mueller 1 1Department of Biochemistry and Molecular Biology, The Chicago Medical School, North Chicago, IL, 60064 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:58-64https://doi.org/10.1093/emboj/18.1.58 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The F1-ATPase is a multimeric enzyme (α3β3γδϵ) primarily responsible for the synthesis of ATP under aerobic conditions. The entire coding region of each of the genes was deleted separately in yeast, providing five null mutant strains. Strains with a deletion in the genes encoding α-, β-, γ- or δ-subunits were unable to grow, while the strain with a null mutation in ϵ was able to grow slowly on medium containing glycerol as the carbon source. In addition, strains with a null mutation in γ or δ became 100% ρ0/ρ− and the strain with the null mutation in γ grew much more slowly on medium containing glucose. These additional phenotypes were not observed in strains with the double mutations: ΔαΔγ, ΔβΔγ, Δatp11Δγ, ΔαΔδ, ΔβΔδ or Δatp11Δδ. These results indicate that ϵ is not an essential component of the ATP synthase and that mutations in the genes encoding the α- and β-subunits and in ATP11 are epistatic to null mutations in the genes encoding the γ- and δ-subunits. These data suggest that the propensity to form ρ0/ρ− mutations in the γ and δ null deletion mutant stains and the slow growing phenotypes of the null γ mutant strain are due to the assembly of F1 deficient in the corresponding subunit. These results have profound implications for the physiology of normal cells. Introduction The mitochondrial (mt) ATP synthase is a multimeric enzyme composed of a water-soluble portion, F1, and a membrane portion, F0. F1 is composed of five subunits with the stoichiometry α3β3γδϵ (Todd et al., 1980; Walker et al., 1985). The catalytic site is largely within the β-subunit, with some participation by the α-subunit, as demonstrated by biochemical and genetic studies (for a review, see Duncan and Cross, 1992) and shown in the crystal structure of bovine F1 (Abrahams et al., 1994). As such, there are three catalytic sites formed by three α/β pairs arranged like segments of an orange. In addition, two gene products, Atp11p and Atp12p, are required for the assembly of the F1-ATPase (Ackerman and Tzagoloff, 1990). The function of these proteins is not known, but they are not subunits of the enzyme and may provide a chaperone-like activity specific for the F1-ATPase. The mechanism of ATP synthesis by the ATP synthase proceeds by the alternating or binding site mechanism initially proposed by Boyer et al. (1973). In this mechanism, the phosphorylation of ADP is isoenergetic and occurs at one of the three catalytic sites, which have a high affinity for ATP. The flow of protons alters the conformation of the active site, lowering its affinity for ATP and thereby allowing the release of newly formed ATP from the enzyme. This conformational change is thought to occur largely by interactions of the γ-subunit with the α- and β-subunits of the enzyme (Abrahams et al., 1994). Indeed, γ is in the center of F1 and makes critical and unique contacts with each of the catalytic domains in F1. Furthermore, γ rotates in the center of F1 in an ATP-dependent fashion (Noji et al., 1997). As such, the evidence indicates that F1 is a molecular motor, which is driven by protons to produce ATP. The crystal structure of bovine F1 did not show the structure or position of the δ- and ϵ-subunits. The roles of these subunits are less certain, but studies suggest they are involved in the coupling of ATP synthesis to proton translocation, with δ being part of the rotor (Jounouchi, 1992; Guelin et al., 1993; Zhang et al., 1994; Capaldi et al., 1996; Aggeler et al., 1997; Schulenberg et al., 1997; Häsle et al., 1998). This study uses yeast Saccharomyces cerevisiae to study the assembly, function and mechanism of the ATP synthase. Null mutants have been made in each of the five genes encoding the subunits of the F1-ATPase. Mutations in all but the gene encoding the ϵ-subunit provide yeast unable to grow on a non-fermentable carbon source, indicative of cells defective in oxidative phosphorylation. Furthermore, strains with null mutations in the genes encoding γ, δ and ϵ provide additional phenotypes which allow epistatic interactions to be explored. One of these additional phenotypes is the cell's increased tendency to lose mtDNA or the ρ factor. Historically, the ρ factor was determined to be a cytoplasmically inherited genetic unit, which was later determined to be the mtDNA (Ephrussi et al., 1949). Cells with ρ− mutations, or cytoplasmic petite mutations, have large deletions in the mtDNA, while the ρ0 mutant strain is devoid of its mtDNA (Slonimski and Ephrussi, 1949). Epistasis means ‘standing above’ and, in this study, mutations in the gene encoding the α- and β-subunit of the ATPase and in ATP11 are epistatic to the mutations in the genes encoding the γ- and δ-subunits of the ATPase. The results of these experiments indicate that the ATP synthase can be assembled into complexes devoid of γ, δ or ϵ and these subunit-deficient complexes are responsible for the secondary phenotypes. This conclusion provides important implications for the physiology of normal cells. Results Null mutations in the genes encoding the α-, β-, γ-, δ- and ϵ-subunits of F1-ATPase were made by homologous recombination with the KanMX resistance module flanked by 40 bp of the target sequence (Güldener et al., 1996). The sites of recombination were targeted to delete the entire coding regions for the mature subunits of the ATPase. In addition, the lox recombination sites flanked the KanMX module, which allowed the subsequent curing of the KanMX module by subsequent expression of the cre recombinase (Güldener et al., 1996). This strategy allowed the sequential deletion of multiple genes without using additional markers for selecting cells for the recombination event. The correct integration events were confirmed by PCR using primers that flanked the sites of recombination in the genome and paired with primers homologous to the KanMX module. Two PCRs were performed which accessed the 5′ and 3′ junctions of the recombination sites. The sizes of the PCR products were consistent with the sizes predicted if the correct integration event had occurred in the genome (data not shown). The deletion mutations were all made initially in the haploid strain, W303-1A. However, as is shown below, since deletion of the genes encoding the γ- and δ-subunits caused the cells to produce ρ0/ρ− mutations (loss of mtDNA, see Introduction) and since deletion of γ provided a slow-growing phenotype, these mutations were made in the diploid strain, W303. Haploid cells with a null deletion mutation in the gene encoding either α or β are unable to grow on complete medium containing glycerol as the carbon source (YPG), indicating that the cells are defective in oxidative phosphorylation (Figure 1). Neither mutation has a large detrimental effect on growth on medium containing glucose. This result is in contrast to a report that suggests that a null mutation in α inhibits cell growth due to its role as a chaperone-like molecule (Yuan and Douglas, 1992). Haploid cells with a mutation in ϵ grew slowly on YPG medium, indicating that ϵ is not essential for oxidative phosphorylation (Figure 1). This result is also contrary to a report that indicated that yeast with a null mutation in ϵ is unable to grow on YPG medium (Guelin et al., 1993). However, consistent with the prior report, cells devoid of ϵ have a strong tendency to produce ρ0/ρ− mutations (Table I). Figure 1.Growth phenotypes of yeast with null mutations in the genes encoding the α-, β- and ϵ-subunits. Growth of the strains on minimal glucose (SD) and complete medium containing glycerol as the carbon source (YPG) is shown. Strains with a null mutation in the gene encoding the α- (A and B), β- (C and D) or ϵ-subunits (E) were transformed with the yeast vector (column 1, V) and the yeast vector containing the corresponding yeast gene (column 2, pY). The cells were grown at 30°C on SD and YPG media for 4 days and at 18°C on YPG medium for 7 days. Download figure Download PowerPoint Table 1. Percentage of ρ0/ρ− formed from strains carrying single and double mutations in the genes encoding subunits of the ATPase Genotype Percentage ρ0/ρ− W303-1A 9 Δα <1 Δβ <1 Δγ 100 Δδ 100 Δϵ 60 ΔATP11 1 ΔγΔα <1 ΔγΔβ <1 ΔγΔϵ 100 ΔγΔATP11 <1 ΔγΔβ, β-E222K.URA3 <1 ΔδΔα <1 ΔδΔβ <1 ΔδΔγ 100 ΔδΔϵ 99 ΔδΔATP11 1 ΔδΔβ, β-E222K.URA3 35 tetrads analyzed using a variety of independent null mutant stains. Rather, the difference is probably due to the differences in how the null mutations were constructed. In the prior study, the null mutant was made by inserting the HIS3 gene into the BclI sites of the yeast ATP3 gene (Paul et al., 1994). This construct results in a fusion protein, with the first 134 amino acids being from the yeast γ-subunit. It is possible that this protein would be able to interfere with the activity responsible for the high ρ0/ρ− formation and the slow-growing phenotype of the Δγ mutants. Our studies indicate that the high rate of ρ0/ρ− mutations formed in the γ and δ mutant strains and the slow growing phenotype of the γ deletion mutant strain are due to the formation of an ATP synthase complex devoid of the corresponding subunit. The genetic data are quite clear on this conclusion. These phenotypes are not due to the absence of the ATP synthase since they are not observed in strains with null mutations in α or β. Nor are these phenotypes due to the absence of either γ or δ per se since, when paired with a mutation in β, the phenotype disappears. This epistatic effect is not limited to a mutation in β, but can also be seen with mutations in α and the ATP11 genes. Thus, a complex containing the α- and β-subunits is formed, which requires Atp11p apparently as an assembly factor, and this complex is required to cause the ρ0/ρ−-forming and slow-growing phenotypes. In addition, a functional active site is required, since the active site mutation β-E222K is also epistatic to the null mutations in γ and δ. A model to explain these results is shown in Figure 4. Normally, proton flow through the ATP synthase is coupled to the synthesis of ATP. However, in a strain with a null mutation in δ or γ, an ATP synthase complex lacking γ or δ is made which is defective in coupling proton transport to ATP synthesis. This is consistent with the genetic and biochemical studies in Escherichia coli which implicate the subunit that corresponds to δ in coupling of proton transport to ATP synthesis in the ATP synthase (Dunn and Futai, 1980; Jounouchi et al., 1992; Zhang et al., 1994). Likewise, missense mutations in E.coli γ have been shown to alter the coupling capacity of the enzyme (Shin et al., 1992). Furthermore, an F1-ATP synthase that was assembled, but lacking γ, would be predicted to be uncoupled since γ acts as the rotor in the molecular motor, ATP synthase (Abrahams et al., 1994; Noji et al., 1997). Figure 4.Model to explain the phenotypes of the single and double mutants. See text for explanation. Download figure Download PowerPoint This model also suggests that passive proton flow through F0 does not occur without a functional complex. As such, mutations that disrupt F1 assembly do not provide a functional proton pore. This is consistent with results using E.coli that indicate that F1 is necessary for proton conductance through F0 (Pati et al., 1991). More surprisingly, since the missense mutation β-E222K was epistatic to mutations in γ and δ, it appears that passive proton flow requires a functional active site. However, this result needs to be studied in more detail since it is possible that the missense mutation produced an enzyme that, though assembled, had an altered conformation. Why are ρ0/ρ− mutations produced? Given, that the mutations in δ and γ result in passive proton flow through the F0 portion of the enzyme, this should result in a decrease or elimination of the protonmotive force across the mitochondrial membrane. Since mitochondrial biogenesis requires a ΔΨ across the membrane, these mutations should be lethal to the cell (Neupert, 1997). To circumvent this problem, the yeast eliminate the proton pore by eliminating the mtDNAs which encode subunits of the proton pore, subunits 6, 9 and 10 of the ATP synthase. There is also a correlation between the formation of ρ0/ρ− mutants and coupling of the ATP synthase. Mutations in ATP5 (Uh et al., 1990) or the ϵ-encoding gene (Guelin et al., 1993) also have the effect of uncoupling the ATP synthase and result in cells that lose their mtDNA, while cells with mutations in ATP11, ATP12, α or β do not make ρ0/ρ− mutants. Thus, the available data suggest that deleterious effects on a cell caused by mutations that uncouple the ATP synthase are at least partially compensated for by secondary ρ0/ρ− mutations. The slow-growing phenotype of cells with a γ null mutation is suggested to occur from the hydrolysis of ATP, which decreases the intracellular ATP level. Since hydrolysis of ATP is normally controlled by the IF1, the inhibitor protein of the ATP synthase (Hashimoto et al., 1990), the model suggests that IF1 is not effective on the complex that is lacking γ. It is possible that IF1 works by blocking γ rotation. This is an appealing hypothesis since it would provide a mechanical explanation of how IF1 could block the hydrolysis but not the synthesis of ATP. Like a ratchet of a wrench, IF1 may allow the unidirectional rotation of γ in the ATP synthase. This is consistent with the placement of the binding site of IF1 near the C-terminal end of the β-subunit, away from the catalytic site (Jackson and Harris, 1988). The suggestion that an active complex is formed in the absence of γ appears to be contradictory to a prior study where the γ-less F1 ATPase could not be detected in a yeast strain with a null mutation in the gene encoding γ (Paul et al., 1994). However, γ is important for forming a stable enzyme complex (reviewed in Gromet-Elhanan, 1992), and the isolation procedure may have disrupted any complex originally present. Additionally, in vitro, γ (and δ) is not essential for forming an active enzyme (Gromet-Elhanan, 1992) and the crystal structure of the bovine enzyme indicates that γ has no direct involvement in forming the active site. As such, a γ-less ATPase complex could form in the mutant cell and this complex may both be active for ATP hydrolysis and activate passive proton translocation through F0. This and other studies (Jounouchi, et al., 1992; Guelin et al., 1993; Zhang et al., 1994; Capaldi et al., 1996; Aggeler et al., 1997; Schulenberg et al., 1997; Häsler et al., 1998) suggest that δ and ϵ are involved in the coupling of the ATP synthase. In the framework of F1 as a molecular motor, it is appealing to suggest that δ and ϵ act as a molecular clutch in the ATP synthase. This is consistent with cross-linking studies which indicate that cross-linking the equivalent of δ to γ does not inhibit the enzyme activity, and other studies, which place it in contact with F1 and F0 (Guelin et al., 1993; Capaldi et al., 1996; Aggeler et al., 1997; Schulenberg et al., 1997; Häsler et al., 1998). As such, the coupling capacity of the ATP synthase could be controlled or altered by changes in the level of the δ-, ϵ- or even the γ-subunit. Possibly, the coupling capacity of the ATP synthase in mammals is variable and this variability might partially account for phenotypic differences, for example, in the tendency to obesity. Materials and methods Yeast strains The yeast S.cerevisiae strains, W303-1A (a, ade2-1, his3-1,15, leu2-3,112, trp1-1, ura3-1) (obtained from B.Trumpower) and W303 (a/α ade2-1/ade2-1, his3-1,15/his3-1,14, leu2-3,112,/leu2-3,112, trp1-1/trp1-1, ura3-1/ura3-1) (obtained from S.Lindquist) were used throughout this study as the parents of the mutant strains. The percentage of ρ0/ρ− cells was determined for the mutant strains by mating colonies to ρ0 tester strains, K289-3A ρ0 and K338-8D ρ0 (Klapholz and Esposito, 1982), followed by testing for growth on YPG plates. Media The yeast media are standard recipes as described previously (Sherman, 1991): YPD, 2% peptone, 1% yeast extract, 2% glucose; YPG, 2% peptone, 1% yeast extract, 3% glycerol; and YPAD, 2% peptone, 1% yeast extract, 2% glucose and 20 mg/l adenine sulfate. Minimal medium (SD) contained 2% glucose and was supplemented with adenine, histidine, arginine, methionine, tyrosine, lysine, leucine, isoleucine and tryptophan (Trp) or uracil (Ura) at 20 mg/l. Genetic analysis Tetrad analysis was performed by standard methods (Sherman and Hicks, 1991). The cells were grown on pre-spore media for 1 day, transferred to sporulation media for 4–6 days, and dissected on YPD medium. Yeast transformation was performed by the lithium acetate method after growth in YPAD medium (Gietz and Schiestl, 1995). For selection of G418-resistant cells, the transformants were allowed to recover for 6–8 h at 30°C or placed at 4°C for 12–48 h and plated on YPD containing 0.2 mg/ml G418 (Gietz and Schiestl, 1995). The null mutants were made by homologous recombination of PCR products using the KanMX resistance module (Güldener et al., 1996). The strains were checked by whole-cell PCR (Niedenthal et al., 1996) to confirm the correct integration event in the chromosome at the 5′ and 3′ junctions. Note added in proof Data discussed as ‘data not shown’ as well as other pertinent materials can be found at http://www.finchcms.edu/biochem/mueller/1999emboj.h.... Acknowledgements Thanks are given to Dr John Keller for critically reading the manuscript. Special thanks are given to Dr Sharon Ackerman for providing a plasmid with the β-E222K mutation. This project was supported by a grant from the NIH (GM44412). References Abrahams JP, Leslie AG, Lutter R and Walker JE (1994) Structure at 2.8 Å resolution of F1-ATPase from bovine heart mitochondria. Nature, 370, 621–628.CrossrefCASPubMedWeb of Science®Google Scholar Ackerman SH and Tzagoloff A (1990) Identification of two nuclear genes (ATP11, ATP12) required for assembly of the yeast F1-ATPase. Proc Natl Acad Sci USA, 87, 4986–4990.CrossrefCASPubMedWeb of Science®Google Scholar Aggeler R, Ogilvie I and Capaldi RA (1997) Rotation of a γ–ϵ subunit domain in the Escherichia coli F1F0-ATP synthase complex. The γ–ϵ subunits are essentially randomly distributed relative to the α3β3δ domain in the intact complex. J Biol Chem, 272, 19621–19624.CrossrefCASPubMedWeb of Science®Google Scholar Boyer PD, Cross RL and Momsen W (1973) A new concept for energy coupling in oxidative phosphorylation based on a molecular explanation of the oxygen exchange reactions. Proc Natl Acad Sci USA, 70, 2837–2839.CrossrefCASPubMedWeb of Science®Google Scholar Capaldi RA, Aggeler R, Wilkens S and Grüber GJ (1996) Structural changes in the γ and ϵ subunits of the Escherichia coli F1F0-type ATPase during energy coupling. J Bioenerg Biomembr, 28, 397–401.CrossrefCASPubMedWeb of Science®Google Scholar Duncan TM and Cross RL (1992) A model for the catalytic site of F1-ATPase based on analogies to nucleotide-binding domains of known structure. J Bioenerg Biomembr, 24, 453–461.CrossrefCASPubMedWeb of Science®Google Scholar Dunn SD and Futai M (1980) Reconstitution of a functional coupling factor from the isolated subunits of Escherichia coli F1-ATPase. J Biol Chem, 255, 113–118.CASPubMedWeb of Science®Google Scholar Ephrussi B, Hottinguer H and Chimenes H (1949) Action de l'acriflavie sur les levures. I. La mutation petitie colonie. Ann Inst Pasteur (Paris), 76, 351–367.Web of Science®Google Scholar Gietz RD and Schiestl RH (1995) Transforming yeast with DNA. Methods Mol Cell Biol, 5, 255–269.Web of Science®Google Scholar Giraud M-F and Velours J (1994) ATP synthase of yeast mitochondria. Isolation of the F1 δ subunit, sequence and disruption of the structural gene. Eur J Biochem, 222, 851–859.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Giraud M-F and Velours J (1997) The absence of the mitochondrial ATP synthase δ subunit promotes a slow growth phenotype of ρ− yeast cells by a lack of assembly of the catalytic sector F1. Eur J Biochem, 245, 813–818.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Gromet-Elhanan Z (1992) Identification of subunits required for the catalytic activity of the F1-ATPase. J Bioenerg Biomembr, 24, 447–452.CrossrefCASPubMedWeb of Science®Google Scholar Guelin E, Chevallier J, Rigoulet M, Guerin B and Velours J (1993) ATP synthase of yeast mitochondria. Isolation and disruption of the ATP ϵ gene. J Biol Chem, 268, 161–167.CASPubMedWeb of Science®Google Scholar Güldener U, Heck S, Fiedler T, Beinhauer J and Hegemann JH (1996) A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acids Res, 24, 2519–2524.CrossrefPubMedWeb of Science®Google Scholar Hashimoto T, Yoshida Y and Tagawa KJ (1990) Regulatory proteins of F1F0-ATPase: role of ATPase inhibitor. J Bioenerg Biomembr, 22, 27–38.CrossrefCASPubMedWeb of Science®Google Scholar Häsler K, Engelbrecht S and Junge W (1998) Three-stepped rotation of subunits γ and ϵ in single molecules of F1-ATPase as revealed by polarized, confocal fluorometry. FEBS Lett, 426, 301–304.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Jackson PJ and Harris DA (1988) The mitochondrial ATP synthase inhibitor protein binds near the C-terminus of the F1 β-subunit. FEBS Lett, 229, 224–228.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Jounouchi M, Takeyama M, Noumi T, Moriyama Y, Maeda M and Futai M (1992) Escherichia coli H (+)-ATPase: role of the δ subunit in binding Fl to the F0 sector. Arch Biochem Biophys, 292, 87–94.CrossrefCASPubMedWeb of Science®Google Scholar Klapholz S and Esposito RE (1982) A new mapping method employing a meiotic rec− mutant of yeast. Genetics, 100, 387–412.CASPubMedWeb of Science®Google Scholar Liang Y and Ackerman SH (1996) Characterization of mutations in the β-subunit of the mitochondrial F1-ATPase that produce defects in enzyme catalysis and assembly. J Biol Chem, 271, 26522–26528.CrossrefCASPubMedWeb of Science®Google Scholar Mukhopadhyay A, Uh M and Mueller DM (1994) Level of ATP synthase activity required for yeast Saccharomyces cerevisiae to grow on glycerol media. FEBS Lett, 343, 160–164.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Neupert W (1997) Protein import into mitochondria. Annu Rev Biochem, 66, 863–917.CrossrefCASPubMedWeb of Science®Google Scholar Niedenthal RK, Riles L, Johnston M and Hegemann JH (1996) Green fluorescent protein as a marker for gene expression and subcellular localization in budding yeast. Yeast, 12, 773–786.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Noji H, Yasuda R, Yoshida M and Kinosita K (1997) Direct observation of the rotation of F1-ATPase. Nature, 386, 299–302.CrossrefCASPubMedWeb of Science®Google Scholar Pati S, Brusilow WS, Deckers-Hebestreit G and Altendorf K (1991) Assembly of the F0 proton channel of the Escherichia coli F1F0 ATPase: low proton conductance of reconstituted F0 sectors synthesized and assembled in the absence of F1. Biochemistry, 30, 4710–4714.CrossrefCASPubMedWeb of Science®Google Scholar Paul M-F, Ackermann S, Yue J, Arselin G, Velours J and Tzagoloff A (1994) Cloning of the yeast ATP3 gene coding for the γ-subunit of F1 and characterization of atp3 mutants. J Biol Chem, 269, 26158–26164.CASPubMedWeb of Science®Google Scholar Schulenberg B, Wellmer F, Lill H, Junge W and Engelbrecht S (1997) Cross-linking of chloroplast F0F1-ATPase subunit ϵ to γ without effect on activity: ϵ and γ are parts of the rotor. Eur J Biochem, 249, 134–141.Wiley Online LibraryCASPubMedWeb of Science®Google Scholar Sherman F (1991) Getting started with yeast. Methods Enzymol, 194, 3–21.CrossrefCASPubMedWeb of Science®Google Scholar Sherman F and Hicks J (1991) Micromanipulation and dissection of asci. Methods Enzymol, 194, 21–37.CrossrefCASPubMedWeb of Science®Google Scholar Shin K, Nakamoto RK, Maeda M and Futai M (1992) F0F1-ATPase γ-subunit mutations perturb the coupling between catalysis and transport. J Biol Chem, 267, 20835–20839.CASPubMedWeb of Science®Google Scholar Slonimiski PP and Ephrussi B (1949) Action de l'acriflavie sur les levures. V. Le système des cytochromes des mutants petite colonie. Ann Inst Pasteur (Paris), 77, 47–64.Google Scholar Todd RD, Griensenbeck TA and Douglas MG (1980) The yeast mitochondrial adenosine triphosphatase complex. Subunit stoichiometry and physical characterization. J Biol Chem, 255, 5461–5467.CASPubMedWeb of Science®Google Scholar Uh M, Jones D and Mueller DM (1990) The gene coding for the yeast oligomycin sensitivity-conferring protein. J Biol Chem, 265, 19047–19052.CrossrefCASPubMedWeb of Science®Google Scholar Walker JE, Fearnley IM, Gay NJ, Gibson BW, Northrop FD, Powell SJ, Runswick MJ, Saraste M and Tybulewicz VLJ (1985) Primary structure and subunit stoichiometry of F1-ATPase from bovine mitochondria. J Mol Biol, 184, 677–701.CrossrefCASPubMedWeb of Science®Google Scholar Yuan H and Douglas MG (1992) The mitochondrial F1 ATPaseα-subunit is necessary for efficient import of mitochondrial precursors. J Biol Chem, 267, 14697–14702.CASPubMedWeb of Science®Google Scholar Zhang Y, Oldenburg M and Fillingame RH (1994) Suppressor mutations in F1 subunit ϵ recouple ATP-driven H+ translocation in uncoupled Q42E subunit c mutant of Escherichia coli F1F0 ATP synthase. J Biol Chem, 269, 10221–10224.CASPubMedWeb of Science®Google Scholar Previous ArticleNext Article Volume 18Issue 14 January 1999In this issue FiguresReferencesRelatedDetailsLoading ...