Stalk segment 5 of the yeast plasma membrane H+-ATPase: mutational evidence for a role in glucose regulation.

In P(2)-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 E(2) conformational state toward E(1). 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.

Stalk segment 4 of the yeast plasma membrane H+-ATPase. Mutational evidence for a role in the E1-E2 conformational change.

In the P(2)-type ATPases, there is growing evidence that four alpha-helical stalk segments connect the cytoplasmic part of the molecule, responsible for ATP binding and hydrolysis, to the membrane-embedded part that mediates cation transport. The present study has focused on stalk segment 4, which displays a significant degree of sequence conservation among P(2)-ATPases. When site-directed mutants in this region of the yeast plasma membrane H(+)-ATPase were constructed and expressed in secretory vesicles, more than half of the amino acid substitutions led to a severalfold decrease in the rate of ATP hydrolysis, although they had little or no effect on the coupling between hydrolysis and transport. Strikingly, mutant ATPases bearing single substitutions of 13 consecutive residues from Ile-359 through Gly-371 were highly resistant to inorganic orthovanadate, with IC(50) values at least 10-fold above those seen in the wild-type enzyme. Most of the same mutants also displayed a significant reduction in the K(m) for MgATP and an increase in the pH optimum for ATP hydrolysis. Taken together, these changes in kinetic behavior point to a shift in equilibrium from the E(2) conformation of the ATPase toward the E(1) conformation. The residues from Ile-359 through Gly-371 would occupy three full turns of an alpha-helix, suggesting that this portion of stalk segment 4 may provide a conformationally active link between catalytic sites in the cytoplasm and cation-binding sites in the membrane.

Epitope-tagged constructs of the yeast plasma-membrane H(+)-ATPase.

In this study, two different epitope tags (HA, c-myc) were introduced near the N terminus of the yeast PMA1 H(+)-ATPase. The resulting proteins were indistinguishable from the wild-type ATPase in their ability to travel through the secretory pathway, as judged by quantitative immunoblotting of isolated secretory vesicles. Furthermore, there were no significant abnormalities in ATPase activity (including K(m) for MgATP, Vmax, pH optimum, and IC50 for inhibition by vanadate) or in ATP-dependent proton pumping. Finally, the epitope-tagged ATPases could support normal growth and displayed the expected activation by glucose.

Functional role of charged residues in the transmembrane segments of the yeast plasma membrane H+-ATPase.

As defined by hydropathy analysis, the membrane-spanning segments of the yeast plasma membrane H(+)-ATPase contain seven negatively charged amino acids (Asp and Glu) and four positively charged amino acids (Arg and His). To explore the functional role of these residues, site-directed mutants at all 11 positions and at Glu-288, located near the cytoplasmic end of M3, have been constructed and expressed in yeast secretory vesicles. Substitutions at four of the positions (Glu-129, Glu-288, Asp-833, and Arg-857) had no significant effect on ATP hydrolysis or ATP-dependent proton pumping, substitutions at five additional positions (Arg-695, His-701, Asp-730, Asp-739, and Arg-811) led to misfolding of the ATPase and blockage at an early stage of biogenesis, and substitutions of Asp-143 allowed measurable biogenesis but nearly abolished ATP hydrolysis and proton transport. Of greatest interest were mutations of Glu-703 in M5 and Glu-803 in M8, which altered the apparent coupling between hydrolysis and transport. Three Glu-703 mutants (E703Q, E703L, E703D) showed significantly reduced pumping over a wide range of hydrolysis values and thus appeared to be partially uncoupled. At Glu-803, by contrast, one mutant (E803N) was almost completely uncoupled, while another (E803Q) pumped protons at an enhanced rate relative to the rate of ATP hydrolysis. Both Glu-703 and Glu-803 occupy positions at which amino acid substitutions have been shown to affect transport by mammalian P-ATPases. Taken together, the results provide growing evidence that residues in membrane segments 5 and 8 of the P-ATPases contribute to the cation transport pathway and that the fundamental mechanism of transport has been conserved throughout the group.

Biogenesis and function of the yeast plasma-membrane H(+)-ATPase.

One of the most abundant proteins in the yeast plasma membrane is the P-type H(+)-ATPase that pumps protons out of the cell, supplying the driving force for a wide array of H(+)-dependent cotransporters. The ATPase is a 100 kDa polypeptide, anchored in the lipid bilayer by 10 transmembrane alpha-helices. It is structurally and functionally related to the P-type Na(+),K(+)-, H(+),K(+)- and Ca(2+)-ATPases of animal cells and the H(+)-ATPases of plant cells, and it shares with them a characteristic reaction mechanism in which ATP is split to ADP and inorganic phosphate (P(i)) via a covalent beta-aspartyl phosphate intermediate. Cryoelectron microscopic images of the H(+)-ATPase of Neurospora crassa and the sarcoplasmic reticulum Ca(2+)-ATPase of animal cells have recently been obtained at 8 nm resolution. The membrane-embedded portion of the molecule, which presumably houses the cation translocation pathway, is seen to be connected via a narrow stalk to a large, multidomained cytoplasmic portion, known to contain the ATP-binding and phosphorylation sites. In parallel with the structural studies, efforts are being made to dissect structure/function relationships in several P-type ATPases by means of site-directed mutagenesis. This paper reviews three phenotypically distinct classes of mutant that have resulted from work on the yeast PMA1 H(+)-ATPase: (1) mutant ATPases that are poorly folded and retained in the endoplasmic reticulum; (2) mutants in which the conformational equilibrium has been shifted from the E(2) state, characterized by high affinity for vanadate, to the E(1) state, characterized by high affinity for ATP; and (3) mutants with altered coupling between ATP hydrolysis and proton pumping. Although much remains to be learned before the transport mechanism can be fully understood, these mutants serve to identify critical parts of the polypeptide that are required for protein folding, conformational change and H(+):ATP coupling.

N-terminal chimeric constructs improve the expression of sarcoplasmic reticulum Ca(2+)-ATPase in yeast.

Wild-type and chimeric constructs comprising rabbit sarcoplasmic reticulum (SR) Ca(2+)-ATPase and the N-terminal cytoplasmic portion of yeast plasma membrane H(+)-ATPase were expressed in yeast under control of a heat-shock regulated promoter. The wild-type ATPase was found predominantly in endoplasmic reticulum (ER) membranes. Addition of the first 88 residues of H(+)-ATPase to the Ca(2+)-ATPase N-terminal end promoted a marked shift in the localization of chimeric H(+)/Ca(2+)-ATPase which accumulated in a light membrane fraction associated with yeast smooth ER. Furthermore, there was a three-fold increase in the overall level of expression of chimeric H(+)/Ca(2+)-ATPase. Similar results were obtained for a chimeric Ca(2+)-ATPase containing a hexahistidine sequence added to its N-terminal end. Both H(+)/Ca(2+)-ATPase and 6xHis-Ca(2+)-ATPase were functional as demonstrated by their ability to form a phosphorylated intermediate and undergo fast turnover. Conversely, a replacement chimera in which the N-terminal end of SR Ca(2+)-ATPase was replaced by the corresponding segment of H(+)-ATPase was not stably expressed in yeast membranes. These results indicate that the N-terminal segment of Ca(2+)-ATPase plays an important role in enzyme assembly and contains structural determinants necessary for ER retention of the ATPase.

Molecular cloning of NHE3 from LLC-PK1 cells and localization in pig kidney.

LLC-PK1 cells, an established line from pig kidney, express basolateral and apical Na+/H+ exchangers that can be distinguished by their differing sensitivities to the amiloride analog N-ethyl-N-isopropylamiloride (EIPA). It has been shown previously that the basolateral exchanger is encoded by NHE1. In the present study, a combination of reverse transcription-PCR, 5' RACE, and genomic library screening was used to clone the coding region of the porcine NHE3 gene. There was significant homology between the LLC-PK1 sequence and the previously reported rabbit and rat NHE3 genes, with nucleotide and deduced amino acid identities of 87 and 85% in rabbit, and 85 and 87% in rat, respectively. To study expression patterns, Northern analysis was carried out using an NHE3 cDNA to probe poly(A)+ RNA isolated from LLC-PK1 cells, and from pig kidney cortex. In all three cases, a major transcript of 6.1 kb was detected along with two minor transcripts of 4.7 and 3.8 kb. In situ hybridization with two different NHE3 probes gave intense labeling of the distal convoluted tubule in pig kidney but (unexpectedly) no detectable labeling of the proximal tubule. These studies suggest that there are marked species differences in NHE3 expression in the distal nephron.

Evidence for a salt bridge between transmembrane segments 5 and 6 of the yeast plasma-membrane H+-ATPase.

The plasma-membrane H+-ATPase of Saccharomyces cerevisiae, which belongs to the P2 subgroup of cation-transporting ATPases, is encoded by the PMA1 gene and functions physiologically to pump protons out of the cell. This study has focused on hydrophobic transmembrane segments M5 and M6 of the H+-ATPase. In particular, a conserved aspartate residue near the middle of M6 has been found to play a critical role in the structure and biogenesis of the ATPase. Site-directed mutants in which Asp-730 was replaced by an uncharged residue (Asn or Val) were abnormally sensitive to trypsin, consistent with the idea that the proteins were poorly folded, and immunofluorescence confocal microscopy showed them to be arrested in the endoplasmic reticulum. Similar defects are known to occur when either Arg-695 or His-701 in M5 is replaced by a neutral residue (Dutra, M. B., Ambesi, A., and Slayman, C. W. (1998) J. Biol. Chem. 273, 17411-17417). To search for possible charge-charge interactions between Asp-730 and Arg-695 or His-701, double mutants were constructed in which positively and negatively charged residues were swapped or eliminated. Strikingly, two of the double mutants (R695D/D730R and R695A/D730A) regained the capacity for normal biogenesis and displayed near-normal rates of ATP hydrolysis and ATP-dependent H+ pumping. These results demonstrate that neither Arg-695 nor Asp-730 is required for enzymatic activity or proton transport, but suggest that there is a salt bridge between the two residues, linking M5 and M6 of the 100-kDa polypeptide.

Phosphorylation region of the yeast plasma-membrane H+-ATPase. Role in protein folding and biogenesis.

Mutations at the phosphorylation site (Asp-378) of the yeast plasma-membrane H+-ATPase have been shown previously to cause misfolding of the ATPase, preventing normal movement along the secretory pathway; Asp-378 mutations also block the biogenesis of co-expressed wild-type ATPase and lead to a dominant lethal phenotype. To ask whether these defects are specific for Asp-378 or whether the phosphorylation region as a whole is involved, alanine-scanning mutagenesis has been carried out to examine the role of 11 conserved residues flanking Asp-378. In the sec6-4 expression system (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949), the mutant ATPases displayed varying abilities to reach the secretory vesicles that deliver plasma-membrane proteins to the cell surface. Indirect immunofluorescence of intact cells also gave evidence for a spectrum of behavior, ranging from mutant ATPases completely arrested (D378A, K379A, T380A, and T384A) or partially arrested in the endoplasmic reticulum to those that reached the plasma membrane in normal amounts (C376A, S377A, and G381A). Although the extent of ER retention varied among the mutants, the endoplasmic reticulum appeared to be the only secretory compartment in which the mutant ATPases accumulated. All of the mutant proteins that localized either partially or fully to the ER were also malfolded based on their abnormal sensitivity to trypsin. Among them, the severely affected mutants had a dominant lethal phenotype, and even the intermediate mutants caused a visible slowing of growth when co-expressed with wild-type ATPase. The effects on growth could be traced to the trapping of the wild-type enzyme with the mutant enzyme in the ER, as visualized by double label immunofluorescence. Taken together, the results indicate that the residues surrounding Asp-378 are critically important for ATPase maturation and transport to the cell surface.

Structure-function relationships in membrane segment 5 of the yeast Pma1 H+-ATPase.

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 alpha-helix.

Substitutions of aspartate 378 in the phosphorylation domain of the yeast PMA1 H+-ATPase disrupt protein folding and biogenesis.

There is strong evidence that Asp-378 of the yeast PMA1 ATPase plays an essential role in ATP hydrolysis by forming a covalent beta-aspartyl phosphate reaction intermediate. In this study, Asp-378 was replaced by Asn, Ser, and Glu, and the mutant ATPases were expressed in a temperature-sensitive secretion-deficient strain (sec6-4) that allowed their properties to be examined. Although all three mutant proteins were produced at nearly normal levels and remained stable for at least 2 h at 37 degrees C, they failed to travel to the vesicles that serve as immediate precursors of the plasma membrane; instead, they became arrested at an earlier step of the secretory pathway. A closer look at the mutant proteins revealed that they were firmly inserted into the bilayer and were not released by washing with high salt, urea, or sodium carbonate (pH 11), treatments commonly used to strip nonintegral proteins from membranes. However, all three mutant ATPases were extremely sensitive to digestion by trypsin, pointing to a marked abnormality in protein folding. Furthermore, in contrast to the wild-type enzyme, the mutant ATPases could not be protected against trypsinolysis by ligands such as MgATP, MgADP, or inorganic orthovanadate. Thus, Asp-378 functions in an unexpectedly complex way during the acquisition of a mature structure by the yeast PMA1 ATPase.

Reactive cysteines of the yeast plasma-membrane H+-ATPase (PMA1). Mapping the sites of inactivation by N-ethylmaleimide.

We have taken advantage of cysteine mutants described previously (Petrov, V. V., and Slayman, C. W. (1995) J. Biol. Chem. 270, 28535-28540) to map the sites at which N-ethylmaleimide (NEM) reacts with the plasma-membrane H+ATPase (PMA)1 of Saccharomyces cerevisiae. When membrane vesicles containing the ATPase were incubated with NEM, six of nine mutants with single cysteine substitutions showed sensitivity similar to the wild-type enzyme. By contrast, C221A and C532A were inactivated more slowly than the wild-type control, and the C221, 532A double mutant was completely resistant, indicating that Cys-221 and Cys-532 are NEM-reactive residues. In the presence of 10 mM MgADP, the wild-type ATPase was partially protected against NEM; parallel experiments with the C221A and C532A mutants showed that the protection occurred at Cys-532, located in or near the nucleotide-binding site. Unexpectedly, the inactivation of the C409A ATPase was approximately 4-fold more rapid than in the case of the wild-type enzyme. Experiments with double mutants made it clear that this resulted from an acidic shift in pKa and a consequent acceleration of the reaction rate at Cys-532. One simple interpretation is that substitution of Cys-409 leads to a local conformational change within the central hydrophilic domain. Consistent with this idea, the reaction of fluorescein 5'-isothiocyanate at Lys-474 was also stimulated approximately 3. 5-fold by the C409A mutation. Taken together, the results of this study provide new information about the reactivity of individual Cys residues within the ATPase and pave the way to tag specific sites for structural and functional studies of the enzyme.