INTRODUCTION

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The secretory compartments of eukaryotic cells require high concentrations of calcium ions (Ca²⁺) for the activities of numerous enzymes that catalyze the folding, modification, processing, and trafficking of secretory proteins. Typically, Ca²⁺ is pumped from the cytosol directly into the endoplasmic reticulum and related secretory compartments by the ATP-dependent SERCA-type Ca²⁺ pumps. Depending on the inherent leakiness of each compartment to Ca²⁺, inhibitors of SERCA pumps can lead to depletion of Ca²⁺ in the secretory pathway and a variety of secretory defects. Most cells express Ca²⁺ release channels in the endoplasmic reticulum that can be activated by second messengers during responses to extracellular stimuli. Rapid Ca²⁺ release lowers Ca²⁺ in the endoplasmic reticulum and elevates free Ca²⁺ concentrations in the cytosol ([Ca²⁺]_c), which then can activate various signaling transduction pathways. Because Ca²⁺ pumps in the plasma membrane (PMCAs) compete with SERCA pumps for substrates, [Ca²⁺]_c can return to basal levels prior to refilling of secretory compartments. Thus, in the absence of Ca²⁺ influx into the cell, repetitive or continuous activation of Ca²⁺ release channels will lead to only transient elevation of [Ca²⁺]_c and sustained depletion of the secretory Ca²⁺ reservoir. To offset the detrimental effects of Ca²⁺ efflux, most cell types employ a regulatory mechanism known as capacitative calcium entry (CCE) which stimulates Ca²⁺ influx specifically in response to depletion of Ca²⁺ from the endoplasmic reticulum (45, 46). Thus, CCE increases the magnitude and duration of calcium signals and also helps replenish the secretory pathway when signaling ceases. Despite the apparent ubiquity and importance of the CCE mechanism, the molecular mechanisms by which secretory organelles communicate with plasma membrane Ca²⁺ channels remains controversial because the critical lumenal, cytoplasmic, and membrane factors have not yet been firmly established in any cell type (see Discussion). Consequently, we sought to develop the budding yeast Saccharomyces cerevisiae as a model system for studies of CCE.

Like animal cells, yeast cells employ a compartmentalized secretory system containing numerous homologs of Ca²⁺-dependent enzymes, including a furin-like protease (Kex2p) in the trans-Golgi network (16), mannosidase I (Mns1p) (27), calnexin (Cne1p), UDP-glucose:glucosyltransferase (Kre5p), protein disulfide isomerases (Pdi1p and Eug1p), and BiP (Kar2p) in the endoplasmic reticulum (12, 14, 34, 41, 48, 56, 57). Yeast lacks a true homolog of SERCA-type Ca²⁺ pumps but does express a related secretory pathway Ca²⁺ pump termed Pmr1p, localized predominantly to the Golgi complex (2, 49). Mutants lacking Pmr1p are viable in standard growth media but concentrate only half as much calcium in the endoplasmic reticulum (55) and exhibit a number of phenotypes attributed to secretory Ca²⁺ depletion, such as decreased retention of foreign proteins in the endoplasmic reticulum by the quality control machinery (49) and defects in pro-alpha-factor processing by Kex2p in the Golgi complex (2). All of these defects can be reversed by elevating Ca²⁺ in the culture medium and in some cases by expression of animal SERCA pumps or by overexpression of Pmc1p (13), the yeast homolog of PMCA, which is localized to the vacuole (7) but may function to some degree in pmr1 mutants while transiting through earlier secretory compartments (32, 38). In spite of some differences in the types and localization of Ca²⁺ pumps, yeast and mammalian cells maintain a similar need and means for concentrating Ca²⁺ in secretory compartments.

Although yeast lacks any proteins related to the inositol triphosphate receptor or the ryanodine receptor, yeast does retain a full repertoire of factors involved in sensing and transducing calcium signals. Yeast homologs of calmodulin, calmodulin-dependent protein kinases, and calmodulin-dependent protein phosphatases (also called PP2B or calcineurin) have been characterized previously (8-10, 26, 29, 37, 43, 62). Growth in very high Ca²⁺ conditions elevates [Ca²⁺]_c and stimulates expression of Pmr1p and Pmc1p through a mechanism requiring Tcn1p/Crz1p, a calcineurin-dependent transcription factor (33, 54). This transcription-dependent response serves to eliminate excess Ca²⁺ from the cytoplasm, permitting growth in high-Ca²⁺ environments. Calcineurin appears to posttranslationally inhibit the function of Vcx1p/Hum1p, an H⁺/Ca²⁺ exchanger in the vacuole that also promotes growth in high-Ca²⁺ conditions (6, 44). Together, Pmr1p, Pmc1p, and Vcx1p control [Ca²⁺]_c and very likely serve to dissipate calcium signals generated in response to external stimuli and conditions.

Sequencing of the yeast genome revealed a gene termed CCH1 that encodes a homolog of voltage-gated Ca²⁺ channels (VGCCs) found in the plasma membrane of electrically excitable animal cells. Recently, cch1 mutants and mid1 mutants were recovered in two separate genetic screens and shown to be partially deficient in Ca²⁺ influx that is stimulated by either pheromone treatment or stress associated with cdc1 mutants (15, 20, 39). MID1 encodes a plasma membrane glycoprotein with no significant similarity to any animal proteins in current databases. How these stimuli evoke Ca²⁺ influx by Cch1p and Mid1p and how the resulting calcium signals are utilized by responding cells remain unanswered.

Here we show that Cch1p and Mid1p are both required for a high-affinity Ca²⁺ influx system that can be stimulated up to 25-fold in situations causing depletion of secretory Ca²⁺ pools. We detected high levels of Cch1p- and Mid1p-dependent Ca²⁺ uptake in pmr1 mutants and in wild-type strains overexpressing either Pmc1p or Vcx1p. Cch1p and Mid1p were also required for growth in low-calcium environments. Therefore, yeast may employ a regulatory mechanism related to CCE in animal cells in order to ensure Ca²⁺ homeostasis over a wide range of environmental conditions.

	MATERIALS AND METHODS

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Genetic methods. All yeast strains used in this study were derived from strain W303-1A (59) by standard molecular and genetic methods (51). The pmc1::TRP1, pmr1::HIS3, pmr1::LEU2, tcn1::G418r, vcx1, and VCX1-D1 mutations have been described elsewhere (6, 33). The mid1::LEU2 null mutation was introduced using plasmid pFB457 (39). The cch1::TRP1 null mutation, which deletes 95% of the CCH1/YGR217w gene, was introduced by transformation of yeast with pKC289 after linearization by EcoRI digestion. pKC289 was constructed using standard procedures (50) by ligating into the XhoI and BamHI sites of pRS304 (52) two segments of DNA flanking CCH1 (from nucleotides 212 to +300 and +6120 to +6900 relative to the predicted start codon) which had been amplified by PCR using primers specific to yeast genomic DNA. DNA encoding 13 repeats of the Myc epitope were inserted into the genome just before the stop codon of CCH1 by homologous recombination of PCR-generated sequences (31). A centromere-based plasmid expressing a hemagglutinin (HA) epitope-tagged Mid1p protein (YCplacMID1-23CA5x2) was a gift from H. Iida (20). Overexpression of PMR1, PMC1, or rabbit SERCA1a was achieved in various strains by transformation with plasmid pKC152 (pRS316-Gal containing a cDNA from PMR1 [28]), pKC302 (a 2µm LEU2 derivative of pKC47 [7]), or pRS316PSA (13), respectively, using empty vectors as controls.

MAT pmr1

Other methods. All assays were performed on log-phase yeast cultures grown in standard synthetic (SC or SCGal) or rich (YPD or YPGal) culture medium containing 2% glucose or galactose (51). For ⁴⁵Ca²⁺ accumulation assays, log-phase yeast cells were collected by centrifugation, washed, resuspended in fresh medium containing tracer amounts of ⁴⁵CaCl₂ (Amersham), incubated at 30°C for 15 s to 4 h with intermittent mixing, then harvested onto Whatman GFF filters, washed, and processed for liquid scintillation counting (6). Total cell-associated Ca²⁺ was determined from specific activities of the media: standard SC and YPD media contained 0.7 and 0.14 mM total Ca²⁺, respectively, as determined by atomic absorption spectroscopy. Extracellular Ca²⁺ concentrations were varied in some experiments by first treating 2×-concentrated YPD medium adjusted to pH 10 using NaOH with 4% (vol/vol) Chelex-100 resin (Bio-Rad) for 1 h at 20°C, removing the resin by sterile filtration, and then adjusting the volume to 1×, the pH to 6.5 with HCl, and MgCl₂ to 1 or 10 mM. This procedure depleted Ca²⁺ to less than 0.5 µM as determined by atomic absorption spectroscopy, allowing accurate determination of ⁴⁵Ca²⁺ uptake over the range from 15 µM to 8.0 mM (Fig. 1). For ⁴⁵Ca²⁺ release assays, log-phase yeast cells were grown for 6 to 9 h in YPD medium supplemented with tracer ⁴⁵CaCl₂, harvested by centrifugation at 4°C, washed four times in ice-cold YPD medium, and then diluted 11-fold into prewarmed YPD medium at 30°C. The cell-free supernatant was collected at intervals by rapid filtration through type HA 0.45-µm-pore-size filters (Millipore), and aliquots were removed for measurement of radioactivity by liquid scintillation counting.

View larger version (20K): [in this window] [in a new window]   FIG. 1.   Stimulation of a high-affinity Ca2+ influx system in pmr1 mutants. Initial rates of 45Ca2+ influx (A) and efflux (B) were determined for wild-type and pmr1 mutant cells growing in YPD medium supplemented with 0.16 and 0.016 µM 45Ca2+ as described in Materials and Methods. (C and D) Accumulation of 45Ca2+ in wild-type and pmr1 mutant cells was determined after 2.5 h of growth in YPD culture medium treated with Chelex-100 resin to remove divalent cations and supplemented with either 1 or 10 mM MgCl2 and the indicated concentrations of 45CaCl2. For the wild type, the data were fit to standard Michaelis-Menten equations for two enzymes by nonlinear regression. The low-affinity Ca2+ uptake systems predominated in 1 mM Mg2+ (Km ~ 500 µM, Vmax ~ 390 nmol/109 cells) but largely disappeared in 10 mM Mg2+, exposing a second high-affinity Ca2+ uptake system (apparent Km ~ 10 µM, Vmax ~ 9 nmol/109 cells). The high-affinity Mg2+-resistant uptake system was enhanced up to 25-fold in pmr1 mutants relative to wild type. Some data from panel C are repeated in panel D for comparison. (E) SERCA1a functionally replaces Pmr1p. Expression of rabbit SERCA1a from plasmid pRS316PSA (13) suppressed Ca2+ uptake in pmr1 mutants but not pmr1 cch1 double mutants or the wild type. Uptake of 45Ca2+ was monitored as described for panel A after 4 h of growth of log-phase cells in YPD medium (0.14 mM Ca2+) supplemented with 10 mM MgCl2.

	RESULTS

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Stimulation of high-affinity Ca²⁺ influx system in pmr1 mutants. If a CCE-like mechanism operates in yeast, we reasoned that mutants lacking one or more of the intracellular Ca²⁺ transporters would accumulate higher levels of ⁴⁵Ca²⁺ from the growth medium. In standard growth media, mutants lacking either Pmc1p, Vcx1p, or both vacuolar transporters accumulated less ⁴⁵Ca²⁺ in the vacuole (6, 7) but exhibited initial rates of ⁴⁵Ca²⁺ influx and [Ca²⁺]_c levels that were indistinguishable from those of the wild-type control strain (35, 36). Depletion of Ca²⁺ from the vacuole, therefore, did not stimulate a Ca²⁺ influx mechanism to a significant degree.

Elevation of [Ca²⁺]_c in pmr1 mutants. CCE in mammalian cells promotes sustained elevation of [Ca²⁺]_c. To test whether [Ca²⁺]_c becomes elevated in pmr1 mutant cells, the fluorescent Ca²⁺ indicator Indo-1 was loaded into the cytoplasm as described previously (18), and the loaded cells were resuspended in synthetic growth medium (~0.7 mM Ca²⁺) for fluorescence measurements. Under these conditions, pmr1 mutants maintained [Ca²⁺]_c at 1.6 ± 0.3 µM, whereas the isogenic wild-type strain maintained [Ca²⁺]_c at 0.1 ± 0.05 µM (n = 3). Similar differences between wild-type and pmr1 mutants were noted previously for nongrowing cells suspended in a glucose buffer (19).

View larger version (22K): [in this window] [in a new window]   FIG. 2.   Involvement of Cch1p and Mid1p function in the pmr1-stimulated high-affinity Ca2+ uptake and signaling system. (A) Expression of the calcineurin-dependent PMC1-lacZ reporter gene in wild-type cells and pmr1 mutants carrying cch1 or mid1 null mutations was monitored after 4 h of growth in YPD medium. Mean ± standard deviation is shown for three replicates. (B) High-affinity Ca2+ uptake in pmr1 mutants requires Cch1p function. 45Ca2+ uptake was monitored after 1 h of growth in Chelex-100-treated YPD medium containing 1 mM MgCl2 as in Fig. 1 except that data were plotted in Eadie-Hofstee format (y intercept = Vmax, slope = Km).

Identification of Cch1p and Mid1p as essential components of the high-affinity Ca²⁺ uptake system stimulated in pmr1 mutants. To identify factors specifically required for high-affinity Ca²⁺ uptake in yeast, pmr1 null mutants were mutagenized and screened for variants that fail to express PMC1-lacZ (see Materials and Methods). Mutants deficient in calmodulin, calcineurin, or Tcn1p/Crz1p function were discarded, and the remaining ~250 variants were subjected to complementation tests. This method revealed 30 and 6 independent mutations in two genes, CCH1 and MID1, which had been reported previously to be important for pheromone-stimulated Ca²⁺ influx (15, 20, 39). MID1 encodes a plasma membrane glycoprotein (20) related to uncharacterized proteins expressed in other fungi. CCH1 encodes a 2,039-amino-acid protein containing four repeated membrane domains, each showing strong similarity to the 1 pore-forming subunit of VGCCs (E value = 10⁵⁴ by BLAST2.0 [1]). Deletion of either CCH1 or MID1 in pmr1 mutants specifically abolished the high-affinity Ca²⁺ uptake system (Fig. 2B and 3) and decreased calcineurin-dependent PMC1-lacZ expression to wild-type levels (Fig. 2A). Simultaneous disruption of both CCH1 and MID1 in pmr1 mutants diminished ⁴⁵Ca²⁺ uptake to the same extent as the individual disruptions (Fig. 3C), demonstrating that both Cch1p and Mid1p were required for the stimulated Ca²⁺ uptake. Thus, Cch1p and Mid1p are necessary components of a high-affinity Ca²⁺ uptake system that can be stimulated in pmr1 mutants grown in moderate- to low-calcium environments. Additional results (see below) suggest that these proteins serve as catalytic and regulatory subunits of a heteromeric Ca²⁺ influx channel.

View larger version (16K): [in this window] [in a new window]   FIG. 3.   Stimulation of the Cch1p- and Mid1p-dependent Ca2+ uptake system by increasing the activity of vacuolar Ca2+ transporters. (A) Accumulation of 45Ca2+ in wild-type and cch1 mutant strains bearing plasmids that overexpress either PMC1, PMR1, or both transporter genes as indicated was determined after 4 h of growth in SCGal-uracil medium (0.7 mM Ca2+). Mean of three replicates (± standard deviation) is shown. Cch1p-dependent Ca2+ uptake was stimulated by overexpression of Pmc1p and partially reversed by simultaneous overexpression of Pmr1p (P < 0.02). (B) Accumulation of 45Ca2+ into wild-type, VCX1-D1 (hyperactive), mid1, and mid1 VCX1-D1 strains (all pmc1 null mutants [6]) bearing plasmids that overexpress PMR1 was determined as for panel A. Overexpression of Pmr1p partially reversed Mid1p-dependent uptake stimulated by overexpression of Vcx1p (P < 0.04). (C) Accumulation of 45Ca2+ into pmr1 and pmr1 tcn1 strains with or without cch1 and mid1 mutations was determined as for Fig. 1B. Mean of three replicates (± standard deviation) is shown.

Excessive Ca²⁺ sequestration into the vacuole stimulates Ca²⁺ accumulation through a Cch1p- and Mid1-dependent process. Depletion of Ca²⁺ from secretory organelles might occur as a consequence of excessive Ca²⁺ sequestration into the vacuole. To test this possibility, ⁴⁵Ca²⁺ accumulation was quantitated in strains either expressing a hyperactive Vcx1p variant or overexpressing Pmc1p. Overexpression of Pmc1p from a high-dosage plasmid greatly stimulated ⁴⁵Ca²⁺ accumulation in wild-type cells but had no significant effect on ⁴⁵Ca²⁺ accumulation in cch1 null mutants (Fig. 3A) or mid1 mutants (not shown). Similarly, expression of a hyperactive Vcx1p mutant termed Vcx1p-D1 (6) strongly stimulated ⁴⁵Ca²⁺ uptake in wild-type strains but not in mid1 mutants (Fig. 3B). In both cases, stimulation of the Ca²⁺ uptake system was partially reversed by simultaneous overexpression of Pmr1p (P < 0.04), suggesting that the vacuolar Ca²⁺ transporters competed with Pmr1p in these conditions. Thus, the Cch1p- and Mid1p-dependent Ca²⁺ uptake system could be stimulated either by Pmr1p inactivation or by Pmr1p insufficiency due to competition with vacuolar Ca²⁺ transporters.

Expression and localization of Cch1p in pmr1 mutants. A possible mechanism for the observed increase in Ca²⁺ influx and accumulation upon depletion of Ca²⁺ stores is increased expression of either Cch1p or Mid1p or both proteins. To test this possibility, quantitative Western blot analyses were performed using epitope-tagged variants of Cch1p and Mid1p. Thirteen repeats of the Myc epitope were inserted at the extreme C terminus of Cch1p by homologous recombination into the chromosomal CCH1 gene (see Materials and Methods). The resulting Cch1p-Myc fusion protein was fully functional in pmr1 mutants (data not shown) but was expressed at identical levels in wild-type and pmr1 mutant strains grown in standard medium (Fig. 4A, top panel). In this experiment, mitochondrial porin was used as a loading control (bottom panel). Furthermore, a functional Mid1p-HA fusion protein expressed from a low-dosage plasmid from its own promoter (20) also accumulated at similar levels in wild-type and pmr1 mutant strains (Fig. 4B). In this case, nonspecific cross-reacting bands served as internal loading controls. These results rule out the possibility that depletion-stimulated Ca²⁺ uptake system involves significant up-regulation of Cch1p or Mid1p.

View larger version (55K): [in this window] [in a new window]   FIG. 4.   Expression, localization, and interaction between Cch1p and Mid1p are unaffected in pmr1 mutants. (A and B) Western blot analysis of epitope-tagged Cch1p and Mid1p variants was performed on total cell protein extracted from wild-type (WT), pmr1 mutant (pmr1), or untagged control () strains after growth in YPD medium to mid-log phase. Various amounts of each sample (either 1, 2, or 4 cell equivalents) were loaded and compared to endogenous cross-reacting proteins or mitochondrial porin as standards to control for slight variations in sample preparation or loading. (C) Immunofluorescence microscopy was performed on wild-type strains containing (top) or lacking (bottom) the epitope-tagged Cch1p-Myc variant. (D) Coimmunoprecipitation of Mid1p and Cch1p. Crude membranes were isolated from wild-type cells carrying Mid1p-HA (lanes 3 and 4) or Cch1p-Myc (lanes 2 and 4), solubilized in buffer containing Triton X-100, and immunoprecipitated using monoclonal antibodies to the HA epitope. Equal portions of the immunoprecipitated pellet and supernatant were then analyzed by Western blotting using either monoclonal antibodies to the Myc epitope (top) or polyclonal antibodies to the Pma1p protein (bottom). Cch1p but not Pma1p coprecipitated with Mid1p-HA (lane 4).

Cch1p and Mid1p supply essential Ca²⁺ to Pmr1p during ion starvation. Previous studies showed that wild-type cells cultured in media supplemented with BAPTA, a potent chelator of Ca²⁺ and other ions, exhibit defects in protein sorting and secretion similar to those observed in pmr1 mutants in standard medium (13). Very high levels of BAPTA even inhibited the growth of wild-type cells, whereas much lower chelator concentrations inhibited the growth of pmr1 mutants (11, 13). It can be inferred from these and other experiments that an essential role of Pmr1p is to supply secretory organelles with the Ca²⁺ necessary to sustain secretion and growth in low-calcium environments. The studies presented above predict that Cch1p and Mid1p would also be important for growth in low calcium conditions due to their ability to provide substrate to Pmr1p. This prediction was tested by comparing the growth of various mutants lacking Cch1p, Mid1p, and/or Pmr1p in media supplemented with increasing amounts of BAPTA.

View larger version (43K): [in this window] [in a new window]   FIG. 5.   A physiological role for the CCE-like mechanism in yeast. (A) Cch1p and Mid1p are essential for growth in low-Ca2+ environments. Yeast strains carrying combinations of cch1, mid1, or pmr1 null mutations were grown in YPD culture medium supplemented with the indicated concentrations of sodium BAPTA for either 24 h (all PMR1 strains) or 48 h (all pmr1 mutant strains). Optical density at 650 nm (OD650) was averaged for three independent cultures. Strains containing the cch1 mutation are shown with open symbols and dashed lines. (B) A working model of Ca2+ homeostasis and signaling in yeast. Depletion of secretory Ca2+ stores during growth in low-Ca2+ environments activates the high-affinity Mg2+-resistant Ca2+ channel composed of Cch1p and Mid1p through a CCE mechanism possibly involving CIF (arrow 1 or 2). Cch1p and Mid1p activation provides more substrate to Pmr1p for transport into secretory compartments and elevate [Ca2+]c, which can stimulate expression of Pmr1p and Pmc1p. Refilling of secretory pathway Ca2+ stores by Pmr1p (and possibly Pmc1p in transit to the vacuole) prevents CIF accumulation activation of Cch1p and Mid1p but does not affect a low-affinity Mg2+-sensitive Ca2+ uptake system. Secretory Ca2+ depletion and stimulation of Ca2+ influx also can be achieved genetically by decreasing Pmr1p activity or increasing Pmc1p or Vcx1p activities.

	DISCUSSION

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The data reported here collectively suggest that depletion of Ca²⁺ from secretory compartments in yeast cells stimulates the activity of a high-affinity Ca²⁺ influx channel composed of Cch1p and Mid1p which helps replenish the depleted organelles (Fig. 5B). We report evidence that the initial rate of Ca²⁺ influx is stimulated in pmr1 mutants and the rate of Ca²⁺ efflux is unaffected, leading eventually to elevated [Ca²⁺]_c, elevated expression of Pmc1p, and elevated accumulation of Ca²⁺ in the vacuole. An earlier study concluded that Ca²⁺ influx was normal in pmr1 mutants but that Ca²⁺ efflux was drastically inhibited, leading to similar long-term effects (19). In that report, Ca²⁺ influx was measured using nongrowing cells incubated in a minimal buffer lacking Mg²⁺, conditions which might have disfavored detection of Cch1p and Mid1p activity. Ca²⁺ efflux was also measured in nongrowing cells over extremely long periods of time in the absence of extracellular Ca²⁺ (up to 30 h) although, like us, they found similar rates of Ca²⁺ efflux in wild-type and pmr1 mutant strains when Ca²⁺ was added to the buffer (19). The results of our long-term ⁴⁵Ca²⁺ accumulation experiments suggested that a novel high-affinity Mg²⁺-resistant Ca²⁺ uptake system was specifically stimulated in pmr1 mutants relative to a low-affinity Mg²⁺-sensitive Ca²⁺ uptake system present at similar levels in both wild-type and pmr1 mutant cells. Remarkably, this high-affinity system in pmr1 mutants depended on the function of both Cch1p and Mid1p and could be suppressed by overexpression of mammalian SERCA pumps or simply raising extracellular Ca²⁺ to 10 mM, conditions known to replenish Ca²⁺ in the secretory pathway independent of Pmr1p function (13). Thus, the stimulation of the high-affinity Cch1p- and Mid1p-dependent Ca²⁺ uptake system appeared to correlate inversely with Ca²⁺ accumulation in secretory compartments, much like CCE in mammalian cells.

Further support for the functional coupling between Ca²⁺ stores and Ca²⁺ influx derives from studies of the vacuolar Ca²⁺ transporters. Abnormal elevation of either Pmc1p or Vcx1p activity stimulated Ca²⁺ accumulation in wild-type cells but not in cells lacking Cch1p or Mid1p (Fig. 3), an effect that was significantly reversed by overexpression of Pmr1p. It could be argued that the elevated vacuolar Ca²⁺ transport decreased Ca²⁺ efflux, but if this were the case, one would predict greater Ca²⁺ accumulation independent of Cch1p and Mid1p since these factors contribute very little to the overall Ca²⁺ influx in wild-type cells. The simplest model consistent with the data is that the vacuolar Ca²⁺ transporters compete with Pmr1p for substrate and can effectively deplete the secretory pathway of Ca²⁺ which stimulates Ca²⁺ influx via Cch1p and Mid1p. This model helps explain why Pmc1p expression and Vcx1p function are so tightly regulated in wild-type cells (6, 33); excessive vacuolar Ca²⁺ transport may deplete Ca²⁺ from secretory organelles especially under conditions of low Ca²⁺ availability from the environment. However, direct measurement of secretory Ca²⁺ concentrations would be necessary to confirm this hypothesis.

Our data also support the hypothesis that Cch1p and Mid1p function together as catalytic and regulatory/accessory subunits of a single high-affinity Ca²⁺ influx channel. In BAPTA tolerance experiments, Ca²⁺ accumulation experiments, and experiments that monitor [Ca²⁺]_c, the cch1 and mid1 single mutants exhibited phenotypes quantitatively similar to those of cch1 mid1 double mutants, suggesting that neither Cch1p nor Mid1p can function without the other. Additionally, mutations in both of these genes were recovered in two genetic screens distinct from ours (20, 39). Cch1p strongly resembles the pore-forming 1 subunit of VGCCs (15, 39) characterized extensively in vertebrate cells. In animals, VGCCs typically comprise several subunits in addition to 1 (4). Mid1p shows no significant homology to any animal proteins in current databases, but surprisingly, expression of Mid1p in CHO cells resulted in the appearance of a nonselective cation channel that responded to membrane stretch (21). Overexpression of Mid1p in either pmr1 mutants (data not shown) or wild-type cells under conditions of membrane stretch (20) had no effect on Ca²⁺ influx rates, as if another factor was limiting for Mid1p activity in yeast. Finally, we show that Cch1p and Mid1p can physically interact in the plasma membrane of wild-type cells.

How closely does CCE in animals resemble the CCE-like process in yeast? Homologs of Pmr1p, the secretory Ca²⁺ ATPases, are expressed ubiquitously in mammalian cells but they have not yet been characterized functionally (17). Their involvement in Ca²⁺ homeostasis and potential for coupling to Ca²⁺ influx mechanisms therefore remain uninvestigated. On the other hand, SERCA-type Ca²⁺ pumps are well known to supply Ca²⁺ to the endoplasmic reticulum and to prevent stimulation of CCE pathway in mammalian cells. Specific inhibitors of SERCA selectively deplete the endoplasmic reticulum of Ca²⁺ and concomitantly enhance Ca²⁺ influx through CCE channels in the plasma membrane. Physiological stimulation of CCE in animal cells occurs after release of Ca²⁺ from the endoplasmic reticulum through the activation of Ca²⁺ release channels such as the IP3 receptors, RyR receptors, or other unidentified channels. Although the Ca²⁺ influx channels activated during CCE have not been conclusively identified, recent evidence suggests that certain members of the TRP family of ion channels (TRPCs) respond to depletion of Ca²⁺ from the endoplasmic reticulum at least when expressed in heterologous systems (46). TRPCs are only distantly related to VGCCs, and the yeast genome contains no clear homologues of TRPC. At least 10 distinct genes encoding VGCC catalytic subunits have been identified in humans, and few of these proteins have been ruled out as CCE channels. Indeed, specific VGCC inhibitors can block Ca²⁺ influx in response to SERCA inhibition in at least some vertebrate cell types (30, 60). Given that the need for Ca²⁺ in secretory organelles is conserved among eukaryotes, it is plausible that a mechanism to ensure Ca²⁺ homeostasis in the secretory pathway arose prior to the divergence of animals and fungi and is largely conserved in both groups of organisms today. In this view, the primary differences between fungal and animal cells would be the types of Ca²⁺ pumps used to supply the secretory organelles and the types of Ca²⁺ channels that release secretory Ca²⁺. The significance of these differences may be relatively minor because stimulation of CCE-like processes in pmr1 mutants could be suppressed by expression of rabbit SERCA1a (Fig. 1D). However, the available data do not yet exclude the alternative possibility of independent origins of CCE in animals and yeast. Mutants lacking Pmr1p exhibit only a twofold decrease of free Ca²⁺ in the endoplasmic reticulum from a level that is already much lower than that of mammalian cells (55), raising the possibility that another secretory organelle such as the Golgi complex couples to the CCE-like process in yeast.

Current hypotheses for the coupling between Ca²⁺ influx channels in the plasma membrane and Ca²⁺ depletion in the endoplasmic reticulum include secretory mechanisms (61), docking mechanisms involving proteins in the plasma membrane and endoplasmic reticulum (42), conformational coupling mechanisms involving IP3 receptors and certain TRPCs (24, 25), various forms of retrograde signal transduction (46), and the production or release of small diffusible molecules that serve as intracellular messengers (40, 47). One such messenger of CCE termed CIF (calcium influx factor) has been described as a membrane-impermeant molecule that rapidly accumulates in human T cells treated with SERCA inhibitors and triggers Ca²⁺ influx in the absence of Ca²⁺ release from the endoplasmic reticulum (23, 58). A molecule with identical properties was recently shown to accumulate in pmr1 mutants but not wild-type yeast cells (5). The contribution of this molecule to Ca²⁺ influx in yeast cells has not been tested because no methods to introduce it into the cytoplasm or to genetically manipulate its biosynthesis have been developed. Additionally, the kinetics of Cch1p stimulation and CIF accumulation after depletion of secretory Ca²⁺ stores could not be determined in yeast because Pmr1p is resistant to the known SERCA inhibitors (53) and no other means of rapidly triggering Ca²⁺ depletion from secretory organelles have been developed. Despite these limitations, the yeast system affords genetic technologies useful for the identification of factors involved in the response to depletion of secretory Ca²⁺ stores. Characterization of the corresponding animal factors together with more mechanistic studies of the yeast system should help resolve the questions of whether the CCE mechanism evolved prior to the divergence of fungi and animals, which features of the mechanism are conserved today, and how the mechanism functions and varies in diverse cell types.