Stalk Segment 5 of the Yeast Plasma Membrane H+-ATPase

MUTATIONAL EVIDENCE FOR A ROLE IN GLUCOSE REGULATION*

Manuel MirandaDagger, Kenneth E. Allen, Juan P. Pardo§, and Carolyn W. Slayman

From the Departments of Genetics and Cellular & Molecular Physiology, Yale University School of Medicine, New Haven, Connecticut 06510

Received for publication, March 15, 2001, and in revised form, April 13, 2001

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In P2-type ATPases, a stalk region connects the cytoplasmic part of the molecule, which binds and hydrolyzes ATP, to the membrane-embedded part through which cations are pumped. The present study has used cysteine scanning mutagenesis to examine structure-function relationships within stalk segment 5 (S5) of the yeast plasma-membrane H+-ATPase. Of 29 Cys mutants that were made and examined, two (G670C and R682C) were blocked in biogenesis, presumably due to protein misfolding. In addition, one mutant (S681C) had very low ATPase activity, and another (F685C) displayed a 40-fold decrease in sensitivity to orthovanadate, reflecting a shift in equilibrium from the E2 conformational state toward E1. By far the most striking group of mutants (F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) were constitutively activated even in the absence of glucose, with rates of ATP hydrolysis and kinetic properties normally seen only in glucose-metabolizing cells. Previous work has suggested that activation of the wild-type H+-ATPase results from kinase-mediated phosphorylation in the auto-inhibitory C-terminal region of the 100-kDa polypeptide. The seven residues identified in the present study are located on one face of the S5 alpha -helix, consistent with the idea that mutations along this face serve to release the auto-inhibition.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The plasma membrane H+-ATPase of yeast belongs to the P2 subfamily of cation-transporting ATPases and is structurally and functionally related to the Na+,K+-, H+,K+-, and Ca2+-ATPases of animal cells (1). All of these enzymes have a 100-kDa catalytic subunit that is anchored in the membrane by four hydrophobic segments (M1-4) at the N-terminal end of the molecule and six hydrophobic segments (M5-10) at the C-terminal end. They split ATP by way of a covalent beta -aspartyl phosphate intermediate and alternate between two major conformational states (E1 and E2) during the reaction cycle.

Recently, the three-dimensional structure of the sarcoplasmic reticulum Ca2+-ATPase has been solved at 2.6 Å (2), furnishing a valuable framework for further work on the transport mechanism of the P2-type ATPases. The cytoplasmic part of the molecule includes the N- and C-terminal segments, the small hydrophilic loop between M2 and M3, and the large hydrophilic loop between M4 and M5 and is organized into three domains: (a) N (ATP-binding domain), (b) P (phosphorylation), and (c) A ("actuator" or anchor for domain N). Within the membrane, the 10 hydrophobic alpha -helices assemble together in a complex way, with 4 of the inner helices (M4, M5, M6, and M8) providing the actual Ca2+-liganding residues.

As seen earlier by cryoelectron microscopy of the sarcoplasmic reticulum enzyme (3) and the plasma membrane H+-ATPase of Neurospora crassa (4), a stalk-like region connects the cytoplasmic part of the molecule to the membrane. Not surprisingly, there is growing evidence that the stalk plays a conformationally active role. In a recent study of the yeast plasma membrane H+-ATPase, our laboratory carried out scanning mutagenesis to examine structure-function relationships in stalk segment 4 (S4), which forms a short, relatively well-conserved link between the phosphorylated aspartate residue (Asp-378) and M4. Kinetic analysis of mutants along the entire length of S4 revealed 13 consecutive positions at which amino acid substitutions led to a shift in equilibrium from the E2 state of the ATPase toward the E1 state (5). Mutagenesis studies of sarcoplasmic reticulum Ca2+-ATPase have revealed conformationally important residues in S4 of that enzyme as well (6, 7).

In the work described here, we have turned to stalk segment 5 (S5), located at the opposite end of the large cytoplasmic loop. In the sarcoplasmic reticulum Ca2+-ATPase, M5 and S5 form a continuous alpha -helix, 60-Å long, stretching from the membrane into the cytoplasm and seeming to serve as a "center mast" around which the rest of the molecule is organized (2). To discover whether S5 of the yeast H+-ATPase contains conformationally active residues, we performed cysteine scanning mutagenesis along the entire segment, from Ala-661 to Tyr-689, followed by kinetic analysis of each of the mutants. Based on the results, only one residue influences the equilibrium between E1 and E2 conformations. By contrast, there are seven residues at which mutations alter the regulation of the ATPase by glucose, leading to a constitutively activated form of the enzyme.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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; Ref. 8) and NY13 (MATa, ura3-52; generously provided by Dr. Peter Novick of the Department of Cell Biology, Yale School of Medicine). In strain SY4, the chromosomal copy of the PMA1 gene has been placed under control of the GAL1 promotor by gene disruption (9) using the integrating plasmid, YIpGAL-PMA1 (8). 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 (10).

Mutagenesis-- Cysteine substitutions were made by polymerase chain reaction (11) in a 519-base pair BglII-SalI restriction fragment of the PMA1 gene that had been subcloned into a modified Bluescript plasmid (Stratagene, La Jolla, CA). After DNA sequencing, the BglII-SalI restriction fragment carrying the mutation was moved into plasmid pPMA1.2 (9). The 3.8-kilobase HindIII-SacI fragment, which contains the entire PMA1 coding region, was then cloned into yeast expression vector YCp2HSE (9), placing the mutant pma1 allele under the control of two tandemly arranged heat-shock elements. Plasmids were then transformed into yeast according to the method of Ito et al. (12).

Isolation of Secretory Vesicles and Quantitation of Expressed ATPase-- Transformed SY4 cells were grown to mid-exponential phase (A600 ~ 1) at 23 °C in 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 secretory vesicles were isolated and suspended in 0.8 M sorbitol, 1 mM EDTA, and 10 mM triethanolamine/acetic acid, pH 7.2 as described previously (13). To determine the level of expressed Pma1 protein, secretory vesicles (5-20 µg) were subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted. Quantitative PhosphorImager (Molecular Dynamics) analysis was carried out at two protein concentrations within the linear range, and the expression level was calculated from the average of two or more determinations.

Chromosomal Integration of pma1 Mutants-- To test for changes in glucose regulation of the ATPase, selected mutations were introduced into the chromosomal copy of the PMA1 gene. For this purpose, a BglII-SalI fragment carrying the mutation was first subcloned into plasmid pGW201 (generously provided by Dr. David Perlin, Public Health Research Institute, New York, NY; Ref. 14) to create a mutant pma1 allele with the URA3 marker at its 3' non-coding end, and the presence of the mutation was confirmed by DNA sequencing. A 6.1-kilobase HindIII fragment containing the mutant allele linked to URA3 was then excised from the plasmid and transformed into yeast strain NY13 using the Alkali-Cation Yeast Transformation kit (Bio 101). Finally, the presence of the mutation was reconfirmed by polymerase chain reaction amplification of chromosomal DNA and DNA sequence analysis.

Preparation of Plasma Membranes-- Glucose-starved and glucose-metabolizing cells were prepared according to the protocol of Serrano (15). Briefly, cells were grown to mid-exponential phase (A600 = 4-6) in minimal medium containing 4% glucose at 30 °C, harvested, washed twice with H2O, and incubated for 1 h in water without glucose (glucose-starved cells); 4% glucose was then added back to an aliquot of the culture for 30 min to obtain glucose-metabolizing cells. In both cases, a microsomal membrane fraction was prepared as described by Perlin et al. (16), and plasma membranes were isolated from this fraction by a modified version of the method of Seto-Young et al. (17). Specifically, the microsomal fraction was suspended in membrane wash buffer containing 10 mM Tris, pH 7.0, 20% (v/v) glycerol, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, and a protease inhibitor mixture (PIC).1 The suspension (2.0 ml) was applied to a discontinuous gradient of 0.9 ml of 65% (w/v) sucrose and 2.0 ml of 54% (w/v) sucrose containing 10 mM Tris, pH 7.0, and 1 mM EDTA. After centrifugation for 2 h at 183,000 × g (SW55 Ti rotor; Beckman) plasma membranes were collected from the interface, diluted 5-fold with 1 mM EGTA/Tris (pH 7.5)/PIC, and centrifuged at 100,000 × g for 1 h. The plasma membrane pellet was resuspended in 1 mM EGTA/Tris (pH 7.5)/PIC and stored at -70 °C. All procedures were carried out at 4 °C.

ATP Hydrolysis and Proton Transport-- Unless otherwise noted, ATP hydrolysis was assayed at 30 °C in 0.5 ml of 50 mM 4-morpholineethanesulfonic acid/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 terminated after 20-40 min, and the release of inorganic phosphate from ATP was measured by the method of Fiske and Subbarow (18). Specific activity was calculated as the difference between ATP hydrolysis in the presence and absence of 100 µM sodium orthovanadate, a potent inhibitor of P-type ATPases. IC50 values for vanadate inhibition were determined by measuring ATP hydrolysis in the presence of increasing concentrations of vanadate. For determination of Km values, 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 by the method of Fabiato and Fabiato (19). To determine the pH optimum for ATP hydrolysis, the pH of the assay medium 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. (20). The specific initial rate of fluorescence quenching for each mutant was adjusted for ATPase expression and reported as a percentage of the wild-type rate.

Protein Determination-- Protein concentrations were assayed by the method of Lowry et al. (21) or as modified by Ambesi et al. (13), with bovine serum albumin as standard.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Selection of Residues for Mutagenesis-- Previous work from our laboratory has examined structure-function relationships in M5 (Ser-690 through Leu-713) of the yeast Pma1 H+-ATPase (22). In the study described here, residues from the adjacent S5 region (Ala-661 through Tyr-689) were subjected to cysteine scanning mutagenesis. As shown in Fig. 1, this region is strongly conserved throughout the P2-type H+-ATPases of fungi, higher plants, and protozoa; there is also recognizable homology with the Na+,K+-, H+,K+-, and Ca2+-ATPases of animal cells.


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Fig. 1.   Sequence alignment of stalk segment 5. The 29-amino acid stretch of stalk segment 5 has been aligned for nine representative P2-type ATPases. Swiss Protein Database sequence accession numbers are (from top to bottom) P05030, P07038, P09627, P54210, P20649, Q00804, P04191, P04074, and P09626. Residues identical to the yeast Pma1 sequence are indicated by a period.

Each of the 29 residues was replaced with Cys, and the mutant alleles were cloned into expression vector YCp2HSE, transformed into yeast strain SY4, and expressed under the control of a heat-shock promoter after turning off the wild-type PMA1 allele (9). Secretory vesicles containing newly synthesized mutant ATPase were then isolated and analyzed (13).

Expression and ATP Hydrolysis-- As measured by quantitative immunoblotting (Table I), only two of the mutant ATPases (G670C and R682C) failed to reach the secretory vesicles, presumably due to protein misfolding; previous studies have shown that such mutant forms are typically arrested in the endoplasmic reticulum (23-25). It is worth noting that Gly-670 is strictly conserved in all known P2-type H+-ATPases, and Arg-682 is found in mammalian Na+,K+-, H+,K+-, and Ca2+-ATPases as well (Fig. 1).

                              
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Table I
Effect of mutations in stalk segment 5 on H+-ATPase expression and activity in secretory vesicles
Values are the mean of at least two determinations with a standard error less than 18%.

The remaining 27 mutant H+-ATPases appeared in the secretory vesicles at 28-109% of the level seen in the wild-type control. Likewise, 26 of the 27 mutants were able to hydrolyze ATP at rates ranging from 40% to 151%, after correction for the level of expression in the secretory vesicles (Table I). Only one mutant (S681C) showed a major reduction in ATP hydrolysis (to 9%), and even here, the uncorrected ATPase activity was 5-fold greater than the background activity observed in empty-plasmid controls.

Proton Transport-- Because S5 links the large hydrophilic loop to M5, a membrane segment known to be involved in cation transport (2, 26-30), it was important to test the effects of the Cys substitutions on the ability of the ATPase to pump protons. This was assayed by fluorescence quenching of the pH-sensitive dye acridine orange (Table I). In mutants A677C and S681C, ATP-dependent quenching was clearly above background, but the rates were below the limit at which they could be measured quantitatively by the acridine orange assay. In another mutant, Y689C, the rate of proton transport (175%) appeared to exceed the rate of ATP hydrolysis (107%), but further experiments showed that this discrepancy could be explained by a shift in the pH optimum of the mutant enzyme (see below). For all of the other mutants, the initial rate of ATP-dependent acridine orange quenching closely paralleled the rate of ATP hydrolysis, indicating that the substitutions had little or no effect on the coupling between transport and hydrolysis.

Kinetic Properties-- The mutant ATPases were next assayed for MgATP dependence, vanadate sensitivity, and the effect of pH on the rate of ATP hydrolysis, parameters that can provide a useful clue to changes in reaction mechanism. As summarized in Table II, there was one mutant (F685C) that displayed an increase in the IC50 for vanadate (to 42 µM) and a decrease in the apparent Km for MgATP (to 0.3 mM); F685C also showed a measurable alkaline shift in pH optimum (to pH 6.0). As pointed out previously, coordinated changes of this kind can readily be accounted for by a shift in conformational equilibrium from the E2 state (high affinity for vanadate; low affinity for MgATP) to the E1 state (low affinity for vanadate; high affinity for ATP) (5, 20, 22). Similar mutants have been described for mammalian Na+,K+-ATPase by Daly et al. (31) and Boxenbaum et al. (32).

                              
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Table II
Kinetic properties of mutant H+-ATPases in secretory vesicles
Values represent the mean of at least two determinations with an average standard error less than 5%.

More striking were seven mutants (F666C, L671C, I674C, A677C, I684C, R687C, and Y689C) with a 2- to 3-fold decrease in the apparent Km for MgATP (to 0.2-0.3 mM) and a rise in pH optimum (to pH 6.0-6.3) but no change (or even a slight decrease) in the IC50 for vanadate (Table II). This constellation of kinetic changes is reminiscent of that seen when wild-type yeast cells, previously starved of glucose, are returned to a glucose-containing medium. Under such conditions, the H+-ATPase undergoes a rapid 10-fold increase in activity, along with a lowering of the Km for MgATP and an alkaline shift of pH optimum (33). There is evidence that glucose activation results from kinase-mediated phosphorylation of the ATPase, very likely in the C-terminal region of the polypeptide (see "Discussion"). Thus, it seemed possible that the seven mutations listed above may have somehow shifted the ATPase into a constitutively activated state.

Glucose Activation-- To test this idea, the seven mutations highlighted in Table I were integrated into the chromosomal copy of the PMA1 gene to see whether the H+-ATPase was still capable of glucose activation at the plasma membrane. For the experiments to be described, wild-type and mutant cells were grown to mid-exponential phase in glucose medium and incubated under two conditions: (a) 90 min of starvation in glucose-free medium, and (b) 60 min of starvation followed by a 30-min incubation in medium containing 4% glucose. Plasma membranes were then isolated and assayed.

Based on quantitative immunoblotting, wild-type and mutant plasma membranes contained roughly equal amounts of Pma1 ATPase, and there was little if any change in the amount when starved cells were incubated briefly with glucose (Table III). Activity measurements revealed a striking difference between the wild-type and mutant enzymes, however. Across a wide range of pH values, the wild-type ATPase had relatively low activity in starved cells but was conspicuously stimulated when the cells were treated with glucose (Fig. 2); the increase in activity ranged from 4.4-fold at pH 5.7 to 9.0-fold at pH 6.25 (Table III). The seven mutant ATPases, on the other hand, were already activated in starved cells and changed very little upon glucose treatment (Fig. 2; Table III).

                              
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Table III
H+-ATPase activity in plasma membranes isolated from glucose-starved and glucose-metabolizing cells
Values are the mean of at least three different membrane preparations with an average standard error less than 16%.


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Fig. 2.   Effect of glucose on ATPase activity as a function of pH. Plasma membranes from yeast cells expressing wild-type (top) or Y689C (bottom) ATPase were isolated from glucose-starved cells () or glucose-metabolizing cells (open circle ). ATPase activity was determined as a function of pH as described under "Experimental Procedures."

Glucose also had a less pronounced effect on the kinetic properties of the mutant ATPases (Table IV). Whereas the Km of the wild-type enzyme for MgATP fell 8-fold (from 3.1 mM to 0.4 mM) upon incubation with glucose, the Km values of the mutant enzymes were already reduced in starved cells (0.2-1.2 mM). Likewise, although the pH optimum of the wild-type ATPase displayed a small but reproducible alkaline shift (from pH 5.7 to pH 6.1) after glucose treatment, the mutants had pH optima between pH 6.0 and pH 6.3, even under starvation conditions. Taken together, the kinetic findings reinforce the notion that single amino acid substitutions in S5 have led to a constitutive activation of the Pma1 ATPase.

                              
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Table IV
Kinetic properties of mutant ATPase in the plasma membrane


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The original goal of this study was to identify amino acid residues in stalk segment 5 that play an important role in the reaction cycle of the yeast Pma1 H+-ATPase. The strategy, as described previously (5, 20), was to screen for mutations that increase the IC50 for vanadate by shifting the conformational equilibrium away from the E2 state (to which vanadate binds) toward E1; typically, such mutants also display a reduced Km for MgATP and an alkaline change in pH optimum. Unlike stalk segment 4, where mutations of this type occurred at 13 consecutive positions, only 1 vanadate-resistant mutant (F685C) was seen in S5. At the corresponding position of the sarcoplasmic reticulum Ca2+-ATPase (Tyr-754), Sorensen and Andersen (34) have also observed kinetic changes upon substitution with Ala, but in this case, there was an increase in apparent affinity for vanadate, reflecting an elevated dephosphorylation rate of E2P to E2.

A closer correspondence between the two ATPases was evident at Arg-682 (Arg-751 in the SERCA Ca2+-ATPase), a residue found in all known P-type ATPases. Here, substitution by Cys in the H+-ATPase or by Ala, Ile, or Glu in the Ca2+-ATPase virtually abolished expression, suggesting that the conserved Arg plays an essential role in protein folding. Indeed, in the recently published crystal structure of the Ca2+-ATPase (2), Arg-751 interacts with several residues in the cytoplasmically exposed M6-M7 loop; it is easy to imagine that these interactions may serve to stabilize the protein.

By far the most significant finding of the present study was that seven periodically spaced residues in S5 play a role in the regulation of yeast H+-ATPase by glucose. Rapid, reversible down-regulation of ATPase activity during glucose starvation was first observed by Serrano (33), who found it to be associated with a reduction in the apparent affinity for MgATP and a shift in pH optimum from pH 6.0 to pH 5.7. Since then, evidence has mounted that down-regulation is mediated by the C terminus of the ATPase, functioning as an auto-inhibitory domain. Consistent with this idea, the yeast ATPase can be activated by deleting 11 or 18 amino acid residues from the C terminus (35, 36). Although the mechanism is not yet fully understood in molecular terms, glucose has been shown to trigger kinase-mediated phosphorylation of the ATPase on one or more Ser/Thr residues (37), and site-directed mutagenesis has identified two potential phosphorylation sites in the C-terminal region (Ser-899 and Thr-912) at which amino acid substitutions modify glucose regulation (38). A recent study by Goossens et al. (39) provides evidence that a novel protein kinase known as Ptk2 acts on the first of the two sites, whereas the second is a potential phosphorylation site for calmodulin-dependent protein kinase II. Based on all of these findings, it is attractive to think that glucose induces the phosphorylation of Ser-899 and Thr-912, somehow displacing the C terminus and releasing the ATPase from auto-inhibition. A more detailed discussion of this hypothesis can be found in a recent review by Portillo (40).

If the C terminus can indeed inhibit the activity of the yeast H+-ATPase, one would like to know how it does so. A previous study by Eraso and Portillo (41) took a useful step toward answering this question by isolating intragenic suppressors of the double mutant S911A/T912A, which exhibits little or no glucose activation and cannot grow on glucose medium. Fourteen second-site suppressor mutations were isolated by their ability to restore growth on glucose. Of them, four mapped in the C-terminal region, three mapped in S2, two mapped in S4, four mapped in the presumed ATP-binding domain of the large cytoplasmic loop, and two mapped toward the end of the cytoplasmic loop in the region that we have defined as S5. Direct assays of isolated plasma membranes showed that all of the suppressor mutations produced significant increases in ATPase activity in glucose-starved cells. However, the kinetic properties of the mutant enzymes varied, with some exhibiting changes in Km and pH optimum characteristic of the activated state, and others not doing so. Most relevant to the present study are the two suppressor mutations mapping in S5. One, G670S, led to a 7-fold increase in the rate of ATP hydrolysis in starved cells, along with a significant drop in Km (from 4.0 to 1.2 mM), a rise in pH optimum (from pH 5.5 to pH 6.3), and a fall in the IC50 for vanadate (from 10.0 to 0.5 µM); it therefore met the criteria for constitutive activation as described above. The other suppressor mutant isolated by Eraso and Portillo (41), P669L, displayed a 10-fold elevation in the rate of hydrolysis in starved cells, accompanied by a smaller rise in pH optimum (to pH 6.0) and a fall in the IC50 (to 0.9 µM) but not by a detectable change in Km; thus, it seems to be an intermediate case. Of the related mutations that were constructed in the present study, G670C blocked biogenesis and could not be characterized, whereas P669C had reasonable specific activity, a slightly lowered Km and IC50, and a slightly elevated pH optimum (see Table II).

At the structural level, it is presumably significant that the seven constitutively activated mutants described in the present study map to one face of an alpha -helix, along with the P669L and G670S mutations of Eraso and Portillo (Ref. 41; Fig. 3). Also on the same face are F685C (a mutation affecting the equilibrium between E1 and E2 conformations; see above) and R682C (one of two mutations leading to arrest in the endoplasmic reticulum). Although it is tempting to think that this face of the S5 alpha -helix may interact directly with the C terminus to bring about auto-inhibition, the interaction may equally well be indirect; high-resolution structures of the ATPase in the glucose-starved and glucose-metabolizing states will be required to settle this question.


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Fig. 3.   Helical wheel analysis of S5. Residues at which mutations lead to constitutive activation are highlighted by a shaded box (this study) or an empty box (41). Residues at which mutations cause a block in biogenesis (R682C) or a shift in the E1-E2 equilibrium (F685C) are marked with an asterisk.

In the meantime, it is interesting to consider glucose regulation of the yeast H+-ATPase in a more general framework. Although N- and C-terminal sequences have been poorly conserved among P2-type ATPases, both termini have taken on regulatory roles in various members of the group. For example, the C terminus of the plant plasma membrane H+-ATPase, which is considerably longer than that of the yeast enzyme, has been well documented to function as an auto-inhibitory domain, and inhibition is reversed by the binding of 14-3-3 proteins to the C terminus (reviewed by Morsomme and Boutry (42)). Once again, mutations elsewhere in the ATPase can also reverse the inhibition (43, 44). The plasma membrane Ca2+-ATPase of animal cells is also regulated by means of the C terminus, where calmodulin binding stimulates the rate of ATP hydrolysis and improves the affinities for Ca2+ and ATP (reviewed by Carafoli and Brini (45)). On the other hand, a recently described plant endomembrane Ca2+-ATPase is thought to be activated by the binding of calmodulin to its very long N terminus (46). Thus, as one might have predicted, the P2-type ATPases have evolved a variety of regulatory mechanisms, each involving its own auxiliary molecules and binding sites. Given the high degree of interest in this area, the next few years are likely to see continued progress toward understanding the molecular mechanisms of regulation.

    ACKNOWLEDGEMENTS

We are grateful to Brett Mason, Valery Petrov, and Anthony Ambesi for helpful discussions.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant GM-15761 and by a Fogarty Postdoctoral Fellowship (to M. M.).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: Depts. of Genetics and Cellular & Molecular Physiology, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-2690; Fax: 203-737-1771.

§ Present address: Universidad Nacional Autonoma de Mexico, Departamento de Bioquimica, Facultad de Medicina, Mexico, D.F., 04510 Mexico.

Published, JBC Papers in Press, April 16, 2001, DOI 10.1074/jbc.M102332200

    ABBREVIATIONS

The abbreviation used is: PIC, protease inhibitor mixture (1 µg/ml leupeptin, 1 µg/ml pepstatin, 1 µg/ml aprotinin, and 2 µg/ml chymostatin).

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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