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• W2024506833 abstract "Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+,K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940–7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13–30-fold decrease in K+affinity seen in corresponding mutants of the Na+,K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764–22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative α-helix. Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+,K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940–7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13–30-fold decrease in K+affinity seen in corresponding mutants of the Na+,K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764–22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative α-helix. There is currently much interest in the molecular mechanism of cation transport by P-type ATPases, which are structurally and functionally related to one another, but pump ions as diverse as H+, Na+, K+, Mg2+, Ca2+, Cu2+, Cd2+, and Hg2+ (reviewed in Refs. 1Fagan M.J. Saier Jr., M.H. J. Mol. Evol. 1994; 38: 57-99Crossref PubMed Scopus (146) Google Scholar and 2Lutsenko S. Kaplan J.H. Biochemistry. 1995; 34: 15607-15613Crossref PubMed Scopus (416) Google Scholar). For two members of the group, the Ca2+-ATPase of sarcoplasmic reticulum and the Na+,K+-ATPase of animal cell plasma membranes, site-directed mutagenesis has identified amino acids in membrane segment 5 that appear to be directly involved in the transport pathway. In the first case, MacLennan and co-workers (3Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (469) Google Scholar, 4Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 6262-6267Abstract Full Text PDF PubMed Google Scholar) found that mutations of Glu-771 abolished Ca2+ transport and Ca2+-dependent phosphorylation from ATP and that substitution of the adjacent residue, Gly-770, by Ala led to a significant decrease in Ca2+ affinity (5Andersen J.P. Vilsen B. MacLennan D.H. J. Biol. Chem. 1992; 267: 2767-2774Abstract Full Text PDF PubMed Google Scholar). Based on these observations, they proposed that Glu-771 serves directly as a Ca2+-liganding residue and that Gly-770 may also contribute to high affinity Ca2+ binding. Subsequently, Vilsen and Andersen (6Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar) were able to distinguish the roles of the two residues by demonstrating that mutations of Glu-771 eliminated the ability of the ATPase to occlude Ca2+, whereas the G770A mutant was still capable of occlusion. Andersen (7Andersen J.P. FEBS Lett. 1994; 354: 93-96Crossref PubMed Scopus (18) Google Scholar) has since raised the possibility that Glu-771 may play a role in H+ countertransport, based on changes in dephosphorylation rate when amino acid replacements are made at this position. Parallel studies of the animal cell Na+,K+-ATPase have been of interest since it also contains a glutamate at the corresponding point in M5 1The abbreviations used are: M5, membrane segment 5; MES, 2-(N-morpholino)ethane sulfonic acid; SERCA, sarcoplasmic reticulum ATPase. 1The abbreviations used are: M5, membrane segment 5; MES, 2-(N-morpholino)ethane sulfonic acid; SERCA, sarcoplasmic reticulum ATPase. (Glu-779 in the canine and sheep α1-isoforms). Arguello and Kaplan (8Arguello J.M. Kaplan J.H. J. Biol. Chem. 1994; 269: 6892-6899Abstract Full Text PDF PubMed Google Scholar) first drew attention to this residue by demonstrating that it reacts with 4-(diazomethyl)-7-(diethylamino)coumarin, causing disruption of K+ and Na+ occlusion. Thus, there was reason to think that Glu-779 in the Na+ pump, like its close relative in the Ca2+ pump, might be a cation-liganding residue. Subsequently, however, mutagenesis studies have shown that this simple interpretation cannot be correct. Whereas replacement of the glutamate in M5 by Leu does inactivate the Na+,K+-ATPase (9Jewell-Motz E.A. Lingrel J.B Biochemistry. 1993; 32: 13523-13529Crossref PubMed Scopus (116) Google Scholar), replacement by Gln, Ala, or Arg leaves a functional enzyme with relatively normal K½ values for both Na+ and K+ (10Feng J. Lingrel J.B Cell. Mol. Biol. Res. 1995; 41: 29-37PubMed Google Scholar, 11Vilsen B. Biochemistry. 1995; 34: 1455-1463Crossref PubMed Scopus (86) Google Scholar, 12Koster J.C. Blanco G. Mills P.B. Mercer R.W. J. Biol. Chem. 1996; 271: 2413-2421Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar). Arguello et al.(13Arguello J.M. Peluffo R.D. Feng J. Lingrel J.B Berlin J.R. J. Biol. Chem. 1996; 271: 24610-24616Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) have suggested that Glu-779 may instead be part of the voltage-dependent cation access channel, based on the voltage independence of the pump current in the E779A mutant. In parallel, Arguello and Lingrel (14Arguello J.M. Lingrel J.B J. Biol. Chem. 1995; 270: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar) have implicated a nearby residue, Ser-775, in K+ binding by describing mutants (S775A and S775C) with large increases in the K½ for stimulation of ATPase activity by K+. Given the intriguing differences in M5 between the Ca2+- and Na+,K+-ATPases, we set out recently to map structure-function relationships in M5 of a third, phylogenetically distant member of the P-type ATPase family, the yeast plasma membrane H+-ATPase. This enzyme is encoded by the PMA1gene, accounts for 10% of plasma membrane protein, and splits as much as one-quarter of the ATP produced by the cell (reviewed in Ref. 15Rao R. Slayman C.W. Brambl R. Marzluf G.A. The Mycota: Biochemistry and Molecular Biology. 3. Springer-Verlag, Berlin1996: 29-56Google Scholar). It generates the proton electrochemical gradient that underlies nutrient uptake and, consistent with its key physiological role, is essential for cell viability. While the mechanism of proton transport by the yeast ATPase has not yet been studied in detail, work on a very closely related pump (the Pma1 ATPase of the filamentous fungusNeurospora crassa) provides evidence for a simple stoichiometry of 1 H+ translocated per ATP split (16Perlin D.S. San Francisco M.J.D. Slayman C.W. Rosen B.P. Arch. Biochem. Biophys. 1986; 248: 53-61Crossref PubMed Scopus (66) Google Scholar). In this study, we have carried out alanine-scanning mutagenesis along the full length of M5 in the yeast ATPase. The results have identified five amino acid residues that play a significant role in the reaction cycle, along with three others that are required for proper protein folding and transit through the secretory pathway. Two related strains of Saccharomyces cerevisiae were used in this study: SY4 (MAT a, ura3-52, leu2-3,112, his4-619, sec6-4ts GAL2, pma1::YIpGAL-PMA1) and NY605 (MAT a, ura3-52, leu2-3,112, GAL2). In strain SY4, the chromosomal copy of the PMA1 gene has been placed under control of theGAL1 promoter by gene disruption (17Cid A. Perona R. Serrano R. Curr. Genet. 1987; 12: 105-110Crossref PubMed Scopus (91) Google Scholar) using the integrating plasmid YIpGAL-PMA1 (18Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar). SY4 also carries the temperature-sensitivesec6-4 mutation, which, upon incubation at 37 °C, blocks the fusion of secretory vesicles with the plasma membrane (19Novick P. Field C. Schekman R. Cell. 1980; 21: 205-215Abstract Full Text PDF PubMed Scopus (1256) Google Scholar). NY605 was generously provided by Dr. Peter Novick (Department of Cell Biology, Yale School of Medicine). Mutagenesis (20Sarkar G. Sommer S.S. BioTechniques. 1990; 8: 404-407PubMed Google Scholar) was performed on a 519-base pair BglII-SalI restriction fragment subcloned into a modified Bluescript plasmid (Stratagene, La Jolla, CA). Following DNA sequencing, the BglII-SalI fragment carrying the mutation was moved into plasmid pPMA1.2 (18Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar). The 3.8-kilobase HindIII-SacI fragment, which contains the entire pma1 coding region, was cloned into the yeast expression vector YCp2HSE (18Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar), placing the mutant allele under heat-shock control. Plasmids were then transformed into yeast according to the method of Ito et al. (21Ito H. Fukuda Y. Murata K. Kimura A. J. Bacteriol. 1983; 153: 163-168Crossref PubMed Google Scholar). Transformed SY4 cells were grown to mid-exponential phase (A600 ∼ 1) at 23 °C in supplemented minimal medium containing 2% galactose, shifted to medium containing 2% glucose for 3 h, and then heat-shocked at 39 °C for an additional 2 h. The cells were harvested and washed, and the secretory vesicles were isolated as described previously (22Ambesi A. Allen K.E. Slayman C.W. Anal. Biochem. 1997; 251: 127-129Crossref PubMed Scopus (26) Google Scholar). To determine the level of expressed Pma1 protein relative to a wild-type control, secretory vesicles (5–20 μg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted (18Nakamoto R.K. Rao R. Slayman C.W. J. Biol. Chem. 1991; 266: 7940-7949Abstract Full Text PDF PubMed Google Scholar), followed by PhosphorImager (Molecular Dynamics) analysis; typically, the analysis was carried out at two protein concentrations within the linear range, and the expression level was calculated from the average of the two determinations. Unless otherwise noted, ATP hydrolysis was assayed at 30 °C in 0.5 ml of 50 mm MES/Tris, pH 5.7, 5 mm KN3, 5 mm Na2ATP, 10 mm MgCl2, and an ATP-regenerating system (5 mm phosphoenolpyruvate and 50 μg/ml pyruvate kinase). The reaction was stopped after 20–40 min, and the release of inorganic phosphate from ATP was determined by the method of Fiske and SubbaRow (23Fiske C.H. SubbaRow Y. J. Biol. Chem. 1925; 66: 375-400Abstract Full Text PDF Google Scholar). Specific activity was calculated as the difference between ATP hydrolysis measured in the absence and presence of 100 μmsodium orthovanadate, an inhibitor of P-type ATPases. For determination of Km values, the concentration of Na2ATP was varied between 0.15 and 7.5 mm, with MgCl2 always in excess of ATP by 5 mm. Actual concentrations of MgATP were calculated as described previously (24Fabiato A. Fabiato F. J. Physiol. ( Paris ). 1979; 75: 463-505PubMed Google Scholar). To determine the effects of pH on hydrolysis, the pH of the assay mixture was adjusted to values between 5.2 and 7.5 with Tris base. ATP-dependent proton transport was determined by measuring the initial rate of acridine orange fluorescence quenching as described by Ambesi et al.(25Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The specific initial rate of fluorescence quenching for each mutant was adjusted for ATPase expression and is reported as a percent of the wild-type rate. To measure the synthesis of mutant ATPases that were unable to reach the secretory vesicles, SY4 cells were shifted from galactose medium at 23 °C to glucose medium at 39 °C as described above and then metabolically labeled with [35S]methionine (26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Total membranes were isolated and immunoprecipitated with anti-Pma1 antibody (26Nakamoto R.K. Verjovski-Almeida S. Allen K.E. Ambesi A. Rao R. Slayman C.W. J. Biol. Chem. 1998; 273: 7338-7344Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), and after SDS-polyacrylamide gel electrophoresis, the gels were fixed, incubated in 1 m sodium salicylate (30 min at 23 °C), dried, and exposed to Hyperfilm-MP (Amersham Pharmacia Biotech). Limited trypsinolysis was performed on both isolated secretory vesicles and 35S-labeled yeast total membranes. Vesicles or membranes were diluted into 1 mmEGTA/Tris, pH 7.5; centrifuged at 100,000 × g for 35 min; and suspended at 0.5 mg/ml in 20 mm Tris-HCl, pH 7.0, and 5 mm MgCl2. Following preincubation in the absence or presence of 100 μm orthovanadate, 10 mm MgADP, or 10 mm MgATP at 30 °C for 5 min, tosylphenylalanyl chloromethyl ketone-treated trypsin was added (trypsin/protein ratio of 1:4 for secretory vesicles or 1:20 for total membranes), and the incubation was continued for 0.5–20 min. The reaction was terminated by the addition of 1 mm diisopropyl fluorophosphate. Reaction products were analyzed either by immunoblotting (secretory vesicles) or by immunoprecipitation and fluorography (total membranes). Protein concentrations were determined by a modification of the method of Lowry et al.(27Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.J. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar) as described by Ambesi et al. (22Ambesi A. Allen K.E. Slayman C.W. Anal. Biochem. 1997; 251: 127-129Crossref PubMed Scopus (26) Google Scholar) using bovine serum albumin as a standard. In this study, alanine-scanning mutagenesis was used to examine the structural and functional role of amino acids in M5 of the yeast plasma membrane H+-ATPase. Residues Ser-690 to Leu-713 were included based on hydropathy analysis of the Pma1 protein sequence (reviewed in Ref. 15Rao R. Slayman C.W. Brambl R. Marzluf G.A. The Mycota: Biochemistry and Molecular Biology. 3. Springer-Verlag, Berlin1996: 29-56Google Scholar). All but two of the residues were replaced with alanine, whereas alanines at positions 697 and 711 were replaced with serine. The mutant alleles were transformed into yeast strain SY4, expressed under control of an inducible heat-shock promoter, and secretory vesicles were isolated and characterized with respect to expression and ATP hydrolysis. Most substitutions allowed reasonable amounts of the ATPase to reach the secretory vesicles, but there were three cases (R695A, H701A, and L706A) in which little or no Pma1 protein could be detected in the vesicles (Fig. 1 A and TableI, part A). Immunoprecipitation from35S-labeled total membranes revealed that each of these mutant proteins was synthesized, but became arrested in an earlier compartment of the secretory pathway, presumably the endoplasmic reticulum (Fig. 1 B).Table IEffect of pma1 mutations on expression, ATP hydrolysis, and proton transportMutationExpressionaCalculations were made from yields of mutant and wild-type H+-ATPase protein/mg of total secretory vesicle protein, as determined by quantitative immunoblotting (see “Experimental Procedures”).ATP hydrolysisbVanadate-sensitive ATP hydrolysis was measured as described under “Experimental Procedures.” One unit is defined as 1 μmol of Pi/min.Proton transportcThe initial rate of fluorescence quenching (proton transport) was determined as previously described (25). One unit is defined as 1% of total fluorescence quenching/min.UncorrectedCorrectedUncorrectedCorrectedunits/mg%units/mg%Part A Wild type1003.603.60100954954100 Vector70.09NDdND, not determined. Corrections for expression have not been made for mutants with measured ATPase activities below 3% of the wild-type value, but have been made for mutants with measured activities between 3 and 10%. In both cases, proton transport could not be detected by the acridine orange assay.NDNDNDND S690A934.154.4612410691149120 Y691A920.09NDNDNDNDND V692A771.501.95549031172123 V693A792.573.25908341055110 Y694A400.320.8022NDNDND R695A140.09NDNDNDNDND I696A841.131.343727032133 A697A761.652.176056874778 L698A902.282.537056963266 S699A900.270.308NDNDND L700A841.621.935331036939 H701A150.12NDNDNDNDND L702A390.591.514212532033 E703A953.583.7710583387792 I704A983.043.108670972376 F705A732.002.747646163166 L706A90.05NDNDNDNDND G707A621.532.476827243946 L708A641.542.406630948350 W709A410.320.7821NDNDND I710A781.722.206135044947 A711S992.022.045638538941 I712A731.862.547032844947 L713A761.862.446833243746Part B Wild type1004.434.43100609609100 Y694G640.781.2228487512 S699C790.560.7116587312 S699T943.994.2496573610100 E703D560.961.713913524140Values represent the mean of at least three determinations with an average S.E. of 11% or less.a Calculations were made from yields of mutant and wild-type H+-ATPase protein/mg of total secretory vesicle protein, as determined by quantitative immunoblotting (see “Experimental Procedures”).b Vanadate-sensitive ATP hydrolysis was measured as described under “Experimental Procedures.” One unit is defined as 1 μmol of Pi/min.c The initial rate of fluorescence quenching (proton transport) was determined as previously described (25Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). One unit is defined as 1% of total fluorescence quenching/min.d ND, not determined. Corrections for expression have not been made for mutants with measured ATPase activities below 3% of the wild-type value, but have been made for mutants with measured activities between 3 and 10%. In both cases, proton transport could not be detected by the acridine orange assay. Open table in a new tab Values represent the mean of at least three determinations with an average S.E. of 11% or less. The remaining 21 mutant ATPases reached the secretory vesicles in amounts ranging from 39 to 99% of the wild-type control (Table I, part A). When these mutants were assayed for their ability to hydrolyze ATP, 17 of them had activities of 37% or better after correction for the level of expression in the secretory vesicle preparations. Three showed significant reductions in activity: Y694A to 22%, W709A to 21%, and S699A to 8% of the wild-type control. One mutant ATPase (Y691A) was expressed well in secretory vesicles (92%), but appeared to be completely inactive. Y691A and S699A, along with R695A, H701A, and L706A, which were blocked in biogenesis, were later examined for structural defects by limited trypsinolysis, as described below. The mutant ATPases were next assayed for vanadate sensitivity, MgATP dependence, and the effect of pH on the rate of ATP hydrolysis. In all but one case, the kinetic properties of the mutants proved to be essentially normal (TableII, part A). The exception was V692A, which displayed an increased Ki for vanadate (11 μm compared with 1.8 μm for the wild-type control), a decreased Km for MgATP (0.1 mm compared with 1.1 mm for the wild type), and a relatively alkaline pH optimum (pH 6.4 compared with pH 5.7 for the wild type). Because Val-692 is presumably buried in the membrane, it is unlikely to contribute in a direct way to the vanadate- and MgATP-binding sites. Rather, the increased Ki, decreased Km, and altered pH optimum can more reasonably be accounted for by a slowing of the E1P-to-E2P conformational change; as a result, the ATPase accumulates in E1, which has a relatively high affinity for ATP and protons and a relatively low affinity for orthovanadate. This idea is supported by the fact that mutations at three positions in M4 lead to a similar set of kinetic changes (25Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar).Table IIKinetic properties of pma1 mutantsMutationKm(MgATP)Ki(van)avan, vanadate.pH optimummmμmPart A Wild type1.11.85.7 S690A0.71.45.8 V692A0.110.96.4 V693A0.71.85.8 I696A0.62.85.7 A697A1.01.85.6 L698A0.91.35.7 L700A1.02.15.5 L702A0.75.35.7 E703A1.32.25.7 I704A1.32.25.6 F705A1.12.85.7 G707A1.21.95.7 L708A1.02.15.7 I710A1.42.55.6 A711S1.32.05.6 I712A1.02.35.7 L713A1.02.25.7Part B Wild type1.11.85.7 Y694G0.959.65.4 S699C0.57.55.4 S699T1.83.65.7 E703D0.87.35.7Kinetic properties for each mutant were determined under standard assay conditions (50 mm MES/Tris, pH 5.7, 5 mmNa2ATP, 10 mm MgCl2, 5 mmKN3, 5 mm phosphoenolpyruvate, and 50 mg/ml pyruvate kinase at 30 °C) with the following exceptions. TheKm was determined by varying Na2ATP from 0.15 to 7.5 mm (MgCl2 in excess of ATP by 5 mm); the Ki was determined by varying the concentration of vanadate from 0 to 200 μm; and the pH optimum was calculated from assays carried out over a pH range of 5.2–7.5 (see “Experimental Procedures”). Values represent the mean of at least three determinations with an average S.E. of <7%.a van, vanadate. Open table in a new tab Kinetic properties for each mutant were determined under standard assay conditions (50 mm MES/Tris, pH 5.7, 5 mmNa2ATP, 10 mm MgCl2, 5 mmKN3, 5 mm phosphoenolpyruvate, and 50 mg/ml pyruvate kinase at 30 °C) with the following exceptions. TheKm was determined by varying Na2ATP from 0.15 to 7.5 mm (MgCl2 in excess of ATP by 5 mm); the Ki was determined by varying the concentration of vanadate from 0 to 200 μm; and the pH optimum was calculated from assays carried out over a pH range of 5.2–7.5 (see “Experimental Procedures”). Values represent the mean of at least three determinations with an average S.E. of <7%. Given the fact that membrane segment 5 is believed to play a direct role in cation translocation in the Ca2+- and Na+,K+-ATPases, it was of particular interest to explore the proton-pumping ability of the yeast M5 mutants. For this purpose, secretory vesicle preparations were assayed for ATP-dependent quenching of acridine orange fluorescence. In seven of the mutants (Y691A, Y694A, R695A, S699A, H701A, L706A, and W709A), ATPase activities were below the limit at which associated proton pumping could have been reliably detected by the acridine orange assay. With one exception, all of the remaining mutants showed a reasonable correlation between the initial rate of ATP-dependent quenching and the rate of ATP hydrolysis (Table I, part A). In V692A, the rate of acridine orange quenching (123% of the wild-type control) appeared to exceed the rate of ATP hydrolysis (54% of the wild-type control). However, separate measurements indicated that the apparent discrepancy could be accounted for by the pH difference between the hydrolysis assay and the quenching assay, together with the above-mentioned alkaline shift in pH optimum. Thus, at pH 6.7, the V692A enzyme split ATP at 119% of the wild-type rate, completely consistent with its relative rate of acridine orange quenching at the same pH (123%; see Table I, part A). There was no evidence for abnormal ATP-dependent proton transport in any of the other 16 mutants that were studied (Table I, part A). Based on comparison with other P-type ATPases, it seemed useful to make additional amino acid replacements at three positions: Tyr-694, Ser-699, and Glu-703. In the case of Tyr-694, Andersen (28Andersen J.P. J. Biol. Chem. 1995; 270: 908-914Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar) has reported that mutation of the corresponding residue to glycine leads to uncoupling of the SERCA Ca2+-ATPase; the other two residues are believed to play a role in cation binding in the Na+,K+- and SERCA Ca2+-ATPases, respectively (3Clarke D.M. Loo T.W. Inesi G. MacLennan D.H. Nature. 1989; 339: 476-478Crossref PubMed Scopus (469) Google Scholar, 4Clarke D.M. Loo T.W. MacLennan D.H. J. Biol. Chem. 1990; 265: 6262-6267Abstract Full Text PDF PubMed Google Scholar, 6Vilsen B. Andersen J.P. J. Biol. Chem. 1992; 267: 25739-25743Abstract Full Text PDF PubMed Google Scholar). At Tyr-694 of the yeast ATPase, substitution by Gly gave better expression (Y694G, 64%; Table I, part B) than substitution by Ala (Y694A, 40%; Table I, part A). Interestingly, the Y694G enzyme was extremely resistant to orthovanadate, with a Ki of 60 μm (Table II, part B). This large increase inKi, with only minor changes in Kmand pH optimum, suggests that the mutation has a relatively specific effect on vanadate binding, raising the possibility that the cytoplasmic end of M5 may somehow interact with the vanadate-binding pocket. Y694G also showed an apparent difference between the rate of ATP hydrolysis (18% before correction for the level of expression in secretory vesicles, 28% after correction) and the rate of acridine orange fluorescence quenching (8% before correction, 12% after correction). Taken at face value, this difference could indicate partial uncoupling, but the rates are below the limit at which a detailed analysis is possible. In the case of Ser-699, both the Cys and Thr mutants were well expressed, but S699T was considerably more active (96% hydrolysis, 100% transport) than S699C (16% hydrolysis, 12% transport). In both cases, the pH profile for ATP hydrolysis was examined to see whether there was any evidence for a decrease in the apparent affinity of the ATPase for protons, similar to the decrease in K+ affinity reported for the corresponding mutants of the Na+,K+-ATPase (14Arguello J.M. Lingrel J.B J. Biol. Chem. 1995; 270: 22764-22771Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). As shown in Fig. 2, the pH curve for S699C did indeed shift in the acid direction, but only modestly (∼0.2 pH units). A similar shift of ∼0.2 pH units was seen in S699A (data not shown), although in this case, the ATPase activity was very low. By contrast, the more conservative substitution in S699T had no effect on the pH profile. Finally, although the substitution of Glu-703 by Asp (56% expression, 39% hydrolysis, 40% transport) was less well tolerated than the substitution by Ala (95% expression, 105% hydrolysis, 92% transport), there was sufficient activity in both cases to carry out a quantitative study of H+ pumping over a range of MgATP concentrations. The results are illustrated in Fig. 3. As reported earlier (25Ambesi A. Pan R.L. Slayman C.W. J. Biol. Chem. 1996; 271: 22999-23005Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar), the relationship between the initial rate of acridine orange fluorescence quenching and the rate of hydrolysis was approximately linear in the wild-type control. Significantly, the data points for E703A and E703D fell along the same straight line, consistent with normal coupling be" @default.
• W2024506833 created "2016-06-24" @default.
• W2024506833 creator A5052155917 @default.
• W2024506833 creator A5081802330 @default.
• W2024506833 creator A5082381694 @default.
• W2024506833 date "1998-07-01" @default.
• W2024506833 modified "2023-09-28" @default.
• W2024506833 title "Structure-Function Relationships in Membrane Segment 5 of the Yeast Pma1 H+-ATPase" @default.
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