Inhibition of the Ca2+-ATPase Pmc1p by the v-SNARE Protein Nyv1p*

Yoko Takita, Laura Engstrom, Christian UngermannDagger , and Kyle W. Cunningham§

From the Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218 and Dagger  Biochemie-Zentrum Heidelberg, Universität Heidelberg, D-69120 Heidelberg, Germany

Received for publication, October 9, 2000, and in revised form, October 27, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Pmc1p, the Ca2+-ATPase of budding yeast related to plasma membrane Ca2+-ATPases of animals, is transcriptionally up-regulated in response to signaling by the calmodulin-calcineurin-Tcn1p/Crz1p signaling pathway. Little is known about post-translational regulation of Pmc1p. In a genetic screen for potential negative regulators of Pmc1p, a vacuolar v-SNARE protein, Nyv1p, was recovered. Cells overproducing Nyv1p show decreased Ca2+ tolerance and decreased accumulation of Ca2+ in the vacuole, similar to pmc1 null mutants. Overexpression of Nyv1p had no such effects on pmc1 mutants, suggesting that Nyv1p may inhibit Pmc1p function. Overexpression of Nyv1p did not decrease Pmc1p levels but decreased the specific ATP-dependent Ca2+ transport activity of Pmc1p in purified vacuoles by at least 2-fold. The effect of Nyv1p on Pmc1p function is likely to be direct because native immunoprecipitation experiments showed that Pmc1p coprecipitated with Nyv1p. Complexes between Nyv1p and its t-SNARE partner Vam3p were also isolated, but these complexes lacked Pmc1p. We conclude that Nyv1p can interact physically with Pmc1p and inhibit its Ca2+ transport activity in the vacuole membrane. This is the first example of a Ca2+-ATPase regulation by a v-SNARE protein involved in membrane fusion reactions.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Ca2+ plays an important role as a signaling molecule for cell growth. In eukaryotic cells, cytosolic free Ca2+ concentration, ([Ca2+]c), is typically maintained at submicromolar levels against millimolar free Ca2+ concentrations in the lumen of secretory organelles and extracellular spaces. Such steep gradients are generated and maintained by Ca2+-pumping ATPases and ion exchangers, which offset the effects of Ca2+ channels and nonspecific leaks in the membrane. The regulated opening and closing of Ca2+ channels in cellular membranes contribute to [Ca2+]c modulation with intricate temporal and spatial resolution, permitting the use of cytosolic Ca2+ as a regulator of numerous cellular processes such as metabolism, gene expression, and membrane fusion.

In the budding yeast Saccharomyces cerevisiae, large lysosome-like vacuoles serve as the major intracellular Ca2+ reservoir, accumulating ~95% of the total cell-associated Ca2+ (1, 2). As listed in Table I, two vacuolar Ca2+ transporters have been identified, the Ca2+-ATPase Pmc1p and the H+·Ca2+ exchanger Vcx1p (3-5). Deletion of the PMC1 gene decreases the ability to grow in high Ca2+ environments, while deletion of VCX1 decreases Ca2+ tolerance only slightly, suggesting that Pmc1p normally plays a more significant role in vacuolar Ca2+ sequestration. Ca2+ stored in the vacuole can bind inorganic polyphosphates in the lumen and precipitate thereby increasing the capacity for Ca2+ sequestration (2). Recently, release of Ca2+ from the vacuole was found to be important for homotypic fusion of vacuole membranes in vitro (6). Homotypic vacuole fusion involves membrane-bound (SNAREs)1 including the vesicle v-SNAREs Nyv1p, Vti1p, and Ykt6p, the target membrane t-SNARE Vam3p, and Vam7p (7-9). At a late step in the homotypic fusion pathway, Ca2+ is released from the vacuole causing a local elevation in [Ca2+]c, which triggers fusion of docked membranes through a calmodulin-dependent process (6). However, vacuoles lacking both Pmc1p and Vcx1p were fully active in homotypic fusion assays (10). No other roles of vacuolar Ca2+ transport have been identified to date.


                              
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Table I
Yeast proteins, functions, and homologs

The endoplasmic reticulum and the Golgi complex of yeast accumulate Ca2+ via the Ca2+/Mn2+ ATPase Pmr1p, which is important for a variety of secretory functions (11-15). Mutants lacking Pmr1p exhibit high rates of Ca2+ influx and elevation of [Ca2+]c due to stimulation of a plasma membrane Ca2+ channel composed of Cch1p and Mid1p (16). This process resembles the capacitative calcium entry mechanisms in animal cells where depletion of secretory Ca2+ pools promotes Ca2+ influx through the plasma membrane channels and refilling of the depleted organelles (17). In yeast, excessive activity of the vacuolar Ca2+ transporters Pmc1p and Vcx1p can compete with Pmr1p for Ca2+ and activate the capacitative calcium entry-like mechanism (16). Therefore, the activity of vacuolar Ca2+ transporters must be balanced with Pmr1p activity to avoid depletion of secretory organelles and inefficient use of energy for Ca2+ sequestration in the vacuole.

Extensive studies have revealed that all three Ca2+ transporters in yeast are regulated by a signaling network involving calcineurin, a protein phosphatase that becomes activated by binding Ca2+ and calmodulin upon elevation of [Ca2+]c. Genetic studies suggest that Vcx1p may be inhibited by calcineurin at a post-translational level (4). Transcription of PMC1 and PMR1 is increased upon calcineurin-dependent activation of the transcription factor Tcn1p/Crz1p (18, 19). In high Ca2+ environments, the strong up-regulation of Pmc1p is necessary for proliferation (18). However, when environmental Ca2+ concentrations subside, the excess Pmc1p activity might inhibit normal maintenance of Ca2+ in secretory organelles. Negative regulation of Pmc1p activity may, therefore, be important under these and other conditions. To date, no negative regulators of Pmc1p have been identified.

In this study, we screened for negative regulators of Pmc1p in vivo and identified Nyv1p, a transmembrane v-SNARE protein in the vacuole membrane, as a likely candidate. We found no obvious role of Pmc1p or Nyv1p in vacuole morphology or fusion in vivo. Rather, Nyv1p bound to Pmc1p and inhibited its Ca2+ transport activity in vivo and in vitro. Thus, a new role of Nyv1p in yeast may be the regulation of Pmc1p activity and Ca2+ homeostasis.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Yeast Strains, Plasmids, and Growth Conditions-- Standard culture media for yeast and Escherichia coli have been described previously (20). All yeast strains listed in Table II are derivatives of strain W303-1A (21), which were constructed through transformation and isogenic crosses using standard techniques (22). The nyv1::HIS3Sp null mutation was introduced into the wild type strain K601 by transformation with a polymerase chain reaction product amplified from the genomic DNA of yeast strain SEY6210Delta nyv1 (7) using flanking primers that hybridize at nucleotide -457 and nucleotide +1109 relative to the initiation codon of NYV1. The vam3::HIS3 null mutation was introduced to strain K601 by transformation with XbaI-digested plasmid pYVQ311 (23). The mutations were confirmed by polymerase chain reaction analysis, Western blot analysis, and/or observation of vacuole morphology.


                              
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Table II
Yeast strains used in this study
All strains are isogenic to W303-1A (21) and carry additional markers: MATalpha ade2-1 can1-100 his3-11, 15 leu2-3, and 112 trp1-1 ura3-1.

A pmr1 cch1 strain (ELY106) was transformed with a YEp13-based library of yeast genomic DNA (gift from Kim Nasmyth, Vienna, Austria), and transformants were replica plated onto Whatman No. 3MM filter papers that had been placed on the surface of agar medium containing YPD medium supplemented with 5 mM succinic acid, 10 mM CaCl2, 10 mM MgCl2, and 0.3 mM adenine. After growth overnight at 30 °C, the filters were removed and stained for beta -galactosidase activity as described previously (18). Among 8000 transformants, 18 colonies expressed PMC1-lacZ at higher levels than in controls. The plasmids were isolated from these strains, and only eight were found to be positive on retesting. Each plasmid was sequenced at both ends of the genomic DNA insert to identify the genes included. One plasmid, pLE66, contained the TCN1/CRZ1 gene encoding the calcineurin-dependent transcription factor involved in PMC1-lacZ induction (18, 19). Another plasmid, pLE8, carried a genomic DNA insert spanning three complete genes, SUL2, NYV1, and GIS3. In a parallel screen using a pmr1 mid1 strain and a low-copy pRS313-based library of genomic DNA (gift from David Levin, Johns Hopkins University), a plasmid carrying SUL2 and NYV1 genes was isolated as a stimulator of PMC1-lacZ expression in the pmr1 mid1 cells. Subcloning showed the NYV1 gene alone conferred the phenotype of increased PMC1-lacZ expression. The first subclone, plasmid pNYV-HIS, carried 2.5 kilobase pairs of genomic DNA surrounding NYV1 (from the SmaI site at nucleotide -191 to a Sau3A site at nucleotide +2315 relative to initiation codon) ligated into the SmaI site of pYO323 (24). The second subclone, plasmid pNYV-LEU, carried a 1.4-kilobase-pair SmaI-EcoRV fragment of the NYV1 locus ligated into the SmaI site of pYO325 (24). Plasmids pPI12 and pSK60 have been described previously (25).

Measurement of Ca2+ Pools-- Total cell-associated Ca2+ was determined for yeast strains grown at 30 °C for 4 h in YPD medium supplemented with 20 µCi/ml as described previously (4). The nonexchangeable Ca2+ pool was measured by a similar protocol except that the cultures were diluted 5-fold with fresh YPD medium containing 20 mM CaCl2 and incubated an additional 20 min at 30 °C prior to harvesting by filtration. The exchangeable Ca2+ pool represents the difference between total Ca2+ and nonexchangeable Ca2+.

Ca2+ Tolerance Assays-- Yeast cells were grown overnight at 30 °C in YPD (pH 5.5) medium or half-concentrated synthetic complete minus leucine medium. Saturated cell suspensions were then diluted 100-fold into 0.2 ml of the same medium supplemented with various concentrations of CaCl2 and incubated in flat-bottom 96-well dishes for 20 h at 30 °C without shaking. The optical density of each culture was measured at 650 nm using a microplate spectrophotometer (Molecular Devices).

Immunoprecipitation and Immunoblotting-- One hundred µg of purified vacuoles prepared as described previously (26) were solubilized with 500 µl of lysis buffer (50 mM Tris-HCl, pH 8.0, 2 mM EDTA, 150 mM NaCl, 0.5% Tween 20, and protease inhibitors), incubated with anti-Nyv1p antiserum (9) for 1 h at 4 °C, and then treated with protein A beads for an additional 1 h at 4 °C. The protein A beads were collected by centrifugation and washed three times with lysis buffer. The proteins bound to the beads were separated by SDS-polyacrylamide gel electrophoresis, transferred to polyvinylidene difluoride membranes (Millipore), decorated with 12CA5 monoclonal antibodies specific for the HA tag (Roche Molecular Biochemicals) or polyclonal antibodies specific for Nyv1p or Vam3p (8). For immunoprecipitations, after priming the reaction with ATP, vacuoles were first suspended in 750 µl of reaction buffer containing cytosol and ATP, incubated at 27 °C, washed with 500 µl of PS buffer (10 mM PIPES-KOH, pH 6.8, and 200 mM sorbitol) and then solubilized, treated with antibodies to Nyv1p or Vam3p, and processed as described (8).

Pmc1p Activity Assay-- Purified vacuoles were suspended at 32 µg/ml in reaction buffer (10 mM PIPES-KOH, pH 6.8, 200 mM sorbitol, 2 mM MgCl2, and 100 mM KCl) containing 10 µCi/ml 45CaCl2 plus varying concentrations of nonradioactive CaCl2 and EGTA. Free Ca2+ concentrations were calculated using MaxChelator.2 Reactions were prewarmed to 30 °C and initiated by the addition of either 1 mM ATP or 2.5 mM ADP. After 1 min of incubation, vacuoles were collected by rapid filtration onto 0.45-µm nitrocellulose filters (type HA, Millipore) and washed three times with the ice-cold buffer A (5 mM HEPES-NaOH, pH 6.5, and 10 mM CaCl2), and the associated radioactivity was determined by liquid scintillation counting. Time course experiments showed that Ca2+ transport reactions were linear for at least 2 min. ATP-dependent Ca2+ transport was calculated by subtracting the values obtained using ADP from those obtained using ATP.

Staining and Microscopy-- Microscopic observation of yeast vacuoles in vivo was carried out by labeling with FM4-64 (Molecular Probes). Log-phase cells were incubated in 50 µl of YPD containing 20 µg/ml FM4-64 for 20 min at 30 °C. Cells were washed three times with fresh YPD, resuspended in 1 ml of YPD, incubated for an additional 60 min, and observed using a Zeiss Axiovert microscope equipped with 100× objectives.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Genetic Analysis of NYV1 Function in Ca2+ Homeostasis-- Mutants lacking the Golgi Ca2+-ATPase Pmr1p maintain [Ca2+]c at elevated levels in part due to increased Ca2+ influx via the Cch1p-Mid1p Ca2+ channel, resulting in elevated expression of the calcineurin-dependent reporter gene PMC1-lacZ (16, 27, 28). The up-regulated Pmc1p helps lower [Ca2+]c to levels that permit proliferation (3). We reasoned that a hypothetical inhibitor of Pmc1p, when overexpressed in pmr1 mutants, would cause further elevation of [Ca2+]c and PMC1-lacZ expression and possibly decrease proliferation. There would be no such effects in wild type cells because Pmr1p can maintain [Ca2+]c and PMC1-lacZ expression at low levels even in the absence of Pmc1p and Vcx1p (4). To identify potential inhibitors of Pmc1p, a high-dosage plasmid library of yeast genomic DNA was screened for clones that elevated PMC1-lacZ expression in colonies of a pmr1 cch1 double mutant grown in optimized medium (see "Experimental Procedures"). The pmr1 cch1 double mutant was used instead of a pmr1 single mutant because the latter strain exhibits high background staining with X-gal, whereas background staining was much lower in the former (16). As expected under these screening conditions, most of the colonies stained light blue with X-gal due to low expression of PMC1-lacZ, although several stained darker blue. One of the plasmids was isolated and found to carry the NYV1 gene plus flanking genes. Subcloning revealed that the NYV1 gene itself, when overexpressed, was necessary and sufficient to increase PMC1-lacZ expression in pmr1 cch1 mutants. A second screen employing pmr1 mid1 double mutants also recovered a plasmid bearing NYV1.3 The NYV1 gene encodes a v-SNARE protein that localizes to the vacuole membrane and is required for homotypic fusion of vacuoles in vitro (7). These findings suggest that Nyv1p might affect vacuolar Ca2+ homeostasis.

To determine whether NYV1 overexpression alters accumulation of Ca2+ in the vacuole, the nonexchangeable Ca2+ pool was quantitated in a variety of yeast strains after prolonged growth in medium containing 45Ca2+ as a tracer. As seen previously (4), the nonexchangeable Ca2+ pool was very large in wild type cells and vcx1 mutants but was greatly diminished in pmc1 mutants and pmc1 vcx1 double mutants (Fig. 1). Overexpression of NYV1 significantly decreased the nonexchangeable Ca2+ pool in wild type and vcx1 mutants but had no significant effect in either pmc1 mutants or pmc1 vcx1 double mutants. In all these strains, there was no significant effect of NYV1 overexpression on the levels of exchangeable Ca2+ (data not shown). These results suggested that Nyv1p overexpression diminishes Pmc1p activity but does not abolish it. To determine whether the effect of Nyv1p required Vam3p, a vacuolar t-SNARE that forms complexes with Nyv1p (8), similar measurements of nonexchangeable Ca2+ pools were performed on vam3 mutants. For unknown reasons, the nonexchangeable Ca2+ pools were consistently elevated ~2-fold in vam3 mutants relative to wild type, independently of Vcx1p and Pmc1p (Fig. 1). Overexpression of NYV1 decreased the nonexchangeable Ca2+ pool in vam3 vcx1 double mutants but increased this pool in pmc1 vam3 vcx1 triple mutants. A close comparison of these two mutant strains indicated that the Pmc1p-dependent activity was significantly diminished by increased NYV1 dosage. Thus, NYV1 overexpression decreased Ca2+ accumulation in the vacuoles of yeast cells by a process that was independent of Vcx1p and Vam3p but dependent on Pmc1p. The results are consistent with a model in which Nyv1p directly or indirectly inhibits the Ca2+ transport activity of Pmc1p in the vacuole, independently of fusion.



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Fig. 1.   Nyv1p overexpression diminishes Pmc1p-dependent Ca2+ accumulation in the vacuole. The nonexchangeable Ca2+ pool was determined in triplicate for a variety of yeast strains with or without overexpression of Nyv1p after long term growth in YPD medium containing 45Ca2+ as a tracer (see "Experimental Procedures"). Relative to control (plasmid pYO325 (vector)), overexpression of Nyv1p (plasmid pNYV-LEU (2µ-NYV1)) significantly decreased the nonexchangeable Ca2+ pool in strains containing Pmc1p (wild type (WT), strain K601; vcx1, strain K661; and vcx1 vam3, strain YTY51) but not in strains lacking Pmc1p (pmc1, K605; vcx1 pmc1, K665; and vcx1 vam3 pmc1, YTY52).

Pmc1p is essential for growth in high calcium environments (3). Therefore, we tested whether NYV1 overexpression could also diminish Ca2+ tolerance in a Pmc1p-dependent fashion. Overexpression of NYV1 greatly decreased Ca2+ tolerance of vcx1 mutants almost to that of pmc1 vcx1 double mutants (Fig. 2A). Overexpression of NYV1 in pmc1 vcx1 double mutants had little effect on Ca2+ tolerance. As a control for specificity, we also tested the effects of overexpressing VTI1 and YKT6 genes encoding v-SNARE proteins related to Nyv1p, recently implicated in vacuole fusion reactions (9). There was no detectable effect of overexpressing the related v-SNARE proteins in either vcx1 mutants or pmc1 vcx1 double mutants (Fig. 2B). Therefore, the decreased Ca2+ tolerance observed upon NYV1 overexpression can be attributed mostly to the effects on Pmc1p function.



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Fig. 2.   Nyv1p diminishes Pmc1p-dependent Ca2+ tolerance. Optical density was measured at 650 nm for cultures grown for 20 h in either synthetic medium (A and B) or rich medium (C) as described under "Experimental Procedures." Yeast strains analyzed were vcx1 (K661), vcx1 pmc1 (K665), vcx1 nyv1 (YTY5), and vcx1 pmc1 nyv1 (YTY7) harboring plasmids pYO323 [-], pNYV-HIS ([2µ-NYV1]), pPI12 [2µ-VTI1], and pSK60 ([2µ-YKT6]) as indicated.

The above experiments show significant effects of NYV1 overexpression on Pmc1p function. To test whether Nyv1p at native levels can also affect Pmc1p function, we analyzed the phenotype of nyv1 knockout mutants in Ca2+ tolerance assays. The nyv1 null mutant strain exhibited slightly greater Ca2+ tolerance than an isogenic wild type strain (Fig. 2C). These results were also reproducible in the s288c strain background obtained from Research Genetics, Inc. (data not shown). Thus, consistent with effects of Nyv1p overexpression, endogenous levels of Nyv1p appeared to limit Pmc1p function in Ca2+ tolerance. Growth in high Ca2+ conditions had no effect on the abundance of Nyv1p or the expression of a NYV1-lacZ reporter gene (data not shown). We suggest that a relatively static level of Nyv1p can partially inhibit Pmc1p function in vivo and that the up-regulation of Pmc1p in high Ca2+ conditions may overcome this inhibition.

Physical Interactions between Nyv1p and Pmc1p-- Because Nyv1p and Pmc1p are both components of the vacuole membrane and interact functionally, we hypothesized that they might also physically interact. To test this possibility, Nyv1p was immunoprecipitated from purified vacuoles dissolved in a nondenaturing detergent, and coprecipitating proteins were analyzed by Western blotting. A polyclonal antibody that specifically recognizes Nyv1p but not Nyv1p-Vam3p complexes was found to coprecipitate Pmc1p-HA, a functional epitope-tagged derivative of Pmc1p (Fig. 3A, lane 1). No Pmc1p-HA coprecipitated with the antibody when nyv1 mutants were employed (lane 2). Incubation of purified vacuoles in conditions that support homotypic fusion slightly increased the abundance of Nyv1p-Pmc1p complexes (Fig. 3B, lanes 4-6). No Vam3p was coprecipitated with the Nyv1p or Nyv1p-Pmc1p complexes using the anti-Nyv1p antibody. Furthermore, antibodies specific for Vam3p coprecipitated Nyv1p but not Pmc1p (lanes 1-3). These results show that Nyv1p-Pmc1p complexes form in the vacuole membrane and can be purified away from other components of the vacuole membrane such as Nyv1p-Vam3p complexes. Therefore, a novel cellular role of Nyv1p may be to inhibit the Ca2+ transport activity of Pmc1p by direct or indirect physical interaction.



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Fig. 3.   Pmc1p coimmunoprecipitates with Nyv1p. A, polyclonal anti-Nyv1p antibodies were employed in native immunoprecipitation experiments after detergent solubilization of isolated vacuoles. Immunoprecipitates derived from the different yeast strains were then analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting using anti-HA monoclonal antibodies to detect Pmc1p-HA (upper panel) or rabbit anti-Nyv1p polyclonal antibodies (lower panel). B, the purified vacuoles were incubated for 0 to 30 min in conditions supporting homotypic fusion, immunoprecipitated using either anti-Vam3p or anti-Nyv1p polyclonal antibodies, and processed for Western blotting using either anti-HA monoclonal antibodies to detect Pmc1p-HA (top two panels, light and dark exposure), anti-Vam3p, or anti-Nyv1p polyclonal antibodies (bottom two panels). Each panel was obtained from the same samples loaded on separate gels. Yeast strains used to isolate vacuoles were PMC1-HA (K699), nyv1 PMC1-HA (YTY25), wild type (WT) (K601), and nyv1 (YTY1) in panel A and PMC1-HA (K699) in panel B.

Effects of Nyv1p on Pmc1p Activity-- The decreased function of Pmc1p observed upon overexpression of Nyv1p may be due to increased Pmc1p degradation, decreased catalytic activity, or a combination of these or other effects. To distinguish the possibilities, we first examined the levels of Pmc1p in whole cell extracts of strains with or without NYV1 overexpression by Western blot analysis. In all cases, the levels of Pmc1p were similar (Fig. 4). Thus, Nyv1p appeared to have little or no effect on Pmc1p expression. Therefore, we examined Ca2+ transport activity of Pmc1p in purified vacuoles isolated from vcx1 mutants carrying or lacking the NYV1 overexpression plasmid. For these experiments, the initial rates of ATP-dependent 45Ca2+ accumulation were determined at a variety of free Ca2+ concentrations set with EGTA buffers, and the data were fit to the Michaelis-Menten equation (see "Experimental Procedures"). ATP-dependent Ca2+ transport was undetectable in vacuoles obtained from a pmc1 vcx1 double mutant (data not shown) but was readily detectable in the vcx1 mutant expressing Pmc1p-HA (Fig. 5A). The apparent Km for Ca2+ was calculated to be ~4.3 µM, somewhat higher than that of Pmr1p (13). Vacuoles isolated from the same strain but overexpressing NYV1 strain also exhibited a Pmc1p- and ATP-dependent Ca2+ transport activity with apparent Km similar to the control; however, the maximal transport activity was diminished to ~43%. Both vacuole preparations contained equivalent amounts of Pmc1p-HA as determined by Western blotting (Fig. 5B). A similar decrease in Pmc1p specific activity was observed in three independent experiments. The results suggest that Nyv1p decreases the Ca2+ transport activity of Pmc1p by at least 2-fold.



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Fig. 4.   Pmc1p levels are unchanged in nyv1, wild type, and Nyv1p-overexpressing strains. Total cell extracts were prepared and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting from strains nyv1 PMC1-HA (YTY25) (lane 1), PMC1-HA (K699 [pYO323]) (lane 2), PMC1-HA 2 µ-NYV1 (K699 [pNYV-HIS]) (lane 3), and wild type (K601 [pYO323]) (lane 4) that had been grown to mid-log phase in synthetic complete medium. Note that the anti-Nyv1p antibodies cross-react with unknown proteins (*) independently of Nyv1p.



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Fig. 5.   ATP-dependent Ca2+ transport activity of Pmc1p in purified vacuoles is inhibited by overexpressed Nyv1p. A, initial rates of ATP-dependent Ca2+ transport were measured at a variety of free Ca2+ concentrations using purified vacuoles from a vcx1 PMC1-HA strain (YTY28) carrying either a pYO323 ([-]) or pNYV-HIS ([2µ-NYV1]) as described under "Experimental Procedures." The apparent Vmax was calculated to be 28.8 and 12.5 pmol Ca2+/min/mg, respectively, by nonlinear regression using the Michaelis-Menten equation (solid and dashed lines). B, the samples used in A were analyzed for Pmc1p-HA abundance by Western blotting. Ten µl of the samples were loaded. A similar decrease in Pmc1p specific activity upon Nyv1p overexpression was observed in three independent experiments.

Vacuole Morphology in Vivo-- The above results suggest that inhibition of Pmc1p activity is a physiological function of Nyv1p. Does Pmc1p also regulate vacuole fusion reactions? To address this question, the morphology of vacuoles in pmc1 mutants was examined after staining the vacuole membranes of live cells with the fluorescent dye FM4-64 (29). In the W303 strain background, the vacuole morphologies of nyv1 mutant cells and pmc1 mutant cells were indistinguishable from that of wild type cells (Fig. 6). In contrast, vam3 mutants exhibited fragmented vacuoles typical of defects in homotypic and heterotypic fusion. Unlike vam3 mutants, pmc1 and nyv1 mutants efficiently targeted carboxypeptidase Y to the vacuole (7, 30) (data not shown). Thus, pmc1 and nyv1 mutants had no obvious defects in either endocytosis of FM4-64 to the vacuole, trafficking of vacuolar proteins, or vacuole fusion and inheritance. That nyv1 mutants have no phenotypes other than those involving Ca2+ homeostasis may suggest that regulation of Pmc1p function is a primary cellular function of this v-SNARE protein.



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Fig. 6.   Vacuole morphology is unaffected by inactivation of Nyv1p or Pmc1p but disrupted by inactivation of Vam3p. Mid-log phase cells were stained with the membrane dye FM4-64 as described under "Experimental Procedures" and then visualized by epifluorescence microscopy (panels a, c, e, and g) and phase contrast microscopy (panels b, d, f, and h). The imaged strains were wild type (WT) (K601), pmc1 mutant (K605), nyv1 mutant (YTY1), and vam3 mutant (YTY49).



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

This study reports the first evidence of a v-SNARE protein interacting physically and functionally with a Ca2+-ATPase in vivo. We found that Pmc1p specifically coprecipitated with Nyv1p and that the ATP-dependent Ca2+ transport activity of Pmc1p in purified vacuole preparations was significantly decreased upon overexpression of Nyv1p. The phenotypes of yeast cells lacking or overexpressing Nyv1p confirm this model. Overexpression of Nyv1p decreased 45Ca2+ accumulation in the nonexchangeable (vacuolar) pool and decreased Ca2+ tolerance only when Pmc1p was present. Likewise, inactivation of Nyv1p increased Ca2+ tolerance in a Pmc1p-dependent fashion. Taken together, the data support a model in which interaction with Nyv1p decreases Pmc1p activity at least 2-fold. Nyv1p appeared to affect the Vmax rather than Km for Ca2+ in this reaction, as if it inactivates a subset of Pmc1p molecules. The data do not rule out the alternative possibility that Nyv1p partially inhibits the activity of all Pmc1p molecules. That Nyv1p levels were constant in low and high Ca2+ environments4 suggests that the strong up-regulation of Pmc1p expression during growth in high Ca2+ conditions may serve to overcome the inhibitory effect of Nyv1p, further promoting Ca2+ tolerance.

The interaction between Pmc1p and Nyv1p raises questions about the role of Ca2+ in fusion of vacuole membranes. Nyv1p and Vam3p were shown to be required in trans for homotypic vacuole-vacuole fusion in a reconstituted system (7). Furthermore, one report has suggested that trans-pairing of Nyv1p and Vam3p on docked vacuoles triggers Ca2+ release and local elevation of [Ca2+]c, which was essential for stimulating calmodulin-dependent reactions leading to bilayer fusion (6). The vacuolar Ca2+ release channel has not yet been identified, but the roles of Pmc1p and Vcx1p in homotypic fusion have been examined. Vacuoles lacking both Pmc1p and Vcx1p were found to be fully competent for homotypic fusion and even more competent for fusion than wild type vacuoles under some conditions (10). However, this observation sheds little light on the role of Ca2+ in fusion because the pmc1 vcx1 mutant vacuoles would likely contain lower lumenal Ca2+ available for triggering fusion but permit higher free Ca2+ levels to be reached in the suspension buffer. Our finding that Pmc1p associates with Nyv1p but not Vam3p (Fig. 3) has several possible implications for homotypic vacuole fusion. First, Pmc1p may serve as a reservoir (or buffer) for unpaired Nyv1p, thus increasing (or decreasing) Nyv1p availability for eventual trans-SNARE pairing and fusion. This role may be analogous to that of synaptophysin in synaptic vesicles, which forms complexes with the v-SNARE protein synaptobrevin/VAMP (see below). Second, trans-SNARE pairing may dissociate active Pmc1p from complexes with Nyv1p thereby increasing Ca2+ transport activity near the sites of membrane fusion. In this view, the cytoplasm in the vicinity of fusion sites may be restored rapidly to resting concentrations of Ca2+ after fusion or possibly depleted of Ca2+ just before fusion. Alternatively, the Pmc1p-Nyv1p interaction might not facilitate the fusion process to any significant degree, but instead it may simply serve to regulate Ca2+ homeostasis. To resolve all these possibilities, it will be necessary to determine the affinity and dynamics of the Pmc1p-Nyv1p interaction under a variety of conditions and to identify its effects on subreactions of the fusion pathway.

Unlike vam3 mutants and all other mutants defective in homotypic fusion, nyv1 mutants fail to exhibit any significant vacuolar phenotype unrelated to Ca2+. Trafficking of proteins to the vacuole by any of three distinct routes was shown to be normal in nyv1 mutants but strongly disrupted in vam3 mutants (30). Furthermore, the morphology and inheritance of vacuoles in nyv1 mutants closely resembles that of wild type cells in contrast to vam3 mutants (Fig. 6) (7). Finally, using an in vivo assay for homotypic fusion during mating we found that fusion of vacuoles in nyv1/nyv1 zygotes was indistinguishable from wild type but disrupted in vam3/vam3 zygotes.4 It is possible that the in vivo assay for vacuole fusion reflects a distinct form of homotypic fusion rather than that which has been reconstituted in vitro. It is also possible that other v-SNARE proteins in yeast, such as Vti1p and Ykt6p (9), functionally substitute for Nyv1p in vivo but somehow fail to do so in vitro. Currently there is little or no evidence from in vivo studies that Nyv1p plays important roles in either homotypic or heterotypic fusion processes. The strongest phenotypes of nyv1 mutants and NYV1-overexpressing strains to date are those reported here involving effects on Pmc1p and Ca2+ homeostasis.

Pmc1p belongs to the family of Ca2+-ATPases that includes the plasma membrane Ca2+-ATPases (PMCAs) of animals (3). The animal PMCAs are localized almost exclusively to the plasma membrane and carry a C-terminal auto-inhibitory extension that can be relieved upon binding of Ca2+/calmodulin (31). The homologous proteins from fungi, plants, and protozoa apparently lack this C-terminal extension and are frequently localized to vacuoles or other intracellular organelles (32, 33). How the nonanimal PMCA-type Ca2+-ATPases are regulated in vivo remains an interesting unanswered question. Sequences homologous to Nyv1p can be found in all the species containing PMCA-type proteins, raising the possibility that the interaction identified here occurs broadly in nature.

Ca2+ fluxes are well known to regulate heterotypic fusion events, such as the fusion of synaptic vesicles to the plasma membrane at synapses. In this case, the N-type and P/Q-type Ca2+ channels in the presynaptic plasma membrane bind the t-SNARE protein syntaxin and the s-SNARE SNAP-25, which can be bound to synaptotagmin, and the v-SNARE synaptobrevin/VAMP, located on docked synaptic vesicles (34). A second pool of synaptobrevin/VAMP in synaptic vesicles binds to synaptophysin, a polytopic membrane protein that is thought to sequester or buffer the v-SNARE from pairing with the t-SNARE proteins (35). The interaction between Pmc1p and Nyv1p in yeast vacuoles may resemble the interaction between synaptophysin and synaptobrevin/VAMP. Currently there is no evidence that synaptobrevin/VAMP interacts with PMCAs in neurons or any other cell type. However, a large fraction of total cellular synaptobrevin/VAMP has recently been localized to the active zones of the presynaptic membrane (36) where PMCAs also reside (37). The generality of the v-SNARE interactions with Ca2+-ATPases in nature remains to be determined.


    ACKNOWLEDGEMENTS

We thank David Levin, Hugh Pelham, Yoh Wada, and Gerry Waters for plasmids and yeast strains; Rajini Rao and Debjani Mandel for technical comments; and all the members of our laboratory for helpful comments and critical reading of the manuscript.


    FOOTNOTES

* 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.

§ To whom correspondence should be addressed: Dept. of Biology, Johns Hopkins University, 3400 N. Charles St., Baltimore, MD 21218. Tel: 410-516-7844; Fax: 410-516-5213; E-mail: kwc@jhu.edu.

Published, JBC Papers in Press, November 15, 2000, DOI 10.1074/jbc.M009191200

2 Contact corresponding author for Web address.

3 E. G. Locke and K. W. Cunningham, unpublished data.

4 Y. Takita and K. W. Cunningham, unpublished observations.

5 Y. Takita, L. Engstrom, C. Ungermann, and K. W. Cunningham, unpublished observations.


    ABBREVIATIONS

The abbreviations used are: SNAREs, soluble N-ethylmoleimide-sensitive fusion protein (NSF) attachment protein reseptors; YPD, yeast extract/peptone/dextrose; HA, hemagglutinin; X-gal, 5-bromo- 4-chloro-3-indolyl-beta -D-galactopyranoside; PMCA, plasma membrane Ca2+-ATPase; PIPES, 1,4-piperazinediethanesulfonic acid.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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