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.     

The cytoplasmic loop located between transmembrane segments 6 and 7 controls activation by Ca2+ of sarcoplasmic reticulum Ca2+-ATPase

During active cation transport, sarcoplasmic reticulum Ca2+-ATPase, like other P-type ATPases, undergoes major conformational changes, some of which are dependent on Ca2+ binding to high affinity transport sites. We here report that, in addition to previously described residues of the transmembrane region (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989) Nature 339, 476-478), the region located in the cytosolic L6-7 loop connecting transmembrane segments M6 and M7 has a definite influence on the sensitivity of the Ca2+-ATPase to Ca2+, i.e. on the affinity of the ATPase for Ca2+. Cluster mutation of aspartic residues in this loop results in a strong reduction of the affinity for Ca2+, as shown by the Ca2+ dependence of ATPase phosphorylation from either ATP or Pi. The reduction in Ca2+ affinity for phosphorylation from Pi is observed both at acidic and neutral pH, suggesting that these mutations interfere with binding of the first Ca2+, as proposed for some of the intramembranous residues essential for Ca2+ binding (Andersen, J. P. (1995) Biosci. Rep. 15, 243-261). Treatment of the mutated Ca2+-ATPase with proteinase K, in the absence or presence of various Ca2+ concentrations, leads to Ca2+-dependent changes in the proteolytic degradation pattern similar to those in the wild type but observed only at higher Ca2+ concentrations. This implies that these effects are not due to changes in the conformational state of Ca2+-free ATPase but that changes affecting the proteolytic digestion pattern require higher Ca2+ concentrations. We conclude that aspartic residues in the L6-7 loop might interact with Ca2+ during the initial steps of Ca2+ binding.     

Evidence for interaction between transmembrane segments in assembly of Kv1.3

Previously, we showed that the N-terminal recognition domain (T1) of Kv1.3 was not required for assembly of functional channels [Tu et al. (1996) J. Biol. Chem. 271, 18904-18911]. Moreover, specific Kv1.3 peptide fragments including regions of the central core are able to inhibit expression of current produced from a channel lacking the T1 domain, Kv1.3(T1-). To elucidate the mechanism whereby Kv1.3 peptide fragments suppress Kv1.3(T1-) current, we have studied the ability of peptide fragments containing the transmembrane segments S1, S1-S2, or S1-S2-S3 to physically associate with the Kv1.3(T1-) polypeptide subunit in vitro in microsomal membranes. Using c-myc (9E10) epitope-labeled peptide fragments and anti-myc antibody as well as antisera to the Kv1.3 C-terminus, we now demonstrate specific association of these peptide fragments with Kv1.3(T1-). Association of peptide fragments with Kv1.3(T1-) was correlated with integration of both proteins into the membrane. Furthermore, the relative strength and kinetics of this association directly correlated with the ability of fragments to suppress Kv1.3(T1-) current. The rate-limiting step in the sequential synthesis, integration, and formation of a complex was the association of integrated polypeptides within the plane of the lipid bilayer. These results strongly suggest that the physical association of transmembrane segments provides the basis for suppression of K+ channel function by K+ channel peptide fragments in vivo. Moreover, the S1-S2-S3 peptide fragment potently suppressed full-length Kv1.3, thus implicating a role for the S1-S2-S3 region of Kv1.3 in the assembly of the Kv1.3 channel. We refer to these putative association sites as IMA (intramembrane association) sites.     

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.     

Folding and intracellular transport of the yeast plasma-membrane H(+)-ATPase: effects of mutations in KAR2 and SEC65

We have developed two independent assays to study the integration, folding, and intracellular transport of the polytopic plasma membrane H(+)-ATPase in yeast. To follow folding, controlled trypsinolysis was used to distinguish between the E1 conformation of the ATPase (favored in the presence of ADP) and the E2 conformation (favored in the presence of vanadate). By this criterion, wild-type ATPase appears to recognize its ligands and assume distinct conformations within a short time after its biosynthesis. To follow intracellular transport, we have exploited the fact that export of newly synthesized ATPase from the endoplasmic reticulum is accompanied by kinase-mediated phosphorylation, leading to a shift in electrophoretic mobility. Because proper folding is required for transport from the endoplasmic reticulum, the mobility shift also serves as a convenient bioassay for correct folding. As a first step toward identifying cell components important in folding of the nascent ATPase, we have used the dual assays to examine the role of KAR2, encoding the yeast homolog of immunoglobulin heavy chain binding protein/78-kDa glucose-regulated protein, and SEC65, encoding a subunit of the yeast signal recognition particle. Although mutation of KAR2 caused defective translocation of several secretory precursors into the endoplasmic reticulum lumen, ATPase folding and intracellular transport were unperturbed. By contrast, in a sec65 mutant, the folding and intracellular transport of newly synthesized ATPase were delayed. Our data suggest that conformational maturation of the ATPase is a rapid process in wild-type cells and that membrane integration mediated by signal recognition peptide is important for the proper folding of this polytopic protein.     

Phosphorylation of yeast plasma membrane H+-ATPase by casein kinase I

The plasma membrane H+-ATPase of Saccharomyces cerevisiae is subject to phosphorylation by a casein kinase I activity in vitro. We show this casein kinase I activity to result from the combined function of YCK1 and YCK2, two highly similar and plasma membrane-associated casein kinase I homologues. First, H+-ATPase phosphorylation is severely impaired in the plasma membrane of YCK-deficient yeast strains. Furthermore, the wild-type level of the phosphoprotein is restored by the addition of purified mammalian casein kinase I to the mutant membranes. We used the H+-ATPase as well as a synthetic peptide substrate that contains a phosphorylation site for casein kinase I to compare kinase activity in membranes prepared from yeast cells grown in the presence or absence of glucose. The addition of glucose results in increased H+-ATPase activity which is associated with a decline in the phosphorylation level of the enzyme. Mutations in both YCK1 and YCK2 affect this regulation, suggesting that H+-ATPase activity is modulated by glucose via a combination of a "down-regulating" casein kinase I activity and another, yet uncharacterized, "up-regulating" kinase activity. Biochemical mapping of phosphorylated H+-ATPase identifies a major phosphopeptide that contains a consensus phosphorylation site (Ser-507) for casein kinase I. Site-directed mutagenesis of this consensus sequence indicates that Glu-504 is important for glucose-induced decrease in the apparent Km for ATP.     

Reversible association between the V1 and V0 domains of yeast vacuolar H+-ATPase is an unconventional glucose-induced effect

The yeast vacuolar H+-ATPase (V-ATPase) is a multisubunit complex responsible for organelle acidification. The enzyme is structurally organized into two major domains: a peripheral domain (V1), containing the ATP binding sites, and an integral membrane domain (V0), forming the proton pore. Dissociation of the V1 and V0 domains inhibits ATP-driven proton pumping, and extracellular glucose concentrations regulate V-ATPase activity in vivo by regulating the extent of association between the V1 and V0 domains. To examine the mechanism of this response, we quantitated the extent of V-ATPase assembly in a variety of mutants with known effects on other glucose-responsive processes. Glucose effects on V-ATPase assembly did not involve the Ras-cyclic AMP pathway, Snf1p, protein kinase C, or the general stress response protein Rts1p. Accumulation of glucose 6-phosphate was insufficient to maintain or induce assembly of the V-ATPase, suggesting that further glucose metabolism is required. A transient decrease in ATP concentration with glucose deprivation occurs quickly enough to help trigger disassembly of the V-ATPase, but increases in cellular ATP concentrations with glucose readdition cannot account for reassembly. Disassembly was inhibited in two mutant enzymes lacking ATPase and proton pumping activities or in the presence of the specific V-ATPase inhibitor, concanamycin A. We propose that glucose effects on V-ATPase assembly occur by a novel mechanism that requires glucose metabolism beyond formation of glucose 6-phosphate and generates a signal that can be sensed efficiently only by a catalytically competent V-ATPase.     

Biochemical identification of transmembrane segments of the Ca(2+)-ATPase of sarcoplasmic reticulum

The transmembrane segments of sarcoplasmic reticulum Ca(2+)-ATPase were determined by trypsinization of cytoplasmic side-out intact sarcoplasmic reticulum vesicles. The membrane portion of tryptic digest comprising the transmembrane fragments, joined by the intravesicular segments, was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis after labeling with fluorescein 5-maleimide in the presence of sodium dodecyl sulfate. In this way, seven fluorescent bands of tryptic fragments below 11 kDa were observed which were derived from 4 pairs of membrane spanning segments and one hydrophobic sequence at the C-terminal end. Two peptides of 10.8 and 10.6 kDa had the identical N-terminal sequence beginning at Glu826, representing the transmembrane segments M7 and M8 and their connecting loop. A band at 8.1 kDa contained one peptide beginning at Tyr36 (M1/loop/M2). A 7.7-kDa peptide starting at Leu253 (M3/loop/M4) and a 7.3-kDa peptide beginning at Ala752 (M5/loop/M6) were also observed. A band at 6.7 kDa contained two peptides, one beginning at Ser48 (M1/loop/M2) and another beginning at Tyr763 (M5/loop/M6). In addition, a 4-kDa peptide beginning at Met925 was observed. The size of this peptide did not allow for a complete pair of transmembrane segments, but this peptide could have been derived from trypsinolysis between the last pair of membrane spanning segments. These data therefore provide biochemical evidence for at least 8 transmembrane segments and perhaps two more at the C-terminal end of the enzyme.     

Complementation of the Saccharomyces cerevisiae plasma membrane H+-ATPase by a plant H+-ATPase generates a highly abundant fusicoccin binding site

Accumulating evidence suggests that the H+-ATPase of the plant plasma membrane is activated by a direct, reversible interaction with 14-3-3 proteins involving the displacement of the C-terminal autoinhibitory domain of the enzyme. The fungal phytotoxin fusicoccin (FC) appears to stabilize this H+-ATPase.14-3-3 complex, thus leading to a persistent activation of the H+-ATPase in vivo. In this study we show that functional replacement of the Saccharomyces cerevisiae H+-ATPase genes by a Nicotiana plumbaginifolia H+-ATPase (pma2) results in the generation of a high affinity fusicoccin binding site that is exceptionally abundant. Acquisition of FC binding capacity is accompanied by a significant increase in the amount of plasma membrane-associated yeast 14-3-3 homologs. The existence of a (plant) PMA2.(yeast)14-3-3 complex was demonstrated using two-dimensional gel systems (native/denaturing). After expression of PMA2 lacking most of its C-terminal region, neither H+-ATPase.14-3-3 complex formation nor FC binding activity could be observed. Furthermore, we obtained direct biochemical evidence for a minimal FC binding complex consisting of the C-terminal PMA2 domain and yeast 14-3-3 homologs. Thus we demonstrated unambiguously the relevance of this regulatory ATPase domain for 14-3-3 interaction as well as its requirement for FC binding.     

Ligand-sensitive interactions among the transmembrane helices of Na+/K+-ATPase

An extensively trypsin-digested Na+/K+-ATPase, which retains the ability to bind Na+, K+, and ouabain, consists of four fragments of the alpha-subunit that contain all 10 transmembrane alpha domains, and the beta-subunit, a fraction of which is cleaved at Arg142-Gly143. In previous studies, we solubilized this preparation with a detergent and mapped the relative positions of several transmembrane helices of the subunits by chemical cross-linking. To determine if these detected helix-helix proximities were representative of those existing in the bilayer prior to solubilization, we have now done similar studies on the membrane-bound preparation of the same digested enzyme. After oxidative sulfhydryl cross-linking catalyzed by Cu2+-phenanthroline, two prominent products were identified by their mobilities and the analyses of their N termini. One was a dimer of a 11-kDa alpha-fragment containing the H1-H2 helices and a 22-kDa alpha-fragment containing the H7-H10 helices. This dimer seemed to be the same as that obtained in the solubilized preparation. The other product was a trimer of the above two alpha-fragments and that fraction of beta whose extracellular domain was cleaved at Arg142-Gly143. This product was different from a similar one of the solubilized preparation in that the latter contained the predominant fraction of beta without the extracellular cleavage. The cross-linking reactions of the membrane preparation, but not those of the solubilized one, were hindered specifically by Na+, K+, and ouabain. These findings indicate that (a) the H1-H2 transmembrane helices of alpha are adjacent to some of its H7-H10 helices both in solubilized and membrane-bound states, (b) the alignment of the residues of the single transmembrane helix of beta with the interacting H1-H2 and H7-H10 helices of alpha is altered by detergent solubilization and by structural changes in the extracellular domain of beta, and (c) the three-dimensional packing of the interacting transmembrane helices of alpha and beta are regulated by the specific ligands of the enzyme.     

Genetic probing of the stalk segments associated with M2 and M3 of the plasma membrane H+-ATPase from Saccharomyces cerevisiae

The stalk region of the H+-ATPase from Saccharomyces cerevisiae has been proposed to play a role in coupling ATP hydrolysis to proton transport. Genetic probing was used to examine the role of stalk segments S2 and S3, associated with M2 and M3, respectively. Saturation mutagenesis was used to explore the role of side group character at position Ile183 in S2, at which an alanine substitution was shown previously to partially uncouple the enzyme (Wang, G., Tamas, M. J., Hall, M. J., Pascual-Ahuir, A., and Perlin, D. S. (1996) J. Biol. Chem. 271, 25438-25445). Diverse side group substitutions were tolerated at this position, although three substitutions, I183N, I183R, and I183Y required second site mutations at the C terminus of the enzyme for stabilization. Substitution of glycine and proline at Ile183 resulted in lethal phenotypes, suggesting that the backbone may be more important than side group at this position. Proline/glycine mutagenesis was used to study additional sites in S2 and S3. The substitution of proline at Gly186 resulted in a lethal phenotype, whereas substitutions in S3 of proline or serine at Gly270 and proline or glycine at Thr287 resulted in viable mutants. Mutations G270P and T287P resulted in mutant enzymes that produced pronounced growth defects and ATP hydrolysis rates that were 35% and 60% lower than wild type enzyme, respectively. The mutant enzymes transported protons at rates consistent with their ATPase activity, suggesting that the growth defects observed were due to a reduced rate of ATP hydrolysis and not to uncoupling of proton transport. The prominent growth phenotypes produced by mutations G270P and T287P permitted the isolation of suppressor mutations, which restored wild type growth. Most of the suppressors either replaced the primary site mutation with alanine or restored the wild type residue by ectopic recombination with PMA2, both of which restore alpha-helical tendency. This study suggests that maintaining alpha-helical character is essential to S2 and may play an important role in S3.     

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.     

Molecular modeling of the transmembrane segment of Na+-K+-ATPase. A pentagonal model]

Retrograde differentiation (or dedifferentiation) has recently been proposed as a pathogenetic mechanism involved also in various renal diseases. Here we studied whether evidence of these mechanisms can be found in the kidneys of patients with congenital nephrotic syndrome of the Finnish type (CNF). These patients show isolated massive proteinuria but no primary symptoms from any other organ systems. For the analysis we used antibody markers of early (fibronectin, stem cell factor, Wilms' tumor gene product, cytokeratin) and later (laminin, midgestation and kidney, heparin binding growth-associated molecule) stages of nephron differentiation as well as for apoptosis (acridine orange staining), rescue from apoptosis (anti-Bcl-2 antibodies) and cell proliferation (antibodies to proliferating cell nuclear antigen). In the peritubular spaces atypically organized areas were found which appeared positive with markers of low stages of differentiation, but neither abnormal cell proliferation nor activation of the apoptotic pathway could be detected. As morphologic signs of abnormal tissue organization, we found clusters of tightly compacted and large glomeruli corresponding to the size of two to three normal glomeruli. However, all individual glomerular cell compartments (mesangial, endothelial, visceral epithelial cells) appeared balanced in relative cell numbers. Together these results may indicate abnormal early mesenchymoepithelial tissue interaction leading to excessive and poorly organized formation of glomeruli. This could be causally related also to the serious functional immaturity of CNF kidneys presented as isolated proteinuria.     

Differential exposure of surface epitopes in the beta-strand region of LOOP1 of the yeast H+-ATPase during catalysis

The plasma membrane H+-ATPase of yeast assumes distinct conformational states during its catalytic cycle. To better understand structural changes in the LOOP1 domain, a catalytically important cytoplasmic loop segment linking transmembrane segments 2 and 3, surface epitopes were examined at different stages of catalysis. A polyclonal rabbit antibody was prepared to a fusion protein consisting of LOOP1 and the maltose binding protein. This antibody was affinity-purified to produce a LOOP1-specific fraction that could be used in competition enzyme-linked immunosorbent assays to assess surface exposure of the LOOP1 epitopes. It was found that in an E1 conformation stabilized with either adenosine 5'-(beta,gamma -imino)triphosphate (AMP-PNP) or ADP, less than 10% of the LOOP1 epitopes were accessible on native enzyme. However, when the enzyme was stabilized in an E2-state with ATP plus vanadate, approximately 40% of the surface epitopes on LOOP1 became accessible to antibody. The remaining 60% of the LOOP1 epitopes were fully occluded in the native enzyme and never showed surface exposure. Enzyme-linked immunosorbent assays utilizing fusion proteins consisting of LOOP1 subdomains demonstrated that all of the available epitopes were contained in the beta-strand region (Glu-195-- Val-267) of LOOP1. The epitopes that were differentially exposed during catalysis were included in regions upstream and downstream of the highly conserved TGES sequence. Our results suggest that during catalysis either the beta-strand region of LOOP1 or an interacting domain undergoes substantial structural rearrangement that facilitates epitope exposure.     

Evidence for a H+ nitrate symporter in Aspergillus nidulans

Nitrate transport in Aspergillus nidulans was dependent upon a consistent proton motive force (delta p) across the cell membrane which was maintained in a range of 105 (+/- 6.7) to 131 (+/- 3.4) mV over an external pH span of 5.5 to 7.5. The membrane potential (delta psi) measured by uptake of [3H]-tetra-phenylphosphonium bromide and the transmembrane pH difference (delta pH) measured by the distribution of 3H2O and [14C]- salicylic acid were used to compute the delta p present during transport of nitrate. Energy dependent accumulation of nitrate was measured in actively assimilating and tungstate inhibited cells. A delta G for nitrate of 14 kJ mol-1 was computed from the results. Cells induced for nitrate transport maintained internal nitrate levels of 6 to 8 mM based on an internal volume of 2.6 microliters/mg dry wt as determined by a conventional dual label procedure. A fivefold higher level of cellular nitrate was observed in tungstate inhibited cells. Nitrate accumulation was dependent upon a H+ gradient which was dissipated by treatment with 2-butanol, the ionophores valinomycin and gramicidin and the proton conductors carbonyl cyanide m-chlorophenyl hydrazone and N,N'-dicyclo-hexylcarbodiimide. Significant ATP and nitrate efflux occurred in cells treated with the above agents. The results suggest that nitrate is transported by symport with H+ on a carrier which is functionally linked to a H+ ATPase pump.     

Intracellular sequestration of sodium by a novel Na+/H+ exchanger in yeast is enhanced by mutations in the plasma membrane H+-ATPase. Insights into mechanisms of sodium tolerance

Sodium tolerance in yeast is disrupted by mutations in calcineurin, a Ca2+/calmodulin-dependent protein phosphatase, which is required for modulation of Na+ uptake and efflux mechanisms. Five Na+-tolerant mutants were isolated by selecting for suppressors of calcineurin mutations, and mapped to the PMA1 gene, encoding the plasma membrane H+-ATPase. One mutant, pma1-alpha4, which has the single amino acid change Glu367 --> Lys at a highly conserved site within the catalytic domain of the ATPase, was analyzed in detail to determine the mechanism of Na+ tolerance. After exposure to Na+ in the culture medium, 22Na influx in the pma1 mutant was reduced 2-fold relative to control, consistent with a similar decrease in ATPase activity. Efflux of 22Na from intact cells was relatively unchanged in the pma1 mutant. However, selective permeabilization of the plasma membrane revealed that mutant cells retained up to 80% of intracellular Na+ within a slowly exchanging pool. We show that NHX1, a novel gene homologous to the mammalian NHE family of Na+/H+ exchangers, is required for Na+ sequestration in yeast and contributes to the Na+-tolerant phenotype of pma1-alpha4.     

Evidence for the interplay between trehalose metabolism and Hsp104 in yeast

Disruption of the HSP104 gene in a mutant which cannot accumulate trehalose during heat shock treatment caused trehalose accumulation (H. Iwahashi, K. Obuchi, S. Fujii, and Y. Komatsu, Lett. Appl. Microbiol 25:43-47, 1997). This implies that Hsp104 affects trehalose metabolism. Thus, we measured the activities of enzymes involved in trehalose metabolism. The activities of trehalose-synthesizing and -hydrolyzing enzymes are low in the HSP104 disruption mutant during heat shock. This data is correlated with intracellular trehalose and glucose levels observed in the HSP104 disruption mutant. These results suggest that during heat shock, Hsp104 contributes to the simultaneous increase in both accumulation and degradation of trehalose.     

The PLC1 encoded phospholipase C in the yeast Saccharomyces cerevisiae is essential for glucose-induced phosphatidylinositol turnover and activation of plasma membrane H+-ATPase

Addition of glucose to glucose-deprived cells of the yeast Saccharomyces cerevisiae triggers rapid turnover of phosphatidylinositol, phosphatidylinositol-phosphate and phosphatidylinositol 4,5-bisphosphate. Glucose stimulation of PI turnover was measured both as an increase in the specific ratio of 32P-labeling and as an increase in the level of diacylglycerol after addition of glucose. Glucose also causes rapid activation of plasma membrane H+-ATPase. We show that in a mutant lacking the PLC1 encoded phospholipase C, both processes were strongly reduced. Compound 48/80, a known inhibitor of mammalian phospholipase C, inhibits both processes. However, activation of the plasma membrane H+-ATPase is only inhibited by concentrations of compound 48/80 that strongly inhibit phospholipid turnover. Growth was inhibited by even lower concentrations. Our data suggest that in yeast cells, glucose triggers through activation of the PLC1 gene product a signaling pathway initiated by phosphatidylinositol turnover and involved in activation of the plasma membrane H+-ATPase.