If internal Ca²⁺ release exists in yeast, the vacuole is likely to be involved in this function, as it plays a major role in Ca²⁺ homeostasis. Indeed, free Ca²⁺ concentration in the yeast vacuole reaches 1.3 mM, compared with only 10 μM in the endoplasmic reticulum (Halachmi and Eilam, 1989; Strayle et al., 1999). Therefore, the yeast vacuole is the functional counterpart of the mammalian endoplasmic and sarcoplasmic reticulum for Ca²⁺ storage. Two transporters play complementary roles in sequestering Ca²⁺ into the vacuole: (a) Vcx1p, a low-affinity Ca²⁺/H⁺ exchanger that rapidly sequesters Ca²⁺ into the vacuole; and (b) Pmc1p, a high-affinity Ca²⁺ ATPase required for maintaining low [Ca²⁺]_cyt (Cunningham and Fink, 1994, 1996; Pozos et al., 1996; Miseta et al., 1999). It has been reported that vacuolar membrane vesicles could release Ca²⁺ in the presence of IP3 (Belde et al., 1993); however, the mechanism and the physiological relevance of this effect have not been addressed. Although Ca²⁺ influx into the vacuole has been well characterized, no protein has been shown to effect vacuolar Ca²⁺ release.

All cells must repeatedly adapt to hypertonic shock caused by variations in water availability or solutes concentration. Yeast cells are particularly exposed to such changes, and therefore have developed multiple responses to hypertonic stress. Within minutes, cells shrink and the cytoskeleton disassembles (Morris et al., 1986; Chowdhury et al., 1992). Adaptation to these new conditions requires transcriptional induction of stress-responsive genes, as well as the accumulation of intracellular glycerol (Brown et al., 1986; Albertyn et al., 1994; Hirayama et al., 1995; Tamás et al., 1999). This transcriptional activation is mediated in part by the high-osmolarity glycerol (HOG) response pathway, which is composed of a mitogen-activated kinase cascade regulated by at least two independent osmosensors (Brewster et al., 1993; Posas et al., 1998).

To further investigate this novel Ca²⁺ response, we examined whether the hyperosmolarity induced Ca²⁺ flux comes from an external or an internal source. We repeated these experiments using media containing the Ca²⁺ chelators EGTA or 1,2-bis(2-aminophenoxy) ethane-N,N,N′,N′-tetraacetic acid (BAPTA), as well as using low Ca²⁺ SD medium (see Materials and methods), and observed no differences under these conditions (unpublished data). This strongly suggests that external Ca²⁺ is not required for the observed cytosolic Ca²⁺ peak. Next, we asked if the Ca²⁺ flux is released from internal stores. Because the vacuole plays an important role in Ca²⁺ storage and homeostasis, we investigated the hyperosmolarity induced Ca²⁺ flux in mutants with defects in vacuolar Ca²⁺ storage. In a pmc1Δvcx1Δ strain lacking both transporters for vacuolar Ca²⁺ storage, Ca²⁺ is not sequestered in the vacuole, and consequently, vacuolar [Ca²⁺] is dramatically reduced (Cunningham and Fink, 1996; Pozos et al., 1996). Therefore, if the hyperosmolarity induced Ca²⁺ flux comes from the vacuole, we expect it to be reduced in this strain. Wild-type, pmc1Δ, vcx1Δ, and pmc1Δvcx1Δ strains were subjected to high osmolarity shock (0.8 M NaCl). Strikingly, the Ca²⁺ increase was completely absent in the pmc1Δvcx1Δ strain (Fig. 1 b). In contrast, the single mutant pmc1Δ had a Ca²⁺ peak comparable to wild-type strain, and the Ca²⁺ response was increased in the vcx1Δ strain (Fig. 1 b). This last observation confirms that Vcx1p plays a critical role in rapidly sequestering a sudden pulse of cytosolic Ca²⁺ into the vacuole. Indeed, vcx1Δ cells also display a delay in restoring low [Ca²⁺]_cyt after addition of extracellular Ca²⁺ (Miseta et al., 1999). Together, these results strongly suggest that the hyperosmolarity induced Ca²⁺ flux is generated by release of Ca²⁺ from the vacuole. This is the first time that Ca²⁺ release from the vacuole has been shown in vivo in yeast in response to a specific signal.

As a next step, we aimed to identify the channel responsible for this Ca²⁺ release. We examined the yeast genome for putative Ca²⁺ channels and found a candidate ORF, recently characterized as YVC1, that shows significant homology to the TRP family of ion channels (Palmer et al., 2001). The first TRP channel was discovered in Drosophila melanogaster and is required for phototransduction (Montell and Rubin, 1989). Multiple homologues have since been identified in mammals, Xenopus, squid, and worms, and are involved in such diverse sensory functions as pain, heat, olfaction, and osmolarity signaling; they may also be involved in replenishing intracellular Ca²⁺ stores (Putney and McKay, 1999; Harteneck et al., 2000; Clapham et al., 2001). TRP channels have been the subject of intense investigation recently, yet their gating mechanisms and biological role are not fully understood (Harteneck et al., 2000; Clapham et al., 2001; Montell, 2001). The discovery of a TRP homologue in Saccharomyces cerevisiae prompted us to search other fungal genomes for YVC1 homologues. We found a single homologue in Candida albicans, Neurospora crassa, and in 5 of the 14 hemiascomycetous yeast genomes that have been partially sequenced (Souciet et al., 2000). Next, we analyzed the phylogenetic relationship between these new fungal TRP channels and animal TRPs from worm and mammals (Fig. 2). The resulting tree shows that the newly defined cluster of fungal TRPs forms a distinct subfamily (Fig. 2), in addition to the previously described Short, Osm-like, and Long subfamilies (Harteneck et al., 2000; Clapham et al., 2001), also defined, respectively, as TRPC, TRPV, and TRPM subfamilies (Montell, 2001).

As a first step toward characterizing yeast Yvc1p, we determined its localization in vivo using a COOH-terminal green fluorescent protein (GFP) fusion. Interestingly, Yvc1–GFP was specifically localized to the vacuolar membrane (Fig. 3 a). This localization of the yeast TRP homologue is in contrast to other TRP channels studied thus far, which localize to the plasma membrane (Pollock et al., 1995; McKay et al., 2000; Xu and Beech, 2001). Next, we characterized the effect of Yvc1p levels on yeast cell growth. Although yvc1Δ had no apparent growth defects, cells expressing high levels of Yvc1p were extremely sensitive to the presence of CaCl₂ in the medium (Fig. 3 b). Furthermore, this sensitivity was Ca²⁺-specific, as MgCl₂ at the same concentration did not affect growth (Fig. 3 b), and cells overexpressing YVC1 did not show increased sensitivity to NaCl or KCl (0.6 to 1.2 M) (unpublished data). This Ca²⁺ sensitivity strongly suggests that Yvc1p, like some other TRP channels, participates in Ca²⁺ homeostasis and acts to increase cytosolic [Ca²⁺]. Based on this finding, as well as its localization to the vacuolar membrane, Yvc1p is a good candidate for a Ca²⁺ channel that mediates vacuolar Ca²⁺ release. This hypothesis is also consistent with the electrophysiological properties of YVC1, which has been shown to be permeable to Ca²⁺, among other cations (Bertl and Slayman, 1990, 1992; Bertl et al., 1992; Palmer et al., 2001).

We tested whether YVC1 was involved in the hyperosmolarity induced Ca²⁺ increase by examining [Ca²⁺]_cyt in cells lacking or overexpressing YVC1. The yvc1Δ strain displayed no significant increase in [Ca²⁺]_cyt after hypertonic treatment (Fig. 3 c). In contrast, YVC1 overexpression greatly enhanced the magnitude of the Ca²⁺ peak induced by high osmolarity (Fig. 3 c). These results indicate that Yvc1p mediates increased [Ca²⁺]_cyt in response to hypertonic shock. To confirm that this YVC1-mediated Ca²⁺ release is dependent on vacuolar Ca²⁺, we examined [Ca²⁺]_cyt in pmc1Δ, vcx1Δ and pmc1Δvcx1Δ strains carrying a yvc1Δ allele or overexpressing YVC1. The pmc1Δvcx1Δ strain showed no vacuolar Ca²⁺ release even when YVC1 was overexpressed (Fig. 3 d). This is likely due to low vacuolar [Ca²⁺], and shows that YVC1-dependent Ca²⁺ release comes from the vacuole. As expected, changes in [Ca²⁺]_cyt observed in the Δpmc1 background lacking or overexpressing YVC1 were equivalent to those seen in the wild-type strain (unpublished data). In a vcx1Δ background, deletion of YVC1 completely eliminated the Ca²⁺ increase induced by hypertonic shock (Fig. 3 e). In contrast, overexpression of YVC1 in the vcx1Δ strain caused a dramatic increase in this Ca²⁺ response (Fig. 3 e). Thus, overexpression of YVC1 and mutational inactivation of VCX1 both increase the amplitude of the hyperosmolarity induced Ca²⁺ peak, and these two effects are additive. These observations underscore the importance of Vcx1p in antagonizing and potentially modulating YVC1-dependent Ca²⁺ release. Together, these results show that following hypertonic shock, Yvc1p effects Ca²⁺ release from the vacuole into the cytosol, and that this release is followed by rapid Ca²⁺ sequestration into the vacuole by Vcx1p (Fig. 4).

Yeast actively sequester Ca²⁺ in their vacuole. In these studies we establish that, as in other eukaryotic cells, this Ca²⁺ can be released into the cytosol in response to external stimuli. We also show that this release is followed by refilling of the internal store. However, two key questions remain: (a) What leads to Yvc1p channel opening?; and (b) What are the physiological consequences of Ca²⁺ release? We investigated whether other environmental changes besides hypertonic shock induced Ca²⁺ release by Yvc1p. First, we found that the Ca²⁺ peak induced by injection of extracellular Ca²⁺ (Fig. 1 a) was not affected by YVC1 deletion or overexpression (unpublished data). Thus, in vivo, a brief increase in [Ca²⁺]_cyt is apparently not sufficient to trigger Yvc1p opening, although the YVC1 cation conductance observed in isolated vacuoles is activated by Ca²⁺ (Wada et al., 1987; Bertl and Slayman, 1990, 1992; Bertl et al., 1992; Palmer et al., 2001). Other conditions, such as hypotonic shock or the addition of 0.03% SDS or 7% ethanol, also induced a transient increase in cytosolic Ca²⁺ (Batiza et al., 1996; unpublished data); however, YVC1 was similarly not required for these Ca²⁺ peaks (unpublished data). Therefore, the response of Yvc1p to hypertonic shock appears to be specific. We are currently investigating the role of YVC1-mediated Ca²⁺ release in hypertonic stress signaling. The signaling pathway activated by hypertonic shock has been well characterized in yeast, and is composed of the HOG mitogen-activated kinase cascade (Posas et al., 1998). Further studies will examine the relationship between components of the HOG pathway and the Ca²⁺ increase mediated by YVC1.