The Molecular Neighborhood of Subunit 8 of Yeast Mitochondrial F1F0-ATP Synthase Probed by Cysteine Scanning Mutagenesis and Chemical Modification
2003; Elsevier BV; Volume: 278; Issue: 20 Linguagem: Inglês
10.1074/jbc.m300967200
ISSN1083-351X
AutoresAndrew N. Stephens, M Khan, Xavier Roucou, Phillip Nagley, Rodney J. Devenish,
Tópico(s)Photosynthetic Processes and Mechanisms
ResumoThe detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F1-F0interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F1F0-ATP synthase. The detailed membrane topography and neighboring polypeptides of subunit 8 in yeast mitochondrial ATP synthase have been determined using a combination of cysteine scanning mutagenesis and chemical modification. 46 single cysteine substitution mutants encompassing the length of the subunit 8 protein were constructed by site-directed mutagenesis. Expression of each cysteine variant in yeast lacking endogenous subunit 8 restored respiratory phenotype to cells and had little measurable effect on ATP hydrolase function. The exposure of each introduced cysteine residue to the aqueous environment was assessed in isolated mitochondria using the fluorescent thiol-modifying probe fluorescein 5-maleimide. The first 14 and last 13 amino acids of subunit 8 were accessible to fluorescein 5-maleimide in osmotically lysed mitochondria and are thus extrinsic to the lipid bilayer, indicating a 21-amino acid transmembrane span. The C-terminal region of subunit 8 was partially occluded by other ATP synthase subunits, especially in a small region surrounding Val-40 that was demonstrated to play an important role in maintaining the stability of the F1-F0interaction. Cross-linking using heterobifunctional reagents revealed the proximity of subunit 8 to subunits b, d, and f in the matrix and to subunits b, f, and 6 in the intermembrane space. A disulfide bridge was also formed between subunit 8(F7C) or (M10C) and residue Cys-23 of subunit 6, demonstrating a close interaction between these two hydrophobic membrane subunits and confirming the location of the N termini of each in the intermembrane space. We conclude that subunit 8 is an integral component of the stator stalk of yeast mitochondrial F1F0-ATP synthase. mitochondrial F1F0-ATP synthase p-azidophenacyl bromide N-(4-p-azidosalicylamidobutyl)-3′-(pyridyldithio)-propionamide central hydrophobic domain fluorescein 5-maleimide yeast mtATPase subunit 8 hemagglutinin polyvinylidene difluoride N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine Mitochondrial F1F0-ATP synthase (mtATPase)1 consists of a membrane-extrinsic sector (F1) linked to a membrane-embedded proton channel (F0) by two protein stalks (1Devenish R.J. Prescott M. Roucou X. Nagley P. Biochim. Biophys. Acta. 2000; 1458: 428-442Crossref PubMed Scopus (83) Google Scholar). Synthesis/hydrolysis of ATP occurs on the structurally well characterized F1 sector comprised of five subunits α3-β3-γ1-δ1-ε1(2Abrahams J.P. Leslie A.G. Lutter R. Walker J.E. Nature. 1994; 370: 621-628Crossref PubMed Scopus (2749) Google Scholar, 3Stock D. Leslie A.G. Walker J.E. 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Yamamoto A. Wada Y. Futai M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 13448-13452Crossref PubMed Scopus (89) Google Scholar). By contrast to that of F1 no high-resolution structure of the F0 sector is available. In yeast mitochondria F0 is comprised of at least 12 different polypeptides: OSCP, b, d, e, f, g, h, i/j, and k that are nuclearly encoded, and subunits 6, 8, and 9 that are mitochondrially encoded (1Devenish R.J. Prescott M. Roucou X. Nagley P. Biochim. Biophys. Acta. 2000; 1458: 428-442Crossref PubMed Scopus (83) Google Scholar). F0 has two important functions. First, some F0subunits form a “stator” stalk anchored in the membrane, which during coupled ATP synthesis/hydrolysis prevents futile rotation of mtATPase subunits relative to the rotor. This structure has been visualized in ATP synthases from several organisms (14Wilkens S. Capaldi R.A. Nature. 1998; 393: 29Crossref PubMed Scopus (135) Google Scholar, 15Karrasch S. Walker J.E. J. Mol. Biol. 1999; 290: 379-384Crossref PubMed Scopus (99) Google Scholar, 16Bottcher B. Graber P. Biochim. Biophys. Acta. 2000; 1458: 404-416Crossref PubMed Scopus (45) Google Scholar). In the structurally less complex ATP synthase of Escherichia colithe stator stalk is composed of subunit δ and two copies of subunit b (17Boyer P.D. Annu. Rev. Biochem. 1997; 66: 717-749Crossref PubMed Scopus (1589) Google Scholar). However, only a single copy of subunit b is present in the mtATPase of the yeast Saccharomyces cerevisiae (18Bateson M. Devenish R.J. Nagley P. Prescott M. J. Biol. Chem. 1999; 274: 7462-7466Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar); thus, in conjunction with OSCP (the mitochondrial homolog of subunit δ), other F0 subunits contribute to form the stator stalk. Second, movement of protons through the membrane-embedded channel of F0 allows conversion of the proton gradient generated by respiratory chain activity into chemical energy, in the form of ATP made by phosphorylation of ADP on F1. Based on molecular genetic and biochemical studies in yeast the assembly and stability of the membrane-embedded proton channel of mtATPase depends on the presence of the three mitochondrially encoded protein subunits 6, 8, and 9 (19Nagley P. Trends Genet. 1988; 4: 46-51Abstract Full Text PDF PubMed Scopus (84) Google Scholar). Subunits 6 and 9 are the homologs of bacterial ATP synthase subunits a and c, respectively, and have well defined roles in proton channel function (20Capaldi R.A. Aggeler R. Trends Biochem. Sci. 2002; 27: 154-160Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Subunit 8 is not found in bacteria but is an additional subunit present in the mtATPase of mammals and fungi. The absence of all but one of these F0 subunits from the yeast F1-c10 crystal structure (3Stock D. Leslie A.G. Walker J.E. Science. 1999; 286: 1700-1705Crossref PubMed Scopus (1085) Google Scholar) highlights the need for additional biochemical approaches aimed at elucidating the structure and function of F0 subunits. In yeast subunit 8 (Y8) is a 48-amino acid intrinsic membrane protein essential for the assembly and function of mtATPase (1Devenish R.J. Prescott M. Roucou X. Nagley P. Biochim. Biophys. Acta. 2000; 1458: 428-442Crossref PubMed Scopus (83) Google Scholar). Whereas divergent in amino acid composition, Y8 and its other eukaryotic homologs exhibit three highly conserved regions: an N-terminal MPQL motif, a central hydrophobic domain (CHD), and a C-terminal positively charged region (21Velours J. Esparza M. Hoppe J. Sebald W. Guerin B. EMBO J. 1984; 3: 207-212Crossref PubMed Scopus (46) Google Scholar, 22Nagley P. Devenish R.J. Law R.H.P. Maxwell R.J. Nero D. Linnane A.W. Kim C.H. Ozawa T. Yagi K. Bioenergetics: Molecular Biology, Biochemistry, and Pathology. Plenum Press, New York1990: 305-325Crossref Google Scholar, 23Devenish R.J. Papakonstantinou T. Galanis M. Law R.H. Linnane A.W. Nagley P. Ann. N. Y. Acad. Sci. 1992; 671: 403-414Crossref PubMed Scopus (19) Google Scholar). Allotopic expression (24Nagley P. Devenish R.J. Trends Biochem. Sci. 1989; 14: 31-35Abstract Full Text PDF Scopus (45) Google Scholar), whereby a mitochondrial gene is recoded for nuclear expression with subsequent delivery of the protein back to mitochondria, has enabled our laboratory to undertake a detailed molecular genetic approach to investigate the structure and function of Y8. Detailed studies on genetically modified Y8 variants, allotopically expressed in yeast lacking endogenous Y8, have suggested that the N-terminal motif of Y8 plays a functional role in mtATPase (25Law R.H. Farrell L.B. Nero D. Devenish R.J. Nagley P. FEBS Lett. 1988; 236: 501-505Crossref PubMed Scopus (29) Google Scholar, 26Galanis M. Law R.H. O'Keeffe L.M. Devenish R.J. Nagley P. Biochem. Int. 1990; 22: 1059-1066PubMed Google Scholar, 27Grasso D.G. Nero D. Law R.H. Devenish R.J. Nagley P. Eur. J. Biochem. 1991; 199: 203-209Crossref PubMed Scopus (23) Google Scholar) while the positively charged amino acid region at the C terminus of Y8 is involved in both the assembly and function of the F0 sector (27Grasso D.G. Nero D. Law R.H. Devenish R.J. Nagley P. Eur. J. Biochem. 1991; 199: 203-209Crossref PubMed Scopus (23) Google Scholar, 28Papakonstantinou T. Galanis M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; 1144: 22-32Crossref PubMed Scopus (23) Google Scholar, 29Papakonstantinou T. Law R.H. Nagley P. Devenish R.J. Biochem. Mol. Biol. Int. 1996; 39: 253-260PubMed Google Scholar). A surprising feature of Y8 is the functional accommodation of charged amino acid residues within the CHD (30Marzuki S. Watkins L.C. Choo W.M. Biochim. Biophys Acta. 1989; 975: 222-230Crossref PubMed Scopus (30) Google Scholar, 31Papakonstantinou T. Law R.H. Manon S. Devenish R.J. Nagley P. Eur. J. Biochem. 1995; 227: 745-752Crossref PubMed Scopus (10) Google Scholar, 32Papakonstantinou T. Law R.H. Nesbitt W.S. Nagley P. Devenish R.J. Curr. Genet. 1996; 30: 12-18Crossref PubMed Scopus (13) Google Scholar, 33Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 1999; 261: 444-451Crossref PubMed Scopus (18) Google Scholar). Analysis by Roucouet al. (33Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 1999; 261: 444-451Crossref PubMed Scopus (18) Google Scholar) has shown that variants bearing either positive or negatively charged amino acids in the CHD display structural destabilization of the F1-F0 interaction but nevertheless retain function. Cysteine scanning mutagenesis has been used extensively to analyze membrane proteins in their native state and provides a powerful alternative for examining the molecular neighborhood surrounding membrane proteins (34Altenbach C. Marti T. Khorana H.G. Hubbell W.L. Science. 1990; 248: 1088-1092Crossref PubMed Scopus (408) Google Scholar, 35Frillingos S. Sahin-Toth M. Wu J. Kaback H.R. FASEB J. 1998; 12: 1281-1299Crossref PubMed Scopus (321) Google Scholar, 36Tamura N. Konishi S. Iwaki S. Kimura-Someya T. Nada S. Yamaguchi A. J. Biol. Chem. 2001; 276: 20330-20339Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). In a previous study, analysis of allotopically expressed Y8 variants having a cysteine substitution at the N- or C-terminal residue of Y8 demonstrated that the CHD spans the inner mitochondrial membrane with Nout-Cinorientation (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Here we have combined cysteine-scanning mutagenesis with allotopic expression to construct a series of single cysteine replacements encompassing the entire length of the Y8 protein (excluding three residues within the C-terminal positively charged region whose replacement would abrogate function (28Papakonstantinou T. Galanis M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; 1144: 22-32Crossref PubMed Scopus (23) Google Scholar)). Without exception each cysteine replacement was tolerated and all of the expressed Y8 variants restored mtATPase function in cells lacking endogenous Y8, demonstrating that none of the substituted amino acid residues of Y8 are essential for either ATP synthesis/hydrolysis activity or proton flow. Specific labeling experiments using the thiol-modifying chemical reagent fluorescein 5-maleimide (FM) showed that the membrane-spanning CHD of Y8 encompasses residues 15 to 35. By assessing the accessibility of introduced cysteine residues to FM in partially dissociated mtATPase complexes, the C terminus of Y8 was found to be occluded by other mtATPase subunits, especially in a small region close to Val-40 demonstrated to play an important role in maintaining the structural interaction between the F1 and F0 sectors. In addition, site-directed cross-linking experiments demonstrated extensive interactions between Y8 and subunits d, f, b, and 6. These results lead us to propose a significant role for Y8 as part of the stator stalk of mtATPase. The gene cassettes N9L-D/Y8-1 and N9L-D/Y8-1-FLAG, yeast expression vector pPD72, and yeast strains YM2 and FTC2 were as described previously (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Briefly, each gene cassette encodes a full-length wild-type Y8 protein bearing a 7-amino acid extension at the N terminus (YSSEISS, numbered from −7 to −1), which is retained following import of Y8 into mitochondria and processing of the N9L mitochondrial import sequence by matrix protease. The N9L-D/Y8-1-FLAG construct expresses the same protein bearing a C-terminal FLAG epitope (DYKDDDDK). Cells expressing the N9L-D/Y8-1 cassette are denoted YM2, those expressing N9L-D/Y8-1-FLAG are denoted FTC2. Site-directed mutagenesis was carried out on the N9L-D/Y8-1 gene cassette as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar) to generate 46 single cysteine replacements in the expressed Y8 proteins. The Y8(M1C) FLAG variant was constructed by ligation of a BamHI/KpnI fragment containing the N9L-D sequence and the first 15 nucleotides of the synthetic Y8 gene bearing the M1C mutation into a similarly digested pUC9 vector containing the FTC2 variant. The (M1C) FLAG cassette was then excised by BamHI/NotI restriction digestion and ligated into the pPD72 vector as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). A DNA sequence encoding an HA epitope (YPYDVPDYA) was introduced into the N9L-D/Y8-1 cassette using two rounds of PCR mutagenesis. The expressed protein is designated NHAY8. First, the upstream and downstream flanking primer pair, 5′-CAGGAAACAGCTATGACC-3′ and 5′-GGAACGTCGTATGGGTAAGACGAGATCTCGGAAGAGTAGGC-3′, respectively, were used to introduce nucleotides into the N9L-D/Y8-1 cassette specifying the amino acid sequence YPYDV inserted between the YSSEISS sequence that was retained following import of allotopically expressed Y8 into mitochondria and the N terminus of the Y8 peptide. A second round of PCR using the flanking primer pair 5′-CAGGAAACAGCTATGACC-3′ and 5′-GGCTCGAGGAGGCGTAGTCAGGAACGTCGTATGGGTAAGACGAGATC-3′ was then used to incorporate adjacent nucleotides into the N9L-D/Y8-1 cassette specifying amino acids PDYA, to form the complete HA epitope, along with three additional serine residues between this epitope and the Y8 passenger protein. Thus, the N9L-D/NHAY8-1 cassette encodes a protein that contains the N9L-D-YSSEISS sequence (as for YM2) followed by the sequence YPYDVPDYA-SSS-subunit 8, with the original matrix protease cleavage site maintained. A BamHI/XhoI restriction fragment bearing the nucleotide sequence encoding N9L-D/NHA was ligated into similarly digested pUC9 containing the gene cassette encoding selected N9L-D/Y8-1 cysteine variants to create constructs NHAY8(L4C), NHAY8(F7C), NHAY8(M10C), NHAY8(F44C), and NHAY8(L48C). The fidelity of all constructs was checked by DNA sequencing. Yeast strain M31 (MATα ade1 his6)[aap1−] lacking endogenous Y8 has been described previously (30Marzuki S. Watkins L.C. Choo W.M. Biochim. Biophys Acta. 1989; 975: 222-230Crossref PubMed Scopus (30) Google Scholar). M31 cells were transformed by the method of Klebe et al. (38Klebe R.J. Harriss J.V. Sharp Z.D. Douglas M.G. Gene (Amst.). 1983; 25: 333-341Crossref PubMed Scopus (371) Google Scholar) with the pPD72 expression vector that carries a functional ADE1 gene (28Papakonstantinou T. Galanis M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; 1144: 22-32Crossref PubMed Scopus (23) Google Scholar). Transformants displaying the Ade+ phenotype were selected and restoration of respiratory-competent phenotype by the expressed Y8 cysteine variants was assessed by testing growth on medium containing ethanol as the sole carbon source. Generation times, ATP hydrolysis activity in isolated mitochondria, and its sensitivity to oligomycin were measured as described previously (39Law R.H. Manon S. Devenish R.J. Nagley P. Methods Enzymol. 1995; 260: 133-163Crossref PubMed Scopus (67) Google Scholar). Comparisons (Student's ttest) were made against strain YM2 (28Papakonstantinou T. Galanis M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; 1144: 22-32Crossref PubMed Scopus (23) Google Scholar), allotopically expressing unmodified Y8. Thiol-specific labeling of mitochondrial proteins by FM was carried out for 4 h on mitochondria osmotically lysed by exposure to a hypotonic solution (denoted lysate) as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Isolated proteolipids were separated by Tricine SDS-PAGE (18% polyacrylamide) according to the method of Schagger and von Jagow (40Schagger H. von Jagow G. Anal. Biochem. 1987; 166: 368-379Crossref PubMed Scopus (10470) Google Scholar). Analysis of protein fluorescence was as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). For Y8 cysteine variants that remained unmodified under these conditions, labeling by FM was repeated as above in the presence of 1% SDS prior to proteolipid extraction. Cross-linking of mitochondrial proteins using the heterobifunctional reagentsp-azidophenacyl bromide (APA-Br) andN-(4-p-azidosalicylamidobutyl)-3′-(pyridyldithio)-propionamide (APDP) was as described, except that mitochondrial membranes equivalent to 0.5 mg of protein were used (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Cross-linking using CuCl2 was performed as follows. Mitochondrial membranes (equivalent to 0.5 mg of protein) were pelleted at 131,440 ×g for 15 min at 4 °C and resuspended in 200 μl of buffer C (50 mm Tris-HCl, 2 mmMgCl2, pH 7.5). CuCl2 was added to 1.5 mm (from a 25 mm stock solution, prepared fresh) and the samples were incubated on ice for 2 h. EDTA was then added to 10 mm to chelate the remaining Cu2+ and the samples were incubated for 5 min. Mitochondrial membranes were then pelleted at 131,440 ×g for 15 min at 4 °C and the pellet resuspended in 125 μl of 4% SDS (w/v) and 125 μl of dissociation buffer (125 mm Tris-HCl, 2% SDS (w/v), 50% glycerol (v/v), 0.02% bromphenol blue (w/v), pH 6.7). To reduce samples 1 μl of 14.3 mm 2-mercaptoethanol was added and the samples were incubated on ice for 30 min, then at 65 °C for 10 min prior to analysis by SDS-PAGE and immunoblotting. Removal of the F1 sector from mitochondrial membranes was achieved using the modified method of McEnery et al. (41McEnery M.W. Hullihen J. Pedersen P.L. J. Biol. Chem. 1989; 264: 12029-12036Abstract Full Text PDF PubMed Google Scholar). Mitochondrial membranes (equivalent to 2 mg of protein) were pelleted at 131,440 × g for 15 min at 4 °C, and then resuspended in 0.8 ml of PAB buffer (0.15 mK2HPO4, 25 mm EDTA, 100 mm 2-mercaptoethanol, pH 7.9). After 5 min incubation on ice, the mitochondrial membranes were centrifuged as before and the pellet resuspended in 1.0 ml of GPAB buffer (0.15 mK2HPO4, 25 mm EDTA, 100 mm 2-mercaptoethanol, 3.0 m guanidine-HCl, pH 7.9). Following incubation on ice for 13 min, membranes were pelleted as before and washed twice in PA buffer (150 mmK2HPO4, 25 mm EDTA, pH 7.9) by resuspension and centrifugation. For fluorescent labeling experiments membrane samples were then resuspended in 0.2 ml of buffer A and labeling performed as described above. Samples not intended for labeling studies were prepared for denaturing glycine SDS-PAGE as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Monomeric mtATPase complexes were isolated using clear native gel electrophoresis according to the method of Arnold et al. (42Arnold I. Pfeiffer K. Neupert W. Stuart R.A. Schagger H. EMBO J. 1998; 17: 7170-7178Crossref PubMed Scopus (364) Google Scholar), but with the omission of Serva Blue G dye from the sample. Mitochondrial membranes (equivalent to 100 μg of protein) were pelleted at 131,440 × g for 15 min at 4 °C and resuspended in 20 μl of native gel sample buffer (50 mmNaCl, 2 mm aminohexanoic acid, 1 mm EDTA, 50 mm imidazole, 5 mm phenylmethylsulfonyl fluoride). Lauryl maltoside was added to a final concentration of 2.86% (w/v) and the samples (total sample volume 26 μl) were incubated on ice for 20 min. Insoluble material was pelleted at 131,440 × g for 15 min at 4 °C, and 20 μl of the sample was applied to a 4–13% polyacrylamide gradient gel (dimensions 13 × 10 × 0.075 cm). Electrophoresis was carried out for 3 h with an initial current of 15 mA and voltage increasing to a maximum of 500 V. On completion of electrophoresis, gels were transferred to small plastic containers and in situ ATP hydrolase activity was assessed using the method of Yoshida et al. (43Yoshida M. Sone N. Hirata H. Kagawa Y. J. Biol. Chem. 1975; 250: 7910-7916Abstract Full Text PDF PubMed Google Scholar). Gels were photographed, then fixed in a solution of methanol (50%) (v/v), acetic acid (10%) (w/v) for 30 min, and then stained in acetic acid (10%) (v/v), Serva Blue G dye (0.025%) (w/v) overnight. Gels were destained in acetic acid (10%) (v/v) for at least 2 h with several changes of solution until protein bands were distinctly blue against a clear background. Coomassie-stained gels were analyzed using a ProXPRESS multiwavelength fluorimager (PerkinElmer Life Sciences) and Phoretix 2D analysis software (Nonlinear Dynamics, Newcastle upon Tyne, United Kingdom). Denaturing second dimension gel electrophoresis analysis of isolated mtATPase complexes was performed as follows. Monomeric mtATPase complexes isolated using native gel electrophoresis and stained with Serva Blue G dye were carefully excised from the gel and each polyacrylamide fragment rinsed in 1 ml of distilled water in a 1.5-ml snap-cap tube. After a 60-s centrifugation at 20,798 × g, the water was aspirated off and 50 μl of SDS (5%) (w/v) was added to the tube. The gel fragment was then finely ground using a thin glass rod and centrifuged at 20,798 × g for 1 min. Grinding and centrifugation were repeated and then 50 μl of 2× dissociation buffer (125 mm Tris-HCl, 2% SDS (w/v), 50% glycerol (v/v), 0.02% bromphenol blue (w/v), 2% 2-mercaptoethanol (v/v), pH 6.7) was added to the SDS-gel slurry and the samples were heated to 65 °C for 5 min. Following incubation at 4 °C overnight, samples were stored at −20 °C until use. Prior to electrophoresis, the samples were heated to 65 °C for 5 min and then centrifuged at 20,798 × g for 5 min. Immediately following centrifugation 20 μl of the sample was removed, taking care to avoid any fragments of acrylamide gel present in the tube. Samples were electrophoresed (glycine SDS-PAGE) in a 15% polyacrylamide denaturing gel and transferred to PVDF membrane for immunoblotting. Membranes prepared as above were probed with mouse monoclonal antibodies against either the FLAG (diluted 1:380) or HA epitopes (diluted 1:2000) (Sigma), or yeast mtATPase subunits α (diluted 1:5000), β (diluted 1:7000), or b (diluted 1:5000) from our library of mtATPase-specific antibodies (44Hadikusumo R.G. Hertzog P.J. Marzuki S. Biochim. Biophys. Acta. 1984; 765: 258-267Crossref PubMed Scopus (42) Google Scholar). Rabbit polyclonal antisera were also employed against yeast mtATPase subunits γ, d, or OSCP (diluted 1:1000) (44Hadikusumo R.G. Hertzog P.J. Marzuki S. Biochim. Biophys. Acta. 1984; 765: 258-267Crossref PubMed Scopus (42) Google Scholar), f, 6, or h (diluted 1:10000, kindly provided by Dr. J. Velours), and e, g, i/j, or k (diluted 1:500, kindly provided by Dr. R. A. Stuart). Secondary antibodies and detection were as described (37Stephens A.N. Roucou X. Artika I.M. Devenish R.J. Nagley P. Eur. J. Biochem. 2000; 267: 6443-6451Crossref PubMed Scopus (17) Google Scholar). Antibody binding was quantified using ImageQuant software (Amersham Biosciences). A total of 46 single cysteine replacements were made in the Y8 protein at positions ranging from the amino acid directly preceding the N-terminal methionine of allotopically expressed Y8 (position −1) to the most C-terminal residue (position 48). The lysine at position 37 and two arginines at positions 42 and 47 were not substituted as each of these residues contributes to the C-terminal positively charged region required for assembly of Y8 into mtATPase (28Papakonstantinou T. Galanis M. Nagley P. Devenish R.J. Biochim. Biophys. Acta. 1993; 1144: 22-32Crossref PubMed Scopus (23) Google Scholar). To test the functionality in vivo of Y8 cysteine variants, M31(aap1−) cells expressing each single cysteine variant were plated onto medium containing ethanol as the sole carbon source. Without exception, expression of each variant was able to restore respiratory function. However, cells expressing either the Y39C or V40C Y8 variants displayed apparently slower growth on solid medium than did control strain YM2 at 37 °C, yet displayed normal growth at either 18 or 28 °C. To further assess functionality of the Y8 variants at the whole cell level, the generation time of each Y8 variant-expressing strain was assessed in liquid medium containing ethanol as the sole carbon source. Of the 46 cysteine variants tested, only three had a significant effect (Student's t test) on whole cell growth rate. Expression of the L38C variant resulted in a significant decrease (p < 0.01) in generation time compared with strain YM2 expressing unmodified Y8 (TableI). The reason for this increased rate of growth is unclear and has not been examined further. Because yeast should have an excess capacity for ATP production, increased ATP synthesis should not promote growth. By contrast, expression of Y8 variants Y39C or V40C, respectively, displayed a significant increase in generation time compared with that of strain YM2 (p< 0.01 in each case) (Table I).Table IGrowth properties and mitochondrial ATP hydrolase activities of cells expressing Y8 variantsStrainGeneration timeSignificance1-aStudent's t test performed on mean generation times. Comparisons were made against strain YM2, expressing unmodified Y8. (Student's ttest)ATP hydrolase activity% Inhibition by oligomycinNo additionOligomycinhμmol ATP min−1mg protein−1YM25.37 ± 0.57NA1-bNA, not applicable.2.21 ± 0.260.54 ± 0.2775.6YM2(L38C)4.62 ± 0.56p < 0.012.25 ± 0.090.58 ± 0.3474.2YM2(Y39C)6.55 ± 0.23p < 0.011.98 ± 0.161.09 ± 0.05
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