From the Departments of Genetics and Cellular and Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510
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ABSTRACT |
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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 -helix.
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INTRODUCTION |
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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. 1 and 2). 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 (3, 4) 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 (5). 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 (6) 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 (7) 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 1 (Glu-779 in the canine
and sheep 1-isoforms). Arguello and Kaplan (8) 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
(9), replacement by Gln, Ala, or Arg leaves a functional enzyme with
relatively normal K1/2 values for both
Na+ and K+ (10-12). Arguello et al.
(13) 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 (14) have implicated a nearby residue,
Ser-775, in K+ binding by describing mutants (S775A and
S775C) with large increases in the K1/2 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 PMA1 gene, accounts for 10% of plasma membrane protein, and splits as much as one-quarter of the ATP produced by the cell (reviewed in Ref. 15). 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 fungus Neurospora crassa) provides evidence for a simple stoichiometry of 1 H+ translocated per ATP split (16).
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.
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EXPERIMENTAL PROCEDURES |
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Yeast Strains-- Two related strains of Saccharomyces cerevisiae were used in this study: SY4 (MATa, ura3-52, leu2-3,112, his4-619, sec6-4ts GAL2, pma1::YIpGAL-PMA1) and NY605 (MATa, ura3-52, leu2-3,112, GAL2). In strain SY4, the chromosomal copy of the PMA1 gene has been placed under control of the GAL1 promoter by gene disruption (17) using the integrating plasmid YIpGAL-PMA1 (18). SY4 also carries the temperature-sensitive sec6-4 mutation, which, upon incubation at 37 °C, blocks the fusion of secretory vesicles with the plasma membrane (19). NY605 was generously provided by Dr. Peter Novick (Department of Cell Biology, Yale School of Medicine).
Mutagenesis-- Mutagenesis (20) 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 (18). The 3.8-kilobase HindIII-SacI fragment, which contains the entire pma1 coding region, was cloned into the yeast expression vector YCp2HSE (18), placing the mutant allele under heat-shock control. Plasmids were then transformed into yeast according to the method of Ito et al. (21).
Isolation of Secretory Vesicles and Measurement of Expressed ATPase-- 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 (22). 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 (18), 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.
ATPase Activity-- 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 (23). Specific activity was calculated as the difference between ATP hydrolysis measured in the absence and presence of 100 µM sodium 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 (24). 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.
Fluorescence Quenching-- ATP-dependent proton transport was determined by measuring the initial rate of acridine orange fluorescence quenching as described by Ambesi et al. (25). 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.
Metabolic Labeling and Immunoprecipitation-- 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 (26). Total membranes were isolated and immunoprecipitated with anti-Pma1 antibody (26), 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).
Trypsinolysis-- Limited trypsinolysis was performed on both isolated secretory vesicles and 35S-labeled yeast total membranes. Vesicles or membranes were diluted into 1 mM EGTA/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 Determination-- Protein concentrations were determined by a modification of the method of Lowry et al. (27) as described by Ambesi et al. (22) using bovine serum albumin as a standard.
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RESULTS |
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Expression and ATP Hydrolysis-- 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. 15). 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. 1A and Table I, part A). Immunoprecipitation from 35S-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. 1B).
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Kinetic Properties-- 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 (Table II, 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 (25).
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ATP-dependent Proton Transport-- 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).
Further Study of Tyr-694, Ser-699, and Glu-703-- 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 (28) 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 (3, 4, 6).
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 in Ki, with only minor changes in Km and 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 (14). 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.
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Conformational Analysis by Limited Trypsinolysis-- As described above, three mutations in M5 led to a virtually complete block in Pma1 biogenesis: R695A, H701A, and L706A. Because it seemed possible that these proteins might be structurally abnormal, they were examined by limited tryptic digestion. Total membranes were isolated from 35S-labeled cells expressing either mutant or wild-type ATPase and incubated at a trypsin/protein ratio of 1:20 for 0, 1, or 5 min (Fig. 4). Under these conditions, the 100-kDa wild-type enzyme underwent minor proteolytic cleavage, yielding a 97-kDa band, whereas the three mutants were completely degraded in <1 min. Thus, alanine substitutions of Arg-695, His-701, and Leu-706 appear to cause severe misfolding, interfering with the ability of the newly synthesized ATPase to move through the secretory pathway.
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Coexpression of M5 Mutants with Wild-type PMA1-- Wach et al. (29) have previously shown that Asp, Gln, and Arg substitutions of His-701 behave as dominant lethal mutations. In a similar series of experiments, we subcloned H701A, R695A, and L706A into plasmid YCplac33 (30) and placed the mutant alleles under GAL1 control. Each of the plasmids was then transformed into yeast strain NY605, which carries a wild-type PMA1 gene on the chromosome. When the resulting cells were plated on glucose-containing medium, where only the constitutive wild-type gene could be expressed, all of them grew normally. By contrast, on galactose-containing medium, where the mutant gene was also expressed, no growth was observed for cells transformed with H701A, R695A, or L706A. This dominant lethal phenotype indicates that the abnormal proteins interfere with the processing of coexpressed wild-type ATPase, as has previously been shown for H701D, H701Q, H701R, and a number of other PMA1 mutations (26, 29, 31-33).
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DISCUSSION |
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In analyzing the data from this study, the sequence alignment of Fig. 6 can serve as a useful guide. It illustrates a modest degree of evolutionary conservation along membrane segment 5 of the P-ATPases and highlights six residues that are discussed in detail below.
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Tyr-694 lies near the cytoplasmic boundary of M5 and is present in all known P-type H+ pumps from fungi, algae, higher plants, and protozoans. Other members of the family have a similar amino acid residue at this position (usually Tyr or Phe; occasionally Trp, Met, or Leu), except for the Cta3 Ca2+-ATPase of Schizosaccharomyces pombe, where there is a His. It is now clear that Tyr-694 plays a key role in the reaction cycle of the yeast Pma1 H+-ATPase since ATP hydrolysis decreased substantially in Y694A and Y694G, and ATP-dependent H+ transport was barely detectable. In the mammalian SERCA ATPase, as mentioned above, mutation of the corresponding Tyr to Gly led to the uncoupling of Ca2+ transport from ATP hydrolysis (28); and in the Na+,K+-ATPase, the same substitution led to inactivation of the enzyme (34). Thus, there appears to be a nearly uniform requirement for a bulky, hydrophobic amino acid at the position of Tyr-694.
Ser-699 is another residue displaying significant, although not
complete, evolutionary conservation (Fig. 6). All of the known P-type
H+-ATPases contain either Ser or Thr at this position, and
there is a Ser only one residue away in the mammalian
Na+,K+- and SERCA Ca2+-ATPases, the
MgtB ATPase of Salmonella typhimurium, and the Cta3 ATPase
of S. pombe. Clear evidence has been put forward for the functional importance of this Ser in the
Na+,K+-ATPase, where mutation to Ala or Cys
produced a severalfold decrease in the Vmax for
ATP hydrolysis and a 13-31-fold increase in the K1/2 for K+; as a result, cells
expressing the altered pump required higher than normal extracellular
K+ for growth (14). By contrast, Ser-to-Ala and Ser-to-Cys
mutations had little effect on either the activity or the
Ca2+ affinity of the SERCA ATPase (35). In this study,
yeast Pma1 ATPase containing the S699A mutation traveled efficiently to
the secretory vesicles, and based on limited trypsinolysis, it was well
folded and able to bind MgATP, MgADP, and orthovanadate; however, it
had exceedingly low ATPase activity and no detectable ATP-dependent proton pumping. The S699C and S699T enzymes
were more active, allowing them to be studied in greater detail. S699T appeared normal in every respect, but S699C displayed a modest acid
shift in the pH dependence of ATP hydrolysis (0.2 units). While the
shift does not necessarily reflect a lowered affinity for
H+ at a cation-liganding site in the translocation pathway,
this is clearly one possibility. If so, the effect is substantially smaller than the change seen in the
Na+,K+-ATPase (14), where recent work by
Blostein et al. (36) has provided direct evidence that
Ser-775 is either a cation-liganding residue or part of a gating
structure close to the liganding sites. The difference between the two
enzymes may be related to the immediate downstream sequences, which are
LHLE in the case of the H+-ATPase and NIPE in the
Na+,K+-ATPase. As emphasized in a recent study
of the NHE-1 Na+/H+ exchanger by Counillon
et al. (37), a proline residue leaves the backbone carbonyl
at position 4 without a hydrogen donor and introduces a kink that
disrupts hydrogen bonding between position
3 (Ser-775) and position
+1. Thus, the presence of a Pro in the Na+,K+-ATPase would free up backbone carbonyls
that might contribute to cation binding; it would also increase the
local flexibility of the polypeptide chain, which in turn could
influence conformational interactions with other parts of the
protein.
Glu-703 is a third residue that deserves careful attention. In the mammalian SERCA ATPase, replacement of the corresponding glutamate (Glu-771) by an uncharged amino acid destroyed Ca2+ occlusion (6) and Ca2+ transport (3, 4), consistent with the idea that a side chain oxygen is needed at this position to ligand Ca2+. Measurements of phosphorylation from ATP and Pi as a function of Ca2+ concentration soon led to the idea that Glu-771 contributes to the deeper (more luminal) of two sequentially filled Ca2+-binding sites (7, 38). In the Na+,K+-ATPase, the corresponding glutamate (either Glu-779 or Glu-781, depending upon the species) plays a less critical role since substitution by Ala, Asp, Gln, or Arg failed to disrupt function (10-12). However, the Ala mutant did display a severalfold decrease in the apparent affinity for K+ and Na+ (11, 12), along with the disappearance of voltage dependence in patch clamp experiments, suggesting that this residue may line the cation access channel (13). In the yeast Pma1 H+-ATPase, on the other hand, there is no demonstrable role for Glu-703. Replacement by an uncharged amino acid (E703A) had little or no effect on biogenesis, ATPase activity, or the rate of ATP-dependent proton pumping. Furthermore, a detailed look at the relationship between H+ pumping and ATP hydrolysis in E703A and E703D gave no evidence for a defect in coupling. It may therefore be significant that Glu-703 is replaced by Val or Cys in the P-type H+-ATPases from Leishmania donovani, Dunaliella bioculata, and Arabidopsis thaliana and by Ala in two Ca2+-ATPases (yeast Pmr1 and mammalian PMCA); it is also replaced by an uncharged amino acid (Gly or Met) in the heavy metal-transporting ATPases (Fig. 6).
Finally, Arg-695, His-701, and Leu-706 are at least structurally important for the Pma1 ATPase since replacement by Ala led to trypsin sensitivity (misfolding), failure to move to the secretory vesicles, and a dominant lethal phenotype. In the case of His-701, this result corroborates earlier work by Wach et al. (29), who showed that H701R, H701Q, and H701E behave as dominant lethal mutations. Furthermore, although Arg-695, His-701, and Leu-706 are poorly conserved among the P-type ATPases, position 706 is occupied by Ile in the SERCA ATPase, where mutation to Ala has been reported to produce a nonfunctional enzyme (35).
To place these and the rest of the data in a useful context, Fig.
7 provides a helical wheel diagram for M5
of the yeast H+-ATPase. It is perhaps significant that the
structurally and functionally important amino acids identified in this
study are seen to lie along one face of the putative -helix.
Included are three residues required for proper ATPase folding and
biogenesis (Arg-695, His-701, and Leu-706), three residues at which Ala
substitutions inactivate the ATPase (Tyr-694, Trp-709, and Ser-699),
and two residues at which mutations lead to complex changes in ligand
binding and/or the equilibrium between E1 and
E2 conformations (Tyr-691, based on the inability of
vanadate to protect against trypsinolysis, and Val-692, based on
simultaneous shifts in several kinetic parameters).
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Taken together, although the results of this study give no firm evidence for a transport site in M5 of the yeast ATPase, they do indicate that M5 plays a significant role in the reaction cycle. As progress is made toward solving the structure of the closely related Neurospora H+-ATPase (40), the mutational data should provide helpful insight into structure-function relationships.
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ACKNOWLEDGEMENTS |
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We thank Dr. Anita Panek for support, Dr. Joab Trajano Silva for helpful discussions, and Kenneth Allen for expert technical assistance.
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FOOTNOTES |
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* This work was supported by NIGMS Research Grant GM15761 from the National Institutes of Health and by a fellowship from the Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (to M. B. D.).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.
Present address: Universidade Federal do Rio de Janeiro, Cidade
Universitária, Centro de Tecnologia, Bloco A, Inst.
Química, Dept. de Bioquimica, 5 Andar, Sala547, CEP 21949-900 Rio de Janeiro, Brasil.
§ To whom correspondence and reprint requests should be addressed: Dept. of Genetics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-2690; Fax: 203-785-7227.
1 The abbreviations used are: M5, membrane segment 5; MES, 2-(N-morpholino)ethane sulfonic acid; SERCA, sarcoplasmic reticulum ATPase.
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REFERENCES |
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