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

Michael Bateson, Rodney J. DevenishDagger , Phillip Nagley, and Mark Prescott

From the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Victoria 3168, Australia

    ABSTRACT
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Abstract
Introduction
Appendix
References

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.

    INTRODUCTION
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Abstract
Introduction
Appendix
References

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 (1). 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 gamma  subunit in the F1 sector of the complex (2). Moreover, the rotary process by which ATP is synthesized is thought to involve a stator that prevents the futile rotation of the alpha 3beta 3 hexamer. There is evidence for such a stator consisting of subunits b and delta  in the bacterial enzyme (3, 4). 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,1 which are homologues of the bacterial subunits b and delta , 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. (5) 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. (6) 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. (7). The stoichiometry of 1 for OSCP reported by Collinson et al. (5) and Hekman et al. (6) contradicts the earlier report of Penin et al. (8), 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 (9), 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.

    EXPERIMENTAL PROCEDURES

Materials-- Ni2+-NTA resin was purchased from QIAGEN Pty. Ltd. (Melbourne, Australia). Dodecyl beta -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).

Construction of Expression Vectors-- 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 (10) 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 (9). Expression cassettes carried by the pAS series of vectors are under the transcriptional control of the PGK1 promoter. 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 (11). All plasmids were introduced into a yeast strain null for expression of the corresponding chromosomal gene. YRD15 (MATalpha his3-11, 2-15 leu2-3, 2-112 ura3-251, 3-373, [rho+])is the parent strain for these null mutants (11). Generation times of strains were determined at 28 °C in SaccE medium, which is a rich medium containing 2% ethanol as a carbon source (12).

Isolation of ATP Synthase from Mitochondrial Lysates-- Mitochondria were prepared from yeast following zymolase digestion of the cell wall (13). Lysates were prepared from isolated mitochondria, and Ni2+-NTA chromatography was performed as described by Bateson et al. (9). Assembled ATP synthase complexes were immunoprecipitated from lysates of mitochondria using an immobilized monoclonal antibody directed against the beta -subunit of the F1 sector (14).

ATPase Assay-- Mitochondria were isolated (13), and ATPase activity was monitored spectrophotometrically by the oxidation of NADH in an enzyme-linked assay containing pyruvate dehydrogenase and lactate dehydrogenase (15). 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-- SDS-PAGE was carried out according to standard protocols (16) using a Bio-Rad minigel apparatus. Gels contained 15% acrylamide and were stained for protein with silver (17).

Western Blotting and Image Analysis-- Proteins were transferred to nitrocellulose membrane after SDS-PAGE by standard procedures (18). 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.

    RESULTS

Principle of the Method for Determining Subunit Stoichiometry-- 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 (9). We have also recovered assembled mtATPase complexes containing a tagged subunit b.2 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 YMB6 are capable of coexpressing wild-type subunits d, OSCP, and subunit b, respectively, with the Hex6-tagged version of the same subunit. In these strains, expression of the wild-type subunit, under the tight transcriptional control of the GAL1 promoter, can be strongly induced by the addition of galactose to the growth medium. Under the conditions used in these experiments, expression of the Hex6-tagged subunit, under the transcriptional control of the PGK1 promoter, was essentially constitutive. The addition of galactose to cultures of these cells in SaccE growth medium would result in the induction of expression of the wild-type subunit, while, at the same time, expression of the Hex6-tagged version would continue. Cells expressing both the untagged wild-type and Hex6-tagged subunits would be expected to contain a mixed population of mtATPase complexes assembled from both forms of each subunit. We reasoned that the stoichiometry of each subunit could then be determined by assessing the recovery of untagged and Hex6-tagged subunits from such mixed mtATPase populations present in mitochondrial lysates using Ni2+-NTA chromatography. The prediction was that if the stoichiometry of the relevant subunit were 1, there would be only two species of mtATPase complexes present, those containing a single copy of either an untagged wild-type subunit or the corresponding Hex6-tagged subunit. Only complexes containing the Hex6-tagged subunit would be adsorbed to the Ni2+-NTA resin from mitochondrial lysates; therefore, only tagged subunit would be detected in the mtATPase complexes recovered. If, however, the stoichiometry for a subunit was >1, then more than two species would be present in the population of mtATPase complexes. A subpopulation of complexes would exist that contains both a wild-type and a Hex6-tagged subunit. Subsequent Ni2+-NTA chromatography of lysates containing such a subpopulation would result in the recovery of untagged wild-type subunit as an integral component of complexes recovered by virtue of the presence of the Hex6-tagged subunit. The presence of the untagged wild-type polypeptide in Ni2+-NTA eluates would therefore indicate that the stoichiometry of a particular subunit was >1.

mtATPase Containing Hex6-tagged Subunits Is Functional-- The ability of the each of the Hex6-tagged subunits to act as a functional replacement for the corresponding endogenous subunit was tested. Yeast cells lacking expression of genes encoding subunit d, OSCP, or b are unable to grow on nonfermentable substrates owing to the absence of a functional ATP synthase. Strains YMB1, YMB2, and YMB3, expressing subunit d-Hex6, OSCP-Hex6, and subunit b-Hex6, respectively, in the absence of the corresponding endogenous subunit, were assessed for growth in liquid SaccE medium containing ethanol as a nonfermentable carbon source. The growth rate of these strains at 28 °C was compared with that of strains A7NP, A5NP, and A4NP, expressing subunits d, OSCP, and b, respectively, each lacking a Hex6 tag at the C terminus. Equivalent generation times were observed for all strains (data not shown).

The function of each of the Hex6-tagged subunits was investigated in more detail. ATPase activity was measured in lysates of isolated mitochondria in the presence and absence of oligomycin, an inhibitor of the F0 proton channel. Such a measurement gives an indication of the degree of functional coupling between the F1 catalytic sector and the F0 membrane sector of the enzyme complex. The ATPase activity of mitochondrial lysates prepared from strains YMB1, YMB2, and YMB3 was found to be closely comparable to the ATPase activity observed in mitochondrial lysates of the corresponding control strains A7NP, A5NP, and A4NP (data not shown). Inhibition by oligomycin of ATPase activity in each of the mitochondrial preparations was found to be in the range of 82-88% of the uninhibited activity. Therefore, the modification of subunit d, OSCP, or b to contain the C-terminal addition of hexahistidine compromised neither the ability of each of these subunits to assemble into mtATPase complexes nor the capacity of the resultant complex to generate adequate ATP synthesis for cellular growth.

Recovery of Assembled mtATPase Complexes from Mitochondrial Lysates by Ni2+-NTA Chromatography-- In preliminary experiments, the relative levels of tagged and untagged forms of each of the subunits d, OSCP, and b, expressed in YMB4, YMB5, and YMB6 cells, respectively, were monitored during growth. Cell lysates were prepared from cells of each strain cultured in growth medium containing ethanol and galactose and subjected to SDS-PAGE. Following transfer of proteins to nitrocellulose membrane, blots were probed with appropriate subunit-specific antisera (data not shown). Mitochondria were prepared from cells with a high content of the untagged subunit and a low content of the Hex6-tagged subunit. The ATPase activity in isolated mitochondria was determined and found to be closely comparable to that in control mitochondria. Inhibition by oligomycin was similar and in the range of 84-92% of the uninhibited activity (data not shown). These results indicate that cells expressing subunit d-Hex6, OSCP-Hex6, or subunit b-Hex6 in the presence of the corresponding untagged subunit were not compromised in their ability to assemble functionally coupled complexes.

Mitochondria were lysed, and assembled mtATPase complexes were isolated by immunoprecipitation with an immobilized F1-beta antibody or by adsorption to Ni2+-NTA resin. Proteins purified by these methods were subjected to SDS-PAGE and visualized by silver staining (Fig. 1; only data for the Ni2+-NTA affinity chromatography are shown). The expected polypeptide profile for a fully assembled ATP synthase (lane 1) was generated using a preparation of the purified complex isolated from control cells (19). Polypeptides corresponding to each of the subunits could be identified in eluates from Ni2+-NTA incubated with lysates of mitochondria containing modified subunits d, OSCP, and b (lanes 2-4). It was concluded that, in each case, assembled mtATPase was efficiently recovered by both Ni2+-NTA chromatography and immunoprecipitation (data not shown). In lane 2 (subunit d-Hex6), a polypeptide corresponding to subunit d was absent and replaced by a polypeptide migrating just below the position for OSCP. In lane 3 (OSCP-Hex6), a polypeptide corresponding to OSCP was absent and replaced by a polypeptide migrating just below subunit b. In lane 4 (subunit b-Hex6), a polypeptide corresponding to subunit b was absent and replaced by a polypeptide of decreased mobility. The change in relative mobility in each case was consistent with the additional mass (823 Da) of six histidine residues.


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Fig. 1.   Isolation of mtATPase complexes containing Hex6-tagged subunits from cells grown in the presence of galactose. ATP synthase isolated from the parental strain YRD15 (lane 1) by the method of Rott and Nelson (19) was included as a standard and is representative of a fully assembled complex. Proteins from lysates of mitochondria isolated after growth in medium containing galactose prepared from yeast strains YMB4 (lane 2), YMB5 (lane 3), and YMB6 (lane 4) were recovered by Ni2+-NTA chromatography. Eluates were subjected to SDS-PAGE analysis and stained with silver. The positions of selected subunits of the standard ATP synthase preparation are identified on the left. Arrowheads (lanes 2-4) indicate the positions of the Hex6-tagged subunits (subunit d-Hex6, OSCP-Hex6, and subunit b-Hex6, respectively).

The Stoichiometry of Subunits d, OSCP, and b Is 1 in Each Case-- Proteins recovered from lysates of YMB4, YMB5, or YMB6 mitochondria by immunoprecipitation with an immobilized F1-beta antibody or by Ni2+-NTA chromatography were subjected to SDS-PAGE under conditions that could resolve both the tagged and untagged forms of each subunit. Separate blots were probed with monospecific antisera against subunit d, OSCP, or b or hexahistidine (Fig. 2).


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Fig. 2.   Recovery and immunological detection of mtATPase subunits. After growth of strains YMB4 (A), YMB5 (B), and YMB6 (C) in medium containing galactose, mitochondria were isolated. Proteins were recovered from mitochondrial lysates by immunoprecipitation with an immobilized F1-beta antibody (lane 1) or by Ni2+-NTA chromatography (lanes 2 and 4). Samples of protein that had not been adsorbed to the Ni2+-NTA resin during the chromatography procedure were also analyzed (lane 3). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Membranes were probed with antisera containing antibodies with the following specificities: subunit d (Sud; A, lanes 1-3), OSCP (B, lanes 1-3), subunit b (Sub; C, lanes 1-3), and hexahistidine (A-C, lane 4 in each case). The blots were developed with Vistra ECF substrate, and bands were visualized by scanning for chemifluorescence using a PhosphorImager.

Two polypeptides were detected in material immunoprecipitated from lysates of each mitochondrial preparation when blots were probed with antisera against untagged wild-type subunits (Fig. 2, A-C, lane 1). In each case, a single polypeptide with similar mobility to the slow migrating polypeptide observed in lane 1 was detected when a portion of the blot was probed with a monoclonal antibody against hexahistidine (Fig. 2, A-C, lane 4). Therefore, the polypeptides of low mobility corresponded to subunit d-Hex6 (panel A), OSCP-Hex6 (panel B), and subunit b-Hex6 (panel C) respectively, whereas the polypeptide of high mobility corresponded to the untagged form of each of the subunits. These results indicate that both untagged and Hex6-tagged subunits were present in assembled mtATPase complexes isolated from the mitochondrial preparations. Quantitative analysis of the signal in lane 1 of the blots is presented in Table I.

                              
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Table I
Quantification of subunits recovered by immunoprecipitation and Ni2+-NTA chromatography
Chemifluorescent signals corresponding to untagged and Hex6-tagged subunits on immunoblots of immunoprecipitations and Ni2+-NTA purifications were quantified using ImageQuant software on the PhosphorImager scan. The amounts of untagged and Hex6-tagged subunits recovered are shown as a percentage of the total population of untagged and Hex6-tagged subunits and represent the mean ± S.D. of three loadings of the sample.

A single polypeptide was detected in eluates of Ni2+-NTA resin incubated with lysates of each mitochondrial preparation when blots were probed with antisera against untagged wild-type subunits (Fig. 2, A-C, lane 2). Quantitative analysis of the signal in lane 2 indicated that, in assembled complexes adsorbed to Ni2+-NTA, the Hex6-tagged subunit represented some 80-95% of the total subunit recovered (Table I). This result indicates that the stoichiometry of each subunit in mtATPase is 1. No significant amounts of the Hex6-tagged subunits were detected in the material not adsorbed to the Ni2+-NTA resin (Fig. 2, A-C, lane 3), indicating that adsorption to the resin of complexes containing the Hex6-tagged subunit was complete in each case. This is an important consideration as it may be expected that complexes having two or more copies of a Hex6-tagged subunit would have a significantly higher affinity for the Ni2+-NTA resin and would, if the resin was present in limiting amounts, displace complexes containing only one copy of a Hex6-tagged subunit.

    DISCUSSION

In this study, 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 cells expressing a Hex6-tagged subunit along with the corresponding wild-type subunit. The question was then asked, which subunits, tagged or untagged, along with other subunits of the mtATPase are adsorbed to Ni2+-NTA resin? The answer to this question provided a definitive statement about the stoichiometry of these subunits. On the basis that only the Hex6-tagged subunit was recovered in eluates, it was concluded that there is no more than one of each of subunits d, OSCP, and b per mtATPase complex.

Although these results are in accordance with a recent stoichiometric determination for bovine ATP synthase (5), the implications of these findings for ATP synthase structure across species become evident when we look at the stoichiometry of these subunits in the bacterial system. In Escherichia coli, the stoichiometry of subunits b and delta  (OSCP homologue) is well established as b2delta (20, 21). Although the bacterial enzyme is considered as the "prototype" of all F1F0-ATPases, knowledge of its structure and subunit composition may not be directly applicable to the prediction of the structure and composition of eukaryotic enzymes.

In E. coli, subunit b is proposed to be part of a stator that holds the F1 alpha 3beta 3 headpiece in place during rotation of the gamma  subunit. The N-terminal portion of subunit b is anchored in the membrane; however, the remainder of the protein is considered to form an alpha -helical segment that extends and interacts with the F1 sector (22). Chemical cross-linking experiments suggest that the C-terminal portion of subunit b interacts with subunit delta , which in turn interacts with subunits of the F1 sector (3, 23). Recently, subunit b has been shown to cross-link with subunit alpha  (4). Homologues of subunit b have been found in all F1F0-ATPases studied so far. Whereas in E. coli there are two copies of subunit b, our results suggest there is only one copy of subunit b per complex in yeast. This difference in stoichiometry means that either another subunit fulfills the role of the second copy of subunit b or the stator stalk has a different composition in the mitochondrial enzyme of eukaryotes. It should be noted that considerable (~30%) homology has been reported between bovine OSCP and the hydrophilic region of E. coli subunit b (25). Thus, OSCP in the eukaryotic complex may fulfill the role of both the second subunit b and subunit delta . Alternatively, a subunit for which there is no homologue in E. coli may fulfill the role of the second copy of subunit b. Such a subunit may perform a role that would be superfluous in the bacterial system or, alternatively, may be the functional equivalent, but not a homologue, in primary amino acid sequence terms, of a bacterial subunit.

We hypothesize that, in yeast, subunit d may take the place of a second subunit b. In yeast cells lacking expression of subunit d, a component of the F0 sector, subunit 6, is not assembled into the inner membrane of the mitochondrion (26). The results of chemical cross-linking experiments in E. coli indicate that subunit b is close to subunit a, the homologue of subunit 6 in the mitochondrial ATP synthase (27, 28). Subunit d of the bovine mtATPase can be cross-linked to subunit A6L, the homologue of subunit 8 in yeast (29). One may further speculate that the function of the small membrane integral and hydrophobic subunit 8 replaces the role of the N-terminal region of subunit b. The C-terminal portion of subunit 8 includes three positively charged residues and extends into the matrix and would be available to make contact with subunit d (24). Thus, in yeast, the combination of the hydrophobic integral membrane protein, subunit 8, and subunit d may serve the function of the second copy of subunit b. We are now engaged in probing the relationship of subunits 8 and d in yeast.

Although the study of the bacterial ATP synthase has provided us with a prototype on which to develop other models, its different subunit stoichiometry and more simple composition mean that some features cannot be simply extended and expected to apply to mtATPase. The use of a yeast model allows a wide range of molecular biological approaches to be used for the study of mtATPase, while, at the same time, providing a more evolutionarily related model on which to develop our detailed understanding of the structure and function of the eukaryotic mtATPase.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 61-3-9905-3782; Fax: 61-3-9905-4699; E-mail: Rodney.Devenish{at}med.monash.edu.au.

2 M. Bateson, R. J. Devenish, P. Nagley, and M. Prescott, unpublished results.

    ABBREVIATIONS

The abbreviations used are: OSCP, oligomycin-sensitivity conferring protein; mtATPase, mitochondrial ATP synthase; NTA, nitrilotriacetic acid; PAGE, polyacrylamide gel electrophoresis.

    APPENDIX

Data not shown relating to generation times of strains and oligomycin-sensitive ATPase activity are included in Tables 1A and 2A.

                              
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Table 1A
Generation times of strains and oligomycin-sensitive ATPase activities of isolated mitochondria
Mitochondria were isolated from cells grown at 28 °C with 2% ethanol as the carbon source. Assays were performed in the presence of 100 µg of oligomycin/mg of protein, where indicated. All measurements were performed in triplicate.

                              
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Table 2A
ATPase activity of mitochondria isolated from coexpression strains induced with galactose
Mitochondria were isolated from cells grown at 28 °C with 2% ethanol as the carbon source. Assays were performed in the presence of 100 µg of oligomycin/mg of protein, where indicated. All measurements were performed in triplicate.

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Abstract
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