Report |
Address correspondence to Martha S. Cyert, Dept. of Biological Sciences, 208B Gilbert Bldg., Stanford University, Stanford, CA 94305-5020. Tel.: (650) 723-9970. Fax: (650) 725-8309. E-mail: mcyert{at}leland.stanford.edu
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Abstract |
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Key Words: calcium signaling; ion channels; osmotic pressure; vacuoles; Saccharomyces cerevisiae
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Introduction |
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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).
Although the response to hypertonic shock has been intensively studied, whether it involves Ca2+ signaling is unknown. In addressing this question, we show that: (a) hypertonic shock induces a transient increase in cytosolic Ca2+ concentrations; (b) the Ca2+ flux comes from the vacuole; and (c) Yvc1p, a homologue of transient receptor potential (TRP) channels, is required for this release. Yvc1p has recently been cloned by Palmer et al. (2001), and has been shown to be a cation-selective channel that can conduct Ca2+, K+, or Na+. This conductance had been previously characterized by electrophysiological methods (Wada et al., 1987; Bertl and Slayman, 1990, 1992; Bertl et al., 1992). The electrophysiological properties of Yvc1p and its presence in the vacuolar fraction suggested that Yvc1p could be a vacuolar Ca2+ channel. In this study we verify this hypothesis using living cells; we demonstrate in vivo, for the first time, that Yvc1p is a vacuolar channel that mediates Ca2+ release in response to hyperosmotic stress.
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Results and discussion |
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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).
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In conclusion, we show that internal Ca2+ release in yeast is mediated by a novel class of Ca2+ release channel, which is unrelated to IP3 or ryanodine receptors. Instead, this release requires a homologue of the TRP family of ion channels, Yvc1p. Like TRP channels in multicellular organisms, YVC1 acts in sensory transduction. However, YVC1 is the first TRP channel homologue shown to mediate Ca2+ release from an intracellular store.
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Materials and methods |
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Vector construction and gene deletion
YVC1GFP plasmid (VDp88) was constructed by cloning a PCR fragment containing YVC1 and 737 pb upstream sequence into the SacI/NheI sites of pGRU2, provided by Bertrand Daignan-Fornier (Institut de Biochemie et Génétique Cellulaires, Bordeaux, France). Hemagglutinin (HA)-tagged YVC1 overexpression plasmids were constructed by a two-step PCR: PCR-amplified 3x (HA) was used as a downstream primer to amplify YVC1 (YOR087/088W). This fragment was cut by Xho/BglII and cloned into Xho/Bam sites in pVT100L or pVT100U (Vernet et al., 1987), leading to pYVC1-HA-L for high expression of YVC1, and pYVC1-HA-U, for moderate expression, respectively. We verified that full-length Yvc1-HA protein (78 kD) was expressed in yeast by Western blotting. pYVC1-U, used for aequorin experiments, is a nontagged version of pYVC1-HA-U: the HA tag was removed by a NotI digest and self-religation. To delete YVC1, a fragment comprising ORF YOR087/088 and 736 bp of upstream sequence was cloned between the XhoI and BamHI sites of pcDNA3.1 (Invitrogen). This plasmid was then cut by EcoRI, which removed the sequence from position -41 to +1780 from the ATG, and a cassette containing the kanr gene was inserted into this site. The resulting plasmid was used to amplify a deletion cassette that was used to transform yeast. Kanamycin-resistant colonies were selected and
yvc1 knockout was checked by PCR as described previously (Güldener et al., 1996).
Aequorin experiments
Yeast carrying the PEVP11/AEQ plasmid, provided by Patrick H. Masson (University of Wisconsin-Madison, Madison, WI) (Batiza et al., 1996) were inoculated from a saturated overnight culture to OD600 = 0.5 in SD media with 2 µM coelenterazine, and were grown overnight at room temperature to reconstitute aequorin from apoaequorin. For each experiment, an aliquot of 250 µl (OD600 = 23) was harvested. Cells were resuspended in 100 µl SD media and transferred to luminometer tubes. The baseline luminescence was recorded every second for 30 s (1-s integration) using a Berthold LB9507 luminometer, and was reported in relative luminescence units/s. Hypertonic shock was performed by adding 100 µl SD containing twice the desired final concentration of sorbitol, KCl, or NaCl. To ensure that total reconstituted aequorin was not limiting in our assays, we measured the maximal luminescence after addition of 0.1% digitonin. The maximal luminescence was 4,000,000 relative luminescence units or more, which is 10x higher than the highest signal observed in our assays. Because light units cannot be accurately converted into intracellular Ca2+ concentrations, our results are presented as relative quantities.
Phylogenetic tree
Evolutionary distances between peptide sequences aligned with ClustalW were calculated with the PHILIP protdist software (Felsenstein, 1993), and the tree was subsequently plotted by the neighbor-joining method (Saitou and Nei, 1987). The GenBank accession numbers for the proteins used are the following (available at GenBank/EMBL/DDBJ accession no.): ScYVC1 (S. cerevisiae, YOR087/088w) (Palmer et al., 2001); CaTRP (sequence 118949852 from Candida albicans genome contig 1.802); NcTRP (sequence 47276751 from N. crassa genome contig 62259); rVR1 (T09054); mOTRPC4 (AAG17543); rVRL-1 (NP_035836); mCaT1 (BAA99538); rCaT2 (BAA99541); CeOTRPC2 (CAA96644); CeOTRPC1 (T37241); CeLTRPC2 (CAA92726); CeLTRPC1 (CAB02303); mLTRPC7 (AAK57433); hChaK2 (AAK31202); hLTRPC2 (NP_003298); hLTRPC4 (NP_060106); mLTRPC5 (AAF98120);CeSTRPC1 (AAA28168); CeSTRPC2 (AAK21447); dTRPL (P48994); dTRP (P19334); mTRPC1 (AAB50622); mTRPC4 (AAC05179); mTRPC5 (AAC13550); mTRPC6 (AAC06146); mTRPC3 (NP_062383); mTRPC7 (AAD42069); and mTRPC2 (AAG29950).
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Footnotes |
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Acknowledgments |
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This work was supported by National Institutes of Health research grant GM48728.
Submitted: 1 November 2001
Revised: 21 November 2001
Accepted: 21 November 2001
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References |
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