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Yeast Yeast 2003; 20: 845–855. Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/yea.1011 Research Article Characterization of vps33+, a gene required for vacuolar biogenesis and protein sorting in Schizosaccharomyces pombe Tomoko Iwaki1 , Fumi Osawa1 , Masayuki Onishi2 , Takako Koga2 , Yasuko Fujita1 , Akira Hosomi1 , Naotaka Tanaka1 , Yasuhisa Fukui2 and Kaoru Takegawa1 * 1 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan 2 Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan *Correspondence to: Kaoru Takegawa, Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan. E-mail: [email protected] Received: 13 March 2003 Accepted: 12 April 2003 Abstract From the fission yeast Schizosaccharomyces pombe we have identified and deleted vps33, a gene encoding a homologue of VPS33, which is required for vacuolar biogenesis in S. cerevisiae cells. When the vps33+ gene is disrupted, Sz. pombe strains are temperature-sensitive for growth and contain numerous small vesicular structures stained with FM4–64 in the cells. Deletion of the Sz. pombe vps33+ gene results in pleiotropic phenotypes consistent with the absence of normal vacuoles, including missorting of vacuolar carboxypeptidase Y, various ion- and drug-sensitivities, and sporulation defects. These results are consistent with Vps33p being necessary for the morphogenesis of vacuoles and subsequent expression of vacuolar functions in Sz. pombe cells. Copyright 2003 John Wiley & Sons, Ltd. Keywords: vacuolar protein sorting; Schizosaccharomyces pombe; vacuolar biogenesis; sporulation Introduction Eukaryotic cells are characterized by a number of different membrane-bound compartments, which serve special roles in the cell. In the yeast Saccharomyces cerevisiae, the vacuole is the most prominent organelle with significant morphology: it is distinguished by its large volume of about onequarter of the total cell volume. The vacuole of S. cerevisiae is an acidic organelle and its internal pH is maintained at 6.2 by a vacuolar-type proteinpumping ATPase. The vacuole is also involved in a variety of physiological processes, such as protein turnover, ion and pH homeostasis and osmoregulation (Jones et al., 1997). To identify the transport components involved in the delivery of proteins to the vacuole, genetic selections in S. cerevisiae were used to isolate mutants that missort and secrete the vacuolar hydrolase carboxypeptidase Y (CPY) (Bankaitis Copyright 2003 John Wiley & Sons, Ltd. et al., 1986; Rothman and Stevens, 1986). Many of the resulting vacuolar protein sorting (vps) mutants were also isolated with screens for vacuolar peptidase deficiencies (pep; Jones, 1977) and vacuolar morphological defects (vam; Wada et al., 1992). Together, these mutants define more than 40 complementation groups and have been categorized into six classes (A–F) with respect to their vacuolar protein sorting, morphology and acidification defects (Banta et al., 1988; Raymond et al., 1992). The four class C vps mutants (pep5/vps11/end1/vam1, vps16/vam9, pep3/vps18/ vam8, and vps33/slp1/vam5/pep14/met27 ) exhibit the most severe vacuolar protein sorting and morphological defects. The class C vps mutants lack any structure resembling a normal vacuole and instead accumulate small vesicles and other aberrant membrane compartments (Banta et al., 1988; Woolford et al., 1990; Preston et al., 1991). Their gene products physically associate together to form 846 the class C Vps protein complex on the vacuolar membrane (Rieder and Emr, 1997). The class C Vps protein complex plays essential roles in the processes of membrane docking and fusion at both the Golgi-to-endosome and endosome-to-vacuole stages of transport (Wurmser et al., 2000; Srivastava et al., 2000; Sato et al., 2000; Peterson and Emr, 2001). The fission yeast Schizosaccharomyces pombe, taxonomically and evolutionally distant from the budding yeast (Russell and Nurse, 1986), is genetically and physiologically well characterized (Egel et al., 1980). Under normal conditions, Sz. pombe has a large number of small vacuoles, unlike S. cerevisiae. Although the functional roles of class C Vps proteins have been extensively investigated in S. cerevisiae, little is known about their Sz. pombe counterparts. Moreover, there are no reports on the isolation and characterization of vacuole-deficient mutants in Sz. pombe. Therefore, we sought to identify and characterize class C Vps proteins that may control vacuolar biogenesis and protein sorting events in Sz. pombe cells. One of the Class C VPS genes, VPS33/SLP1, encodes a Sec1p homologue of 691 amino acids (Banta et al., 1990; Wada et al., 1990). The genome sequence of fission yeast has recently been reported (Wood et al., 2002). We found by a BLAST search of protein databases that Sz. pombe contains a gene in its chromosome II (SPBC1703.15c) that is homologous to Vps33p of S. cerevisiae. In an effort to understand the role of vacuoles in fission yeast cells better, we have constructed a strain with a disrupted vps33 gene and characterized its physiological and morphological phenotypes. This is the first report on the isolation and characterization of a vacuolar-deficient mutant from the fission yeast. Based on our findings, we suggest that the Sz. pombe Vps33 protein is required for multiple vacuolar functions, including vacuolar fusion and biogenesis events. Materials and methods Strains, media, and genetic methods Escherichia coli XL1-blue (Stratagene) was used for all cloning procedures. The wild-type Sz. pombe strains TP4–5A (h- leu1 ura4-D18 ade6-M210 ) and TP4–1D (h + leu1 ura4-D18 his2 ura4 ade6M216 ) (Ohkura et al., 1989) were obtained from Copyright 2003 John Wiley & Sons, Ltd. T. Iwaki et al. Dr T. Toda (ICRF, UK), and KJ100–7B (h 90 leu1 ura4 ) was obtained from Dr K. Tanaka (Tokyo University, Japan). The cpy1 (h + leu1–32 his2 ura4-D18 ade6-M216 cpy1::ura4+ ) and vps34 (h + leu1–32 ura4-D18 ade6-M216 vps34::ura4+ ) mutants were constructed as described previously (Tabuchi et al., 1997; Takegawa et al., 1995). Standard rich medium containing 5 g/l yeast extract, 30 g/l glucose and 250 mg/l adenine sulphate (YEA), 10 g/l yeast extract, 5 g/l peptone and 20 g/l glucose (YPD), synthetic minimal medium (MM) and sporulation medium (ME) for Sz. pombe cells were used, as described elsewhere (Moreno et al., 1991). Sz. pombe cells were transformed by the lithium acetate method or electroporation, as described previously (Okazaki et al., 1990; Suga et al., 2000; Suga and Hatakeyama, 2001). The general genetic methods have been described previously (Alfa et al., 1993). Plasmid constructions pREP41-Hmt1–GFP was constructed as follows. The hmt1 cDNA clone was obtained from Dr D.W. Ow (UC Berkeley, USA). XhoI and NotI sites were introduced at the 5 and 3 ends, and two oligonucleotides were used to amplify hmt1 by PCR. The corresponding PCR product was digested with XhoI and NotI and cloned into the corresponding site of pTN197 derived from pREP41 (Nakamura et al., 2001). The cells were observed with an Olympus BX-60 fluorescence microscope using a U-MGFPHQ filter set (Olympus, Tokyo, Japan). Images were captured with a Sensys Cooled CCD camera using a MetaMorph (Roper Scientific, San Diego, CA), and were saved as Adobe Photoshop files on a Macintosh G4 computer. Gene disruptions The vps33+ locus was disrupted in the wildtype Sz. pombe strain by replacing an internal vps33 gene fragment with the Sz. pombe ura4 gene. To amplify the DNA fragment of vps33 from chromosomal DNA of Sz. pombe, the following oligonucleotides were synthesized: sense, 5 -GCAGACGTCCTAAAGAGTTGGTGG-3 and antisense, 5 -AATCTCTCCTTCCACCTTCCC-3 . A fragment of 0.7 kbp was recovered, and ligated into pGEM-T EASY vector (Promega). A Yeast 2003; 20: 845–855. Characterization of Sz. pombe vacuole-deficient mutants HindIII site within the cloned vps33+ open reading frame was digested and a 1.6 kb ura4+ cassette (Grimm et al., 1988) was inserted. A linearized DNA fragment carrying this disrupted vps33+ gene was used to transform a wild-type haploid TP4–1D (h + ) and KJ100–7B (h 90 ), and ura+ transformants were selected. To confirm that one of the vps33+ genes had been disrupted, ura+ transformants were analysed by Southern blotting and PCR to verify correct integration of the deletion constructs. The Sz. pombe ypt7+ gene (SPBC405.04c) was amplified from chromosomal DNA, and the Bgl II site within the ypt7+ open reading frame was digested and the ura4+ gene was inserted. A linearized DNA fragment carrying this disrupted ypt7+ gene was used to transform a wild-type haploid TP4–1D, and the disruption was analysed by PCR. Vacuole staining Vacuoles were labelled with FM4–64 (Vida and Emr, 1995). Cells were grown to exponential phase in YES medium at 27 ◦ C. Samples of 250 µl cells were incubated with medium containing 80 µM FM4–64 for 30 min at 27 ◦ C. The cells were then centrifuged at 13 000 × g for 1 min, washed by resuspending them in YES to remove free FM4–64, and collected by centrifugation at 13 000 × g for 1 min. Cells were then resuspended in YES and incubated for 90 min at 27 ◦ C before microscopic observation. Stained cells were observed under a fluorescence microscope (Model BX-60; Olympus) and Sensys Cooled CCD camera. Pulse-chase analysis and immunoblot analysis of the Sz. pombe Cpy protein Pulse-chase analysis and immunoprecipitation of the vacuolar carboxypeptidase Y from Sz. pombe (SpCPY) were carried out as previously described (Tabuchi et al., 1997). Antibody incubations were carried out using rabbit polyclonal antibody against Sz. pombe Cpy1p (Tabuchi et al., 1997). The SpCPY colony blot assay was performed by replica-plating freshly grown spots onto nitrocellulose for 3 days of growth, as previously described (Cheng et al., 2002). Copyright 2003 John Wiley & Sons, Ltd. 847 Results Isolation and disruption of the fission yeast VPS33 homologue gene An examination of the Sz. pombe genome database revealed that one gene in chromosome II (SPBC1703.15c) is predicted to encode a protein homologous to the S. cerevisiae Vps33 protein. Therefore we designated this gene vps33. The vps33+ gene encodes a hydrophilic protein of 593 amino acids with three introns. A BLAST search comparing the sequences of Sz. pombe and S. cerevisiae Vps33 proteins to the GenBank database revealed that the protein is highly homologous to proteins found in higher eukaryotes, e.g. human (Carim et al., 2000; Huizing et al., 2001), rat (Pevsner et al., 1996), fly, nematode and plant (Huizing et al., 2001). Figure 1A shows the result of a sequence alignment through the region of highest homology for Vps33p. Notably, the degree of homology was highest at residues 214–243 of Sz. pombe Vps33p, and this region is also highly homologous to another Sec1 family protein, Vps45p from budding yeast (Piper et al., 1994; Cowles et al., 1994), plant (Bassham and Raikhel, 1998) and human (Pevsner et al., 1996) (Figure 1A). The conservation of the Vps33 protein between organisms as widely divergent as yeast and humans indicates that this protein may play a fundamental role in maintaining proper protein trafficking within eukaryotic cells. To examine the phenotypic consequences of a null allele of vps33+ , we performed a gene disruption of this locus. A linear fragment of the vps33+ gene inserted with the Sz. pombe ura4 gene was used to transform haploid strain TP4–1D (Figure 1B). Several transformants (slow-growing colonies) were isolated and the structure of the disrupted allele was verified by Southern blot and PCR analyses (data not shown). Sz. pombe vps33 disruption affects cell growth and vacuole morphology Although the Sz. pombe vps33+ is not essential at low temperatures, the growth of the vps33disrupted cells was very slow. Therefore the growth properties of vps33 cells were analysed further. In liquid YES medium at 27 ◦ C, the cells had a doubling time of ∼8 h, in contrast to 2 h 30 min for wild-type cells. The disruptants showed only slight Yeast 2003; 20: 845–855. 848 T. Iwaki et al. A 28% 196 276 305 29% Identity 526 A 642 689 691aa ScVps33p 138 214 243 465 539 vps33 ∆ 582 592aa SpVps33p (51%) 214 WT 243 26°C B Nomarski 37°C FM4-64 WT B P H EH EHHH vps33 ∆ ura4+ 0.5 kb Figure 1. (A) The Vps33 protein is highly conserved in eukaryotes. The most highly conserved residues 214–243 of Sz. pombe Vps33p are aligned to show the residues of greatest homology between the budding yeast (ScVps33p), rat (RnVps33p), plant (AtVps33p) and human (HsVps33p) homologues. These residues are also aligned between the budding yeast (ScVps45p), plant (AtVps45p) and human (HsVps45p) Sec1 family Vps45 protein. (B) Disruption of vps33+ . A restriction map of the vps33+ gene is shown with the open reading frame indicated by an open arrow. Restriction sites: H, HindIII; E, EcoRI; P, PstI Figure 2. (A) The growth phenotypes of wild-type and vps33 cells on YES plates at 26 ◦ C and 37 ◦ C. Plates were incubated at the indicated temperature for 5 days. (B) Vacuolar morphology in wild-type and vps33 cells. Wild-type (WT) and vps33 cells were grown in YES at 27 ◦ C to mid-log phase and stained with FM4–64 by the method described in Materials and methods. The stained cells were then visualized using Nomarski optics (left panels) and fluorescence microscopy (right panels) growth on synthetic minimal medium. We found that addition of excess NH4 Cl (final concentration of 94 mM; Alfa et al., 1993) to MM medium inhibits the growth of vps33 cells, and additions of 40 mM NH4 Cl were appropriate for the growth (data not shown). This mechanism is unknown but for vps33 mutants it appears to correlate with defects of vacuolar functions. Colonies of wild-type (TP4–1D) and vps33 cells were streaked onto YES plates and incubated at 26 ◦ C and 37 ◦ C for 4 days. While both wildtype and vps33 cells grew at 26 ◦ C, vps33 cells exhibited a temperature-sensitive growth at 37 ◦ C (Figure 2A). This result is similar to that observed in S. cerevisiae vps33 strains (Banta et al., 1990); disruption of the VPS33 gene renders cells unable to survive at 37 ◦ C, which may result from decreased tolerance to temperature stress due to abnormal vacuolar function. In S. cerevisiae, cells in the vps33 mutant exhibited extreme defects in vacuole biogenesis and accumulated vesicles and membrane-enclosed compartments that bore no resemblance to a normal vacuole. To determine the effect of the vps33+ null mutation on vacuole structure in Sz. pombe cells, a vital stain for the vacuolar membrane, FM 4–64, was used in microscopic examination of wild-type and vps33 cells. The cell-shape and septum formation of the vps33 cells were found to be normal using Nomarski optics. Wild-type Sz. pombe cells contained fragmented vacuoles, and these vacuoles appeared to be randomly dispersed throughout the cells. In contrast, cells carrying the vps33 null mutation lacked any structures resembling the vacuoles seen in the wild-type cells under fluorescence optics. The vps33 mutant cells contained numerous small vesicular structures stained with FM4–64 (Figure 2B). These small vesicular structures may be reminiscent of prevacuolar or prelysosomal compartments like S. cerevisiae vps33 mutants (Rieder and Emr, 1997; Vida and Gerhardt, 2000). Together, these experiments Copyright 2003 John Wiley & Sons, Ltd. Yeast 2003; 20: 845–855. Characterization of Sz. pombe vacuole-deficient mutants demonstrate that loss of Sz. pombe Vps33p function in the vps33 strain had a significant impact on the growth and vacuolar morphology of these cells. 849 Nomarski FM4-64 WT The Sz. pombe Vps33 protein is required for vacuolar fusion in vivo Isolated vacuoles from S. cerevisiae can undergo fusion in vitro (Wickner, 2002; Wickner and Haas, 2000). One factor required for this process is the class C Vps complex (Sato et al., 2000). Under normal conditions, Sz. pombe has a large number of small vacuoles, and hypotonic stress caused transitory fusion of vacuoles (Bone et al., 1998). Wild-type and vps33 cells, as well as ypt7 cells, which are deficient in vacuolar fusion (Bone et al., 1998), were grown in YES media, stained with FM4–64, and shifted into water to observe the vacuolar morphology (Figure 3). Wild-type cells had a smaller number of larger vacuoles that resulted from vacuolar fusion. Vacuoles of ypt7 cells are smaller than those of wild-type cells grown in YES medium and vacuolar fusion was not induced by osmotic stress, indicating that Ypt7p is required for the vacuolar fusion process like in S. cerevisiae. The vacuolar structures of vps33 cells were not detected after the shift into water, suggesting that the vacuolar fusion in vivo induced by hypotonic stress is dependent on Vps33p. The vps33 mutants show pleiotropic phenotypes Disruption of S. cerevisiae VPS33 genes causes pleiotropic phenotypes, i.e. hypersensitivity to various divalent cations including calcium ion (CLS14 is allelic to VPS33 ; Wada et al., 1992). We examined the sensitivity to divalent cations, finding that the Sz. pombe vps33 cells show strong sensitivity to 100 mM CaCl2 (Figure 4A), 10 mM MnCl2 , 5 mM ZnCl2 and 300 mM MgCl2 . The vps33 cells also show sensitivity to various drugs, e.g. nystatin at 5 µg/ml and 4 mM sodium orthovanadate. Disruption of the S. cerevisiae VPS33 gene results in a methionine requirement phenotype (Jacquemin-Faure et al., 1994). To determine whether Sz. pombe vps33 strains show methionine auxotropic phenotype, wild-type and vps33 cells were grown on MM media in the presence and absence of methionine at 75 µg/ml. The vps33 cells grew well on both MM + Met and MM − Met Copyright 2003 John Wiley & Sons, Ltd. ypt7∆ vps33∆ Figure 3. The vps33 cells are defective in vacuole fusion in vivo. Wild-type (WT), ypt7 and vps33 cells were grown in YES at 27 ◦ C and stained by FM4–64. Cells were shifted to water for 60 min and the cells were then visualized using Nomarski optics and fluorescence microscopy media at 27 ◦ C for 4 days (Figure 4B). This result shows that Sz. pombe vps33 mutants do not require methionine for growth, unlike S. cerevisiae vps33 mutants. Defects in the ade6+ gene of Sz. pombe (equivalent to the ADE2 gene of S. cerevisiae) leads to accumulation of the purine nucleotide intermediate, phosphoribosylaminoimidazole (Chaudhuri et al., 1996) and consequently their colonies are red. In Sz. pombe and S. cerevisiae cells, this red pigment is accumulated in the vacuoles when limiting amounts of adenine are supplied (Chaudhuri et al., 1996; Weisman et al., 1987; Pringle et al., 1989). This pigment is known to be fluorogenic and so the vacuolar compartments can be seen with a fluorescence microscope. The wild-type TP4–1D (ade6M216 ) cells accumulated red pigment in their vacuoles when grown on YPD medium, especially in the stationary phase (Figure 4C). In contrast, the vps33 mutant cells did not show any compartments stained with ade fluorochrome (Figure 4C). The fission yeast exists in two mating types, h + (plus) and h − (minus) (Gutz et al., 1974), and Yeast 2003; 20: 845–855. 850 T. Iwaki et al. After incubation for 3 days, >90% of the wild-type (h 90 ) cells showed the formation of zygotes and sporulation, while the vps33 cells showed no formation of zygotes (Figure 5). These results suggest that the vps33 gene is required for efficient mating. A WT vps33∆ CaCl2 Localization of vacuolar proteins in the vps33 mutants B WT vps33∆ -Met Nomarski C +Met We have previously reported the isolation and characterization of a vacuolar marker protein, carboxypeptidase Y from Sz. pombe (SpCpy1p) (Tabuchi et al., 1997). We examined the sorting of SpCpy1p in the vps33 strain. During Fluorescence WT WT vps33∆ Figure 4. (A) The vps33 cells are sensitive to calcium ion. Wild-type (WT) and vps33 cells were incubated on YES plates containing 100 mM CaCl2 for 4 days at 28 ◦ C. (B) Growth of wild-type and vps33 cells on minimal plates in the absence (MM — Met) or presence (MM + Met) of 75 µg/ml methionine. (C) Adenine intermediates did not accumulate in vps33 cells. Wild-type and vps33 cells were grown to stationary phase in YPD at 27 ◦ C, and then visualized using fluorescence microscopy vps33∆ when haploid cells of opposite types are shifted to nitrogen-free medium they conjugate to form a diploid zygote, which then undergoes meiosis and sporulation (Egel, 1989). Many sporulationdeficient mutants have been isolated and characterized in Sz. pombe (Bresch et al., 1968; Kishida and Shimoda, 1986) and most of these have exhibited defects in the regulation of the distinct steps of sporulation or meiosis. To confirm the influence of the single vps33+ disruption, it was introduced into a homothallic (h 90 ) strain, which was then plated onto MEA media and observed microscopically. Copyright 2003 John Wiley & Sons, Ltd. Figure 5. Mating deficiency of the homothallic strain with a disrupted vps33 gene. Wild-type (WT) KJ100–7B (h90 leu1 ura4) and vps33 (h90 leu1 ura4 vps33::ura4+ ) cells were cultured on ME plates at 28 ◦ C for 3 days, and then observed using Nomarski optics Yeast 2003; 20: 845–855. Characterization of Sz. pombe vacuole-deficient mutants the synthesis, SpCpy1p undergoes characteristic modifications and changes in its apparent molecular mass; after the 15 min pulse period, the ERand Golgi-specific precursor form (proCPY) and a small amount of the vacuole-specific mature form (mCPY) were labelled in the wild-type cells, and after the 30 min chase, all SpCPY had been transported to the vacuole and matured (Figure 6A). The vps33 null mutant showed a severe sorting defect for SpCPY. After the 30 min chase, mCPY was not detected in the vps33 cells (Figure 6A). To confirm the missort of SpCPY to the cell surface in the vps33 cells, we employed the CPY colony blot assay that directly tests cells for secretion of SpCPY. In wild-type cells, SpCPY is efficiently sorted to the vacuole and therefore is not secreted. In contrast, the vps33 cells showed strong secretion of SpCPY (Figure 6B). These results indicate that the Vps33 protein is required for SpCPY delivery to the vacuole in Sz. pombe. The Sz. pombe cells detoxify excessive cadmium by synthesizing phytochelatins, which bind cadmium and mediate its sequestration into the vacuole (Cobbett, 2000). Cadmium-sensitive mutants deficient in the accumulation of a phytochelatin–cadmium complex were isolated from Sz. pombe (Ortiz et al., 1992). One of the genes, hmt1+ , encodes an ABC-type transporter protein required for cadmium tolerance (Ortiz et al., 1992). The Hmt1-β-galactosidase fusion protein was localized to the Sz. pombe vacuole (Ortiz et al., 1992), and the phytochelatin–cadmium complex is accumulated in the vacuole by the Hmt1 transporter (Ortiz et al., 1995). We examined the cadmium sensitivity of the vps33 strain, and found that the mutant shows strong sensitivity (Figure 7A). This result suggests that the Hmt1 cadmium transporter may not properly localize to the vacuolar membrane. We have constructed the fusion protein GFP–Hmt1 and observed its localization in the vps33 cells. In wild-type cells, the fluorescent pattern of GFP–Hmt1 was found to overlap with the staining of the vacuolar membrane by FM4–64 (Figure 7B), indicating that GFP-Hmt1 resides predominantly on the vacuolar membrane. The staining pattern of the fusion protein was diffuse throughout the vps33 cells, but overlapped with FM4–64 localization (Figure 7B). These results suggest that the vacuolar membrane protein Hmt1p is transported to Copyright 2003 John Wiley & Sons, Ltd. 851 vps33∆ WT A 0 30 0 30 Chase (min) proCPY mCPY B W T ∆ y1 cp 4∆ s3 vp 3∆ s3 vp Figure 6. (A) Processing of carboxypeptidase Y in vivo. Wild-type (WT) and vps33 cells were pulse-labelled with Express-35 S-label for 10 min at 28 ◦ C and chased for 30 min. The immunoprecipitates were separated on an SDS-10% polyacrylamide gel. The autoradiograms of the fixed dried gels are shown. The positions of proCPY (110 kDa) and mature Cpy (mCPY; 32 kDa) are indicated. (B) Immunoblot analysis of carboxypeptidase Y. Cells were grown on a nitrocellulose filter overnight at 30 ◦ C and the filter was processed for immunoblotting using rabbit polyclonal antibody against Sz. pombe Cpy1p. cpy1 was used as a negative control and vps34 was used as a positive control for Cpy1p missorting the prevacuolar compartment; however, the vacuolar pool for the phytochelatin–cadmium complex is significantly reduced because the volume of the vacuolar compartment is reduced by the vps33 deletion. Discussion In an effort to determine proteins involved in vacuolar biogenesis, we identified the Sz. pombe vps33+ gene homologous to S. cerevisiae VPS33. Yeast 2003; 20: 845–855. 852 T. Iwaki et al. A WT vps33∆ 0.1 mM CdCl2 WT B vps33∆ Nomarski Hmt1-GFP FM4-64 Merge Figure 7. (A) The vps33 cells are sensitive to cadmium ions. Wild-type (WT) and vps33 cells were incubated on YES plates containing 0.1 mM CdCl2 for 4 days at 28 ◦ C. (B) Intracellular localization of Hmt1–GFP. Wild-type and vps33 cells were grown in MM supplemented with 5 µg/ml thiamine medium at 27 ◦ C. The cells were harvested and grown in MM — thiamine medium for 12 h, and stained by FM4–64. The cells were incubated with water for 2 h to induce vacuolar fusion. The Hmt1–GFP fusion protein and vacuolar morphology stained by FM4–64 were visualized using Nomarski optics and fluorescence microscopy According to the Sz. pombe GeneDB operated by the Sanger Institute (http://www.genedb.org/ Copyright 2003 John Wiley & Sons, Ltd. genedb/pombe/index.jsp), the Sz. pombe vps33+ gene is constitutively expressed throughout the cell cycle. The constitutive expression of Vps33 suggests that this protein may play a fundamental role in maintaining protein trafficking within Sz. pombe cells. Disruption of the Sz. pombe vps33+ gene results in severe and pleiotropic phenotypes consistent with the absence of normal vacuoles, including temperature-sensitive growth defects, various ion- sensitivities and sporulation defects. Although the vacuolar-deficient vps33 cells are viable at low temperatures, the severity of the vps33 phenotype demonstrates that the presence of a normal vacuole and correct targeting of cellular material to the vacuole is essential for the fission yeast cells. The vacuole participates in the maintenance of cytoplasmic homeostasis by transporting ionic molecules across the vacuolar membrane. Therefore, the large volume of the vacuole probably endows it with a high capacity for the chemiosmotic work that directs ionic homeostasis in the cytoplasm. Our results indicate that the vacuolardeficient vps33 mutant shows strong sensitivity to various cations that are subject to vacuolar sequestration in S. cerevisiae cells (Bode et al., 1995). Although the soluble vacuolar protease SpCPY was missorted to the cell surface, the vacuolar membrane ABC transporter Hmt1 protein was directed to the prevacuolar compartment in the vps33 cells (Figures 6 and 7). This result suggests that the vacuolar membrane transporters may be correctly sorting proteins to the vacuolar membrane in the vps33 cells. Efforts are under way to identify the sorting mechanism of putative vacuolar membrane transporters, including V-ATPase and metal transporters in Sz. pombe cells. Conjugation of the haploid cells of opposite mating types did not occur in the Sz. pombe vps33 disruptant (Figure 4). Genetic analyses indicate that vacuolar protease activities play an important role in sporulation, and vacuolar proteases are required for the nitrogen starvation-induced protein degradation that accompanies sporulation (Jones, 1977, 1984). Our data show that the vacuolar protease SpCPY was missorted and secreted to the cell surface in Sz. pombe vps33 cells (Figure 5). We have also confirmed that a nitrogen starvation-specific protein Isp6p (Sato et al., 1994), a homologue of Yeast 2003; 20: 845–855. Characterization of Sz. pombe vacuole-deficient mutants vacuolar proteinase B from S. cerevisiae, was significantly secreted to the cell surface in vps33 cells (our unpublished results). Disruption of isp6 arrested the cell cycle prior to conjugation and caused a drastic blocking effect on spore formation (Sato et al., 1994). Therefore, the secretion of vacuolar proteases suggests that disruption of vps33 may have marked blocking effects on mating and spore formation. The Sz. pombe vps33 cells do not require methionine for growth, unlike S. cerevisiae vps33 mutants (Figure 3B). The S. cerevisiae met27 mutant was originally isolated as impairing the transcriptional regulation of the MET genes. Disruption of the MET27 gene leads to a methionine requirement and affects S-adenosyl methionine (AdoMet)-mediated transcriptional control of genes involved in sulphur metabolism. The cloning of MET27 showed that it is identical to VPS33 (Jacquemin-Faure et al., 1994). In S. cerevisiae, the biosynthesis of methionine is controlled by AdoMet. AdoMet has been shown to be distributed in exchangeable cytosolic and vacuolar pools, with the latter accounting for up to 70% of the total vacuolar pools in S. cerevisiae (Schwencke and de Robichon-Szulmajster, 1976). The lost of AdoMet storage capacity and increase in the cytosolic AdoMet concentration modify MET gene expression, such that addition of methionine to the growth medium would be required to overcome the imbalance of sulphate flux in S. cerevisiae vps33 cells (Jacquemin-Faure et al., 1994). Unlike the S. cerevisiae vps33 cells, the Sz. pombe vps33 cells do not require methionine for growth (Figure 3B), suggesting that the system of transcriptional regulation may be different between these two yeasts. The budding yeast class C Vps complex genetically and biochemically interacts with other proteins: (a) the class B Vps39p (Vam6p) and Vps41p (Vam2p); (b) Ypt7p, an orthologue to the mammalian Rab7 GTPase; and (c) a syntaxin homologue Vam3p (Wickner, 2002). Vps39p is a guanine–nucleotide exchange factor for Ypt7p (Wurmser et al., 2000), and the class C Vps complex is also an effector of Ypt7p (Seals et al., 2000). The Sz. pombe Ypt7 homologue (SpYpt7p) was identified by PCR on the basis of sequence conservation between S. cerevisiae Ypt7p and mammalian Rab7p (Bone et al., 1998). When Sz. pombe ypt7 and vps33 cells were transferred from Copyright 2003 John Wiley & Sons, Ltd. 853 YES medium to water, vacuolar fusion was not induced by osmotic stress (Figure 3). These results indicate that both Ypt7p and Vps33p are required for the vacuolar fusion process, as in S. cerevisiae. Although both proteins are required for the vacuolar–membrane fusion, the vacuolar morphology of ypt7 cells is relatively normal (Figure 3). This result suggests that the Vps33 protein plays additional roles at other intracellular transport steps. Members of the Sec1p family of proteins are thought to regulate membrane fusion through their direct interaction with syntaxins. The S. cerevisiae Vps33p genetically and physically associates with the vacuolar syntaxin homologue Vam3p, and the class C Vps complex may function in maintaining Vam3p in an unpaired and active state by preventing non-productive associations on the vacuole (Sato et al., 2000). It is worth pointing out that no homologue of Vam3p has been found in Sz. Pombe, although the sequence of the Sz. pombe genome is complete (Wood et al., 2002). The identification of Sz. pombe syntaxin homologues involved in vacuolar biogenesis is currently under way. It is also likely that Sz. pombe Vps33p also associates with other class C Vps protein-like molecules. We then inspected the complete genome database for Sz. pombe in order to search for genes encoding class C VPS homologues. This inspection revealed the presence of genes [SPAC823.12 and SPAC16A10.03c (VPS11 ); SPAC824.05 (VPS16 ); SPCC790.02 (VPS18 )] in Sz. pombe chromosomes. We are currently examining the role of other Sz. pombe class C Vps homologues in the events of vacuolar biogenesis. Characterization of these mutants will reveal a mechanism for the sorting of vacuolar protein and vacuolar biogenesis in Sz. pombe. Acknowledgements We would like to thank Drs Takashi Toda, Chikashi Shimoda, David Ow and Taro Nakamura for providing the Sz. pombe strains and plasmids. We thank Masaru Ikeuchi for his excellent technical assistance. 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