Single Copies of Subunits d, Oligomycin-sensitivity Conferring Protein, and b Are Present in the Saccharomyces cerevisiaeMitochondrial ATP Synthase

Abstract

In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14–18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case. In the mitochondrial ATP synthase (mtATPase) of the yeast Saccharomyces cerevisiae, the stoichiometry of subunits d, oligomycin-sensitivity conferring protein (OSCP), and b is poorly defined. We have investigated the stoichiometry of these subunits by the application of hexahistidine affinity purification technology. We have previously demonstrated that intact mtATPase complexes incorporating a Hex6-tagged subunit can be isolated via Ni2+-nitrilotriacetic acid affinity chromatography (Bateson, M., Devenish, R. J., Nagley, P., and Prescott, M. (1996) Anal. Biochem. 238, 14–18). Strains were constructed in which Hex6-tagged versions of subunits d, OSCP, and b were coexpressed with the corresponding wild-type subunit. This coexpression resulted in a mixed population of mtATPase complexes containing untagged wild-type and Hex6-tagged subunits. The stoichiometry of each subunit was then assessed by determining whether or not the untagged wild-type subunit could be recovered from Ni2+-nitrilotriacetic acid purifications as an integral component of those complexes absorbed by virtue of the Hex6-tagged subunit. As only the Hex6-tagged subunit was recovered from such purifications, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast is 1 in each case. ATP synthase (F1F0-ATPase) uses energy produced from the electrochemical gradient to produce ATP from ADP and Pi. Production of ATP is thought to occur by the binding change mechanism (1Boyer P.D. Biochim. Biophys. Acta. 1993; 1140: 215-250Crossref PubMed Scopus (924) Google Scholar). The F1 sector has three sites that catalyze the production of ATP and that are repetitively and sequentially driven through defined conformational states. Recent studies have demonstrated that one element of the mechanism driving these sequential events is the rotation of the γ subunit in the F1 sector of the complex (2Noji H. Yasuda R. Yoshida M. Kinosita Jr., K. Nature. 1997; 386: 299-302Crossref PubMed Scopus (1965) Google Scholar). Moreover, the rotary process by which ATP is synthesized is thought to involve a stator that prevents the futile rotation of the α3β3hexamer. There is evidence for such a stator consisting of subunits b and δ in the bacterial enzyme (3Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, 4Rodgers A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Conservation of the general mechanism between the F1F0-ATPases might suggest that a stator of similar structure exists in mitochondrial enzymes. In eukaryotes, subunits b and OSCP, 1The abbreviations used are: OSCP, oligomycin-sensitivity conferring protein; mtATPase, mitochondrial ATP synthase; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis 1The abbreviations used are: OSCP, oligomycin-sensitivity conferring protein; mtATPase, mitochondrial ATP synthase; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis which are homologues of the bacterial subunits b and δ, respectively, would be prime candidates for components of a stator in the mitochondrial ATP synthase (mtATPase). However, mtATPase contains additional subunits that do not have a bacterial homologue, which means that the composition of the stator in higher organisms may be more complex. For an understanding of the structure and function of this stator in the eukaryotic mtATPase, it is important to know the identity and number of each subunit within the enzyme complex. However, the stoichiometry and composition of several subunits in mtATPase enzymes remain ill-defined. It is generally agreed that bacterial ATP synthase contains two identical copies of subunit b per complex and that the corresponding chloroplast enzyme contains one copy each of two non-identical but homologous subunits, b and b′ (I and II). By contrast, in the mammalian system, the stoichiometry of subunit b and other possible stator stalk components such as subunits OSCP and d and coupling factor 6 varies according to the report. Collinson et al. (5Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar) determined the molar ratio of b:OSCP:d:F6(where F6 is coupling factor 6) to be 1:1:1:1 using three independent methods. Using a different approach, Hekman et al. (6Hekman C. Tomich J.M. Hatefi Y. J. Biol. Chem. 1991; 266: 13564-13571Abstract Full Text PDF PubMed Google Scholar) proposed a stoichiometry of 2:1:1:2 for the same group of subunits (b:OSCP:d:F6). Some support for a stoichiometry of 2 for subunit b comes from the earlier studies of Lippe et al. (7Lippe G. Dabbeni Sala F. Sorgato M.C. J. Biol. Chem. 1988; 263: 18627-18634Abstract Full Text PDF PubMed Google Scholar). The stoichiometry of 1 for OSCP reported by Collinsonet al. (5Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar) and Hekman et al. (6Hekman C. Tomich J.M. Hatefi Y. J. Biol. Chem. 1991; 266: 13564-13571Abstract Full Text PDF PubMed Google Scholar) contradicts the earlier report of Penin et al. (8Penin F. Archinard P. Moradi-Ameli M. Godinot C. Biochim. Biophys. Acta. 1985; 810: 346-353Crossref PubMed Scopus (19) Google Scholar), who proposed a stoichiometry of 2 for OSCP in the porcine enzyme. Remarkably, the stoichiometry of subunits b, OSCP, and d in mtATPase from the yeast Saccharomyces cerevisiae has received little attention. In light of the discrepancies between reports on the stoichiometry of subunits in mammalian mtATPase complexes and differences in subunit composition of bacterial and eukaryotic enzymes, a resolution of these issues is required. The stoichiometry of subunits b, OSCP, and d in yeast cannot be merely predicted using models of the bacterial complex. Therefore, we have set out to establish the stoichiometry of subunits b, OSCP, and d in yeast. To achieve this, we exploited a technique initially used to isolate, by immobilized metal ion affinity (Ni2+-NTA) chromatography, mtATPase complexes containing individual subunits tagged with hexahistidine (Hex6), namely, subunit d, OSCP (9Bateson M. Devenish R.J. Nagley P. Prescott M. Anal. Biochem. 1996; 238: 14-18Crossref PubMed Scopus (20) Google Scholar), or subunit b. Using such hexahistidine tagging technology, we demonstrate that the stoichiometry of subunits d, OSCP, and b in yeast mtATPase is 1 in each case. Ni2+-NTA resin was purchased from QIAGEN Pty. Ltd. (Melbourne, Australia). Dodecyl β-maltoside, complete protease inhibitors, and anti-hexahistidine monoclonal antibodies were purchased from Boehringer Mannheim (Sydney, Australia).N-Octyl glucoside was purchased from Sigma. Vistra ECF substrate was purchased from Amersham Pharmacia Biotech (Sidney, Australia). The ATP4 gene cassette, encoding subunit b, was modified by polymerase chain reaction to encode a C-terminal addition of hexahistidine and cloned into the BamHI expression site of the vector pAS2 to form pAS2-SUB-Hex6. Plasmid pAS2 is a derivative of pAS1 (10Prescott M. Higuti T. Nagley P. Devenish R.J. Biochem. Biophys. Res. Commun. 1995; 207: 943-949Crossref PubMed Scopus (17) Google Scholar) and differs in that the LEU2 selectable marker is replaced by HIS3. Genes encoding proteins containing hexahistidine are denoted by the suffix -Hex6. The vector pAS2-SUB, carrying the expression cassette encoding subunit b without a hexahistidine addition, was constructed in a similar fashion. Construction of the plasmids pAS1-OSCP-Hex6, pAS3-SUD-Hex6, pAS1-OSCP, and pAS3-SUD has been described previously (9Bateson M. Devenish R.J. Nagley P. Prescott M. Anal. Biochem. 1996; 238: 14-18Crossref PubMed Scopus (20) Google Scholar). Expression cassettes carried by the pAS series of vectors are under the transcriptional control of the PGK1promoter. The plasmids pRJ21-SUD, pRJ21-OSCP, and pRJ21-SUB, expressing subunits d, OSCP, and b, respectively, under the transcriptional control of the GAL1 promoter, have been described (11Straffon A.F.L. Prescott M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1998; 1371: 157-162Crossref PubMed Scopus (24) Google Scholar). All plasmids were introduced into a yeast strain null for expression of the corresponding chromosomal gene. YRD15 (MATαhis3-11, 2-15 leu2-3, 2-112 ura3-251, 3-373,[rho +])is the parent strain for these null mutants (11Straffon A.F.L. Prescott M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1998; 1371: 157-162Crossref PubMed Scopus (24) Google Scholar). Generation times of strains were determined at 28 °C in SaccE medium, which is a rich medium containing 2% ethanol as a carbon source (12Gray R.E. Law R.H. Devenish R.J. Nagley P. Methods Enzymol. 1996; 264: 369-389Crossref PubMed Scopus (23) Google Scholar). Mitochondria were prepared from yeast following zymolase digestion of the cell wall (13Law R.H. Manon S. Devenish R.J. Nagley P. Methods Enzymol. 1995; 260: 133-163Crossref PubMed Scopus (67) Google Scholar). Lysates were prepared from isolated mitochondria, and Ni2+-NTA chromatography was performed as described by Bateson et al. (9Bateson M. Devenish R.J. Nagley P. Prescott M. Anal. Biochem. 1996; 238: 14-18Crossref PubMed Scopus (20) Google Scholar). Assembled ATP synthase complexes were immunoprecipitated from lysates of mitochondria using an immobilized monoclonal antibody directed against the β-subunit of the F1 sector (14Hadikusumo R.G. Meltzer S. Choo W.M. Jean-Francois M.J. Linnane A.W. Marzuki S. Biochim. Biophys. Acta. 1988; 933: 212-222Crossref PubMed Scopus (64) Google Scholar). Mitochondria were isolated (13Law R.H. Manon S. Devenish R.J. Nagley P. Methods Enzymol. 1995; 260: 133-163Crossref PubMed Scopus (67) Google Scholar), and ATPase activity was monitored spectrophotometrically by the oxidation of NADH in an enzyme-linked assay containing pyruvate dehydrogenase and lactate dehydrogenase (15Roberts H. Choo W.M. Murphy M. Marzuki S. Lukins H.B. Linnane A.W. FEBS Lett. 1979; 108: 501-504Crossref PubMed Scopus (39) Google Scholar). Assays were performed at 28 °C and contained 30 μg of mitochondrial protein. Oligomycin sensitivity of the ATPase was determined by the addition of oligomycin (100 μg/mg mitochondrial protein) SDS-PAGE was carried out according to standard protocols (16Hames B.D. Hames B.D. Rickwood D. Gel Electrophoresis of Proteins: A Practical Approach. IRL Press, Oxford1990: 30-49Google Scholar) using a Bio-Rad minigel apparatus. Gels contained 15% acrylamide and were stained for protein with silver (17Wray W. Boulikas T. Wray V.P. Hancock R. Anal. Biochem. 1981; 118: 197-203Crossref PubMed Scopus (2501) Google Scholar). Proteins were transferred to nitrocellulose membrane after SDS-PAGE by standard procedures (18Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 73: 4350-4354Crossref Scopus (44911) Google Scholar). Membranes were probed with rabbit polyclonal antisera against subunit d or OSCP (diluted 1:1000), mouse monoclonal antibodies against subunit b (diluted 1:7000), and monoclonal antibodies directed against the hexahistidine tag (diluted 1:500). Secondary antibodies were alkaline phosphatase-conjugated anti-rabbit and anti-mouse IgG. Signals were generated by covering the nitrocellulose filter with a 50 μl/cm2 concentration of the chemifluorescent Vistra substrate (diluted 1:4 with water) and incubating for 5 min at room temperature. Chemifluorescence at 540–560 nm was detected using a Storm 820 PhosphorImages (Molecular Dynamics Australia Ltd., Pty., Melbourne, Australia) using excitation at 450 nm. Signals were quantified using ImageQuant software (Molecular Dynamics Australia Ltd., Pty.). In separate experiments, a linear response of the detection technique was established for each polypeptide/antibody combination by analyzing a serial dilution of each sample. We have previously demonstrated that when OSCP-Hex6 or subunit d-Hex6 is expressed in a strain lacking the corresponding endogenous subunit, assembled mtATPase complexes can be adsorbed from a mitochondrial lysate to Ni2+-NTA resin by binding of the Hex6-tagged subunit to the resin (9Bateson M. Devenish R.J. Nagley P. Prescott M. Anal. Biochem. 1996; 238: 14-18Crossref PubMed Scopus (20) Google Scholar). We have also recovered assembled mtATPase complexes containing a tagged subunit b. 2M. Bateson, R. J. Devenish, P. Nagley, and M. Prescott, unpublished results. Recovery of assembled mtATPase complexes via a Hex6-tagged subunit has now been exploited in this study to determine the stoichiometry of the three subunits, d, OSCP, and b. Strains YMB4, YMB5, and are of wild-type subunits d, OSCP, and subunit b, respectively, with the Hex6-tagged of the same subunit. In these expression of the wild-type subunit, under the transcriptional control of the GAL1 promoter, can be by the addition of to the the used in these experiments, expression of the Hex6-tagged subunit, under the transcriptional control of the was The addition of to of these in SaccE medium would in the of expression of the wild-type subunit, at the same expression of the Hex6-tagged would expressing the untagged wild-type and Hex6-tagged subunits would be to a mixed population of mtATPase complexes assembled from of each subunit. We that the stoichiometry of each subunit could then be determined by the of untagged and Hex6-tagged subunits from such mixed mtATPase in mitochondrial lysates using Ni2+-NTA The was that the stoichiometry of the subunit were would be only two of mtATPase complexes those containing a copy of an untagged wild-type subunit or the corresponding Hex6-tagged subunit. complexes containing the Hex6-tagged subunit would be adsorbed to the Ni2+-NTA resin from mitochondrial only tagged subunit would be detected in the mtATPase complexes the stoichiometry for a subunit was then more two would be in the population of mtATPase A of complexes would that contains a wild-type and a Hex6-tagged subunit. Ni2+-NTA chromatography of lysates containing such a would in the of untagged wild-type subunit as an integral component of complexes recovered by virtue of the of the Hex6-tagged subunit. The of the untagged wild-type in Ni2+-NTA would that the stoichiometry of a subunit was The of the each of the Hex6-tagged subunits to as a for the corresponding endogenous subunit was lacking expression of encoding subunit d, OSCP, or b are to on to the of a ATP Strains and expressing subunit and subunit respectively, in the of the corresponding endogenous subunit, were assessed for in SaccE medium containing ethanol as a carbon The of these strains at 28 °C was with that of strains and expressing subunits d, OSCP, and b, respectively, each lacking a tag at the times were for strains not The function of each of the Hex6-tagged subunits was investigated in more ATPase activity was in lysates of isolated mitochondria in the and of an of the a an of the of coupling between the F1 sector and the membrane sector of the enzyme complex. The ATPase activity of mitochondrial lysates prepared from strains and was to be to the ATPase activity in mitochondrial lysates of the corresponding control strains and not by oligomycin of ATPase activity in each of the mitochondrial was to be in the of of the Therefore, the of subunit d, OSCP, or b to the C-terminal addition of hexahistidine the of each of these subunits to into mtATPase complexes the of the complex to ATP for In experiments, the of tagged and untagged of each of the subunits d, OSCP, and b, expressed in YMB4, YMB5, and respectively, were monitored lysates were prepared from of each strain in medium containing ethanol and and to of proteins to nitrocellulose were probed with antisera not Mitochondria were prepared from with a of the untagged subunit and a of the Hex6-tagged subunit. The ATPase activity in isolated mitochondria was determined and to be to that in control by oligomycin was similar and in the of of the activity not that expressing subunit or subunit in the of the corresponding untagged subunit were not in to Mitochondria were and assembled mtATPase complexes were isolated by with an immobilized antibody or by to Ni2+-NTA Proteins by these were to SDS-PAGE and by silver only for the Ni2+-NTA affinity chromatography are The for a assembled ATP synthase was generated using a of the complex isolated from control R. J. Biol. Chem. 1981; Full Text PDF PubMed Google Scholar). corresponding to each of the subunits could be in from Ni2+-NTA with lysates of mitochondria containing modified subunits d, OSCP, and b It was in each assembled mtATPase was recovered by Ni2+-NTA chromatography and not In 2 a corresponding to subunit d was and replaced by a the for In a corresponding to OSCP was and replaced by a subunit b. a corresponding to subunit b was and replaced by a of The change in in each was with the additional of Proteins recovered from lysates of YMB4, YMB5, or mitochondria by with an immobilized antibody or by Ni2+-NTA chromatography were to SDS-PAGE under that could the tagged and untagged of each subunit. were probed with antisera against subunit d, OSCP, or b or hexahistidine were detected in immunoprecipitated from lysates of each mitochondrial when were probed with antisera against untagged wild-type subunits In each a with similar to the in 1 was detected when a of the was probed with a monoclonal antibody against hexahistidine Therefore, the of to subunit d-Hex6 OSCP-Hex6 and subunit respectively, the of to the untagged form of each of the subunits. that untagged and Hex6-tagged subunits were in assembled mtATPase complexes isolated from the mitochondrial of the in the is in of subunits recovered by and Ni2+-NTA corresponding to untagged and Hex6-tagged subunits on of and Ni2+-NTA purifications were quantified using ImageQuant software on the The of untagged and Hex6-tagged subunits recovered are as a of the population of untagged and Hex6-tagged subunits and the of three of the sample. in a corresponding to untagged and Hex6-tagged subunits on of and Ni2+-NTA purifications were quantified using ImageQuant software on the The of untagged and Hex6-tagged subunits recovered are as a of the population of untagged and Hex6-tagged subunits and the of three of the sample. A was detected in of Ni2+-NTA resin with lysates of each mitochondrial when were probed with antisera against untagged wild-type subunits of the in 2 in assembled complexes adsorbed to the Hex6-tagged subunit of the subunit recovered This that the stoichiometry of each subunit in mtATPase is of the Hex6-tagged subunits were detected in the not adsorbed to the Ni2+-NTA resin that to the resin of complexes containing the Hex6-tagged subunit was complete in each case. This is an important as it may be that complexes two or more copies of a Hex6-tagged subunit would have a higher affinity for the Ni2+-NTA resin and the resin was in complexes containing only one copy of a Hex6-tagged subunit. In this the stoichiometry of subunits d, OSCP, and b in the yeast S. cerevisiae was defined using hexahistidine technology. Ni2+-NTA chromatography was performed on a population of mtATPase complexes that were isolated from expressing a Hex6-tagged subunit with the corresponding wild-type subunit. The was then which subunits, tagged or with other subunits of the mtATPase are adsorbed to Ni2+-NTA The to this a the stoichiometry of these subunits. the that only the Hex6-tagged subunit was recovered in it was that is more one of each of subunits d, OSCP, and b per mtATPase complex. these are in with a for ATP synthase (5Collinson I.R. Skehel J.M. Fearnley I.M. Runswick M.J. Walker J.E. Biochemistry. 1996; 35: 12640-12646Crossref PubMed Scopus (72) Google Scholar), the of these for ATP synthase structure when we at the stoichiometry of these subunits in the bacterial In the stoichiometry of subunits b and δ is established as R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus (76) Google Scholar). the bacterial enzyme is as the of of structure and subunit composition may not be to the of the structure and composition of eukaryotic enzymes. In subunit b is proposed to be of a stator that the F1 in rotation of the γ subunit. The of subunit b is in the the of the protein is to form an that and with the F1 sector J. Biol. Chem. Full Text PDF PubMed Google Scholar). suggest that the C-terminal of subunit b with subunit δ, which in with subunits of the F1 sector (3Ogilvie I. Aggeler R. Capaldi R.A. J. Biol. Chem. 1997; 272: 16652-16656Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar, J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). subunit b has been to with subunit A.J.W. Capaldi R.A. J. Biol. Chem. 1998; 273: 29406-29410Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). of subunit b have been in F1F0-ATPases in are two copies of subunit b, suggest is only one copy of subunit b per complex in yeast. This in stoichiometry means that subunit the of the copy of subunit b or the stator stalk has a different composition in the mitochondrial enzyme of It be that has been reported between OSCP and the subunit b T. FEBS Lett. PubMed Scopus Google Scholar). OSCP in the eukaryotic complex may the of the subunit b and subunit a subunit for which is in may the of the copy of subunit b. a subunit may a that would be in the bacterial may be the but not a homologue, in acid of a bacterial subunit. We in subunit d may the of a subunit b. In yeast lacking expression of subunit d, a component of the subunit is not assembled into the membrane of the D. J. J. Biol. Chem. 1991; 266: Full Text PDF PubMed Google Scholar). The of in that subunit b is to subunit the of subunit 6 in the mitochondrial ATP synthase J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. J. R.H. J. Biol. Chem. Full Text PDF PubMed Google Scholar). d of the mtATPase can be to subunit the of subunit in yeast Tomich J.M. Hatefi Y. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar). may that the function of the membrane integral and subunit the of the of subunit b. The C-terminal of subunit three and into the and would be to with subunit d T. M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; PubMed Scopus (23) Google Scholar). in the combination of the integral membrane subunit and subunit d may the function of the copy of subunit b. We are now in the of subunits and d in yeast. the study of the bacterial ATP synthase has with a on which to other different subunit stoichiometry and more composition that cannot be and to to The of a yeast a of to be used for the study of mtATPase, at the same a more on which to understanding of the structure and function of the eukaryotic not to times of strains and ATPase activity are in and times of strains and ATPase of isolated ATP were isolated from at 28 °C with 2% ethanol as the carbon Assays were performed in the of μg of of All were performed in in a activity of mitochondria isolated from coexpression strains with ATP were isolated from at 28 °C with 2% ethanol as the carbon Assays were performed in the of μg of of All were performed in in a Mitochondria were isolated from at 28 °C with 2% ethanol as the carbon Assays were performed in the of μg of of All were performed in Mitochondria were isolated from at 28 °C with 2% ethanol as the carbon Assays were performed in the of μg of of All were performed in