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1 Laboratory of Biological Chemistry, Graduate School of Agricultural and Life Science, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
2 Department of Integrated Bioscience, Graduate School of Frontier Science, University of Tokyo, Chiba 277-8562, Japan
3 Department of Biology, Graduate School of Science, Osaka City University, Osaka 558-8585, Japan
4 Department of Life Sciences, Faculty of Agriculture, Kagawa University, Miki-cho, Kagawa 761-0795, Japan
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Abstract |
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pik3 cells. Vps5p and Vps17p with mutations in the PX domains failed to suppress the defects of their relevant disruptants. Vps5p and Vps17p migrated toward the the forespore membrane in a pik3+-dependent manner, suggesting that these proteins may interact with PtdIns(3)P. Electron-microscopic analysis revealed that the forespore membrane fails to engulf the nucleus in some of these cells, accumulating vesicle-like bodies similar to those seen in spo3 cells. These results suggest that Vps5p and Vps17p are the targets of PtdIns(3)P in vesicle transport required for onset of the forespore membrane formation. |
Introduction |
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PtdIns 3-kinase is an enzyme that phosphorylates the 3' position of phosphoinositides (Haukins et al. 1992; Stephens et al. 1991). Whereas mammalian cells have various types of PtdIns 3-kinases, which are capable of phosphorylating PtdIns, PtdIns(4)P, and PtdIns(4,5)P2, yeast cells have only one PtdIns 3-kinase, PtdIns-specific PtdIns 3-kinase (Volinia et al. 1995). In Saccharomyces cerevisiae, PtdIns 3-kinase was originally identified as Vps34p (Schu et al. 1993). VPS mutants missort a vacuolar protein, carboxypeptidase Y (CPY). These cells have been shown to have some defects in the vesicle transport system, suggesting that selection of VPS mutants is a good assay for identifying the genes required for membrane trafficking (for review, see Bryant & Stevens 1998). In Schizosaccharomyces pombe, PtdIns 3-kinase is coded by the pik3+gene (formerly called p3k+ or vps34+ gene), and is also involved in vesicle transport. Disruption of the gene results in sensitivity of the cells to various stresses and missorting of CPY, as well as a low efficiency of conjugation and loss of viability of the spores (Kimura et al. 1995; Takegawa et al. 1995; Onishi et al. 2003).
Besides disorientated growth of the forespore membranes, many vesicles accumulated in the ascus in pik3 cells, which are similar to spo3 cells (Nakamura et al. 2001). The cell cycle of these cells was normal. A careful analysis of these observations suggests that there may be multiple steps in which PtdIns 3-kinase is involved, including onset of the forespore membrane formation, growth orientation of the forespore membranes, and closure of forespore membranes. A challenge to isolate suppressor mutants which restored all the phenotypes in sporulation was not successful, suggesting that there may be multiple downstream factors relevant to each phenotype. For further analysis on role of PtdIns 3-kinase in sporulation, we searched for targets of PtdIns 3-kinase in sporulation of S. pombe by examining the phenotypes of the clones lacking putative PtdIns(3)P binding proteins.
The product of PtdIns 3-kinase, PtdIns (3)P, has been shown to bind to FYVE and phox homology (PX) domains (for review, see Wurmser et al. 1999; Xu et al. 2001). PX domains were identified as 130-amino acid-long homologous sequences found in two proteins, p40phox and p47phox, the components of the phagocytotic NADPH oxidase (phox) complex. Many of the PX domain-containing proteins are involved in vesicular trafficking, protein sorting, and lipid modification. Isolated PX domains from p40phox and SNX3 were suggested to be capable of localizing to endosomal structures in vivo, in a PI 3-kinase-dependent manner (Xu et al. 2001). Similarly, it has been suggested that the presence of the PX domain is sufficient for localization of Vam7p to vacuoles and endosomes in S. cerevisiae (Trey et al. 1998). Recent studies revealed that PX domains indeed bind specifically to PtdIns(3)P (Yu & Lemmon 2001).
Sorting nexins are identified as an EGF receptor-binding protein by the yeast two-hybrid method (Kurten et al. 1996). Over-expression of the protein enhanced down-regulation of the receptor (Kurten et al. 1996; Haft et al. 1998). There are many members in this family, all of which contain PX domains. Vps5p in S. cerevisiae is one such protein. It has been shown that this protein is involved in vesicle transport, especially in membrane trafficking from endosome to the Golgi apparatus. This protein is a membrane protein complexed with another PX domain-containing protein, Vps17p, which is homologous to Vps5p (Nothwehr & Hindes 1997). Both of these proteins have been proved to bind directly to the PtdIns(3)P in vitro (Burda et al. 2002). In mammalian cells, sorting nexin 3 has been shown to be required for endosome function in a PtdIns(3)P-dependent manner (Xu et al. 2001). These results suggest that sorting nexins are important membrane components for formation of vesicles targeting a specific organelle.
In this paper, we report that sorting nexins, PX domain-containing putative PtdIns(3)P binding proteins, are the targets of PtdIns(3)P in sporulation of S. pombe.
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Results |
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PX domain-containing proteins were searched in the genomic database of S. pombe. Most of them were cloned, and gene disruption was performed. Some of the genes were named after most homologous genes or loci in S. cerevisiae. The cells were grown on SSA plates, and sporulation was monitored. As shown in Fig. 1, only vps5 and vps17 cells were sporulation-defective. Vps5p and Vps17p are the homologues of Vps5p and Vps17p in S. cerevisiae and are also homologous to sorting nexins in mammalian cells. The alignment of the amino acid sequences of these proteins is shown in Fig. 2A. As shown in Fig. 2B, all of the sorting nexins contain PX domains. Vps5p and Vps17p in S. cerevisiae were most homologous to SNX1 and SNX2 (Haft et al. 1998). These proteins differed in the sizes of the N-terminal regions, lengths of coiled-coil-rich regions, and sizes of carboxyl-terminal non-conserved regions. The two homologous proteins in S. pombe were named after these features.
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Sporulation of vps5, vps17 and pik3
Diploid vps5/vps5, vps17/vps17, and pik3/pik3 were cultured on YPD plates for 18 h and then transferred on to SSA plates. After culturing for 24 h, the cells were observed under the microscope. About 60% of vps5/vps5 and vps17/vps17 cells formed spores. However, after further incubation for 48 h, these spores became dark and wrinkled. These phenotypes were very similar to those of pik3/pik3 cells (Fig. 3A, arrows). After treatment with 30% ethanol, no colonies were obtained from these strains, suggesting that the spores were not viable.
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vps5/vps5, vps17/vps17, and pik3/pik3 cells underwent meiotic nuclear division normally, indistinguishable from wild-type cells. These results suggest that the defects of sporulation in these cells were not in meiotic nuclear division.
Vegetative growth of vps5 and vps17 cells
To confirm that these proteins were functionally homologous to those from S. cerevisiae, we tested sorting of CPY in S. pombe deficient in these proteins. As shown in Fig. 4A, CPY was missorted in these cells. These cells failed to fuse the vacuoles to yield large vacuoles after incubation in H2O, under the condition in which large vacuoles were seen in the wild-type cells (Fig. 4B). This phenotype was similar to that observed in vps5 and vps17 mutants in S. cerevisiae, which is typical of the class B vps mutants.
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pik3 cells are sensitive to high temperature and high ion concentration. Therefore, vps5 and vps17 cells were tested for these sensitivities. They were unable to grow at 37 °C and in the presence of 1.2 M KCl (Fig. 4C). The presence of 0.5 M KCl allowed minimum growth of these cells, whereas wild-type cells grew normally under these conditions. These results suggest that these proteins are required for normal vegetative growth as well as for sporulation. The sensitivity of the cells lacking both vps5+ and vps17+ genes remained unaltered compared with those of vps5 or vps17 cells, suggesting that the functioning points of the two proteins may overlap. This result is conceivable because Vps5p and Vps17p have been shown to form a complex in S. cerevisiae. Migration of GFP-Vps5p and GFP-Vps17p to forespore membranes
The behaviour of Vps5p and Vps17p during sporulation was analysed by use of GFP-Vps5p and GFP-Vps17p proteins. These proteins were expressed in vps5/vps5 and vps17/vps17 cells. These proteins complemented the defects of these cells, confirming that these proteins were functional (data not shown). The vps5/vps5 and vps17/vps17 cells harbouring the expression vectors for GFP-Vps5p or GFP-Vps17p were cultured on SSA plates for 8 h and observed under a confocal laser microscope. As shown in Fig. 5A, at the early stage of sporulation when the spores were yet to be seen under the phase contrast microscope, the signals of GFP-fusion proteins were found diffusely at the periphery of the nuclei. When the shapes of the spores became clearer, the signals of the GFP-fusion proteins became tightly associated with the spore membrane. Similar results were obtained when these proteins were expressed in the vps5+/vps5+ or vps17+/vps17+ cells (data not shown). As the spores became older, the fusion proteins disappeared. This distribution of the fusion proteins was not observed in pik3 cells (Fig. 5A).
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To test whether the function of Vps5p and Vps17p depends on interaction of the PX domain and PtdIns(3)P, we introduced mutations that are expected to diminish the affinity of the PX domains to PtdIns(3)P. Recent studies showed that substitution of two arginine residues (Arg57 and Arg58) in the PX domain of p40phox, which are the most highly conserved basic residues among the PX domains (indicated by asterisks in Fig. 2A), to glutamine residues abolished the ability of the protein to bind to PtdIns(3)P in vitro and in vivo (Noack 2001; Kanai et al. 2001). Similar mutations were introduced to yield Vps5pRR and Vps17pRR by substitution of Arg243 and Arg244 in Vps5p, and Arg141 and Arg142 in Vps17p to glutamine residues. These mutant proteins are expressed in the vps5/vps5 or vps17/vps17 strains, and the resulting clones were allowed to sporulate on SSA plates. After treatment with 30% ethanol, viable spores of vps5/vps5 or vps17/vps17 cells harbouring Vps5pRR or Vps17pRR protein were 1.8% and 0.17% compared with the cells harbouring wild-type protein, respectively (Fig. 5C), although the immunoblot of the whole cell lysate showed equivalent expression levels of these proteins (data not shown). These mutant proteins failed to migrate on the spore membrane (Fig. 5B).
Taken together, it is likely that Vps5p and Vps17p approach the forespore membrane at the early stage of the sporulation in a Pik3p-dependent manner, probably through interaction of the PX domain and PtdIns(3)P.
Over-expression of Vps5p and Vps17p partially suppresses defects of pik3 cells
The effect of over-expression of Vps5p and Vps17p on the formation of spores in pik3/pik3 cells was tested. As previously reported, spores of pik3 cells were not viable after treatment with 30% ethanol (Fig. 6A) (Onishi et al. 2003). In contrast, the cells over-expressing Vps5p and/or Vps17p yielded some colonies, whereas the pik3/pik3 cells with the expression vector only did not, suggesting that over-expression of Vps5p or Vps17p overcomes the defect of pik3/pik3 to some extent. Over-expression of Vps17p was more effective than Vps5p in 5 independent experiments (Fig. 6A). The cells over-expressing both Vps5p and Vps17p by the plasmid harbouring vps5+ and vps17+ genes, both driven by the nmt1 promoters in tandem, gave slightly more colonies than did the cells that over-expressed Vps17p only.
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pik3 cells which could not be suppressed by the expression of Vps5p or Vps17p (Fig. 6C). The precise reason why over-expression of Vps5p or Vps17p could improve the viability of the spores from cells lacking Ptdins(3)P is not known. In S. cerevisiae, it has been shown that the N-terminal region of Vps5p containing the PX domain is required for the binding of these proteins to the other components of the retromer complex, and this binding correlates with the sorting efficiency of CPY (Seaman & Williams 2002). Although the PX domain of Vps17p has been shown to be required for efficient formation of the retromer complex, over-expression of the protein could partially compensate for the function of the retromer complex without enhancement of the activity by binding to Ptdins(3)P.
Formation of the forespore membrane in vps5 and vps17 cells
In order to visualize the spore formation in vps5 and vps17 cells, we monitored the migration of Spo3p-GFP. To allow enough expression of Spo3p-GFP, we used haploid strains, in which sporulation occurs later than in diploid strains. vps5 and vps17 cells harbouring an expression vector for Spo3p-GFP were cultured on SSA plates for 12 h and observed under a fluorescence microscope. As shown in Fig. 7A, many of the forespore membranes in vps5 and vps17 cells did not engulf the nuclei and were found as dots or diffuse spheres when monitored with Spo3p-GFP, similar to the pattern seen in pik3 cells (Onishi et al. 2003).
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vps5 cells cultured for 8 h on SSA plates. Occasionally, forespore membranes in these cells failed to engulf nuclei, leaving bare nuclei (Fig. 8A). Even when vps5 cells succeeded in enclosing their forespore membrane, the spores were smaller than wild-type cells, and the cell walls were misshaped (Fig. 8B). These cells also contained many vesicle-like bodies, which were rarely seen in wild-type cells (Fig. 8C). Similar results were obtained with vps17 cells. This accumulation of vesicle like bodies and the failure of forespore membranes to engulf the nuclei were similar to the sporulation of spo3 cells, although the incidence of this abnormality was much higher in spo3 cells than in vps5 and vps17 cells.
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vps5, vps17, pik3, and spo3 strains, we monitored the effects of over-expression of GFP-Psy1p. As we reported previously, defects in spore formation of pik3 and spo3 cells were partially suppressed by the over-expression of GFP-Psy1p (Nakamura et al. 2001; Onishi et al. 2003). As shown in Fig. 7B, the forespore membranes in most of the cells expressing GFP-Psy1p (86% and 74%, respectively) engulfed four nuclei. The percentages of cells with four nuclei engulfed by the forespore membranes were much larger than the percentage of cells expressing Spo3p-GFP (Fig. 7C). For examination of the relationship between Spo3p and Psy1p, these proteins with different tags were coexpressed in the cells and stained (data not shown). Because they completely comigrated, we concluded that defects in sporulation of vps5 and vps17 strains were partly suppressed by the over-expression of GFP-Psy1p. These results support the idea that defects of pik3 and spo3 cells are shared by vps5 and vps17cells. |
Discussion |
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pik3. These phenotypes were very similar to those of spo3, suggesting that one of the functions of Pik3p may be similar to that of Spo3p in the sporulation procedure.
Pik3p may be involved in vesicle transport in S. pombe, as well as in S. cerevisiae. It is possible that some proteins required for the proper growth of forespore membranes may not be delivered to the forespore membrane in pik3 cells. On the basis of this hypothesis, we searched for targets of PtdIns(3)P.
We found two PX domain-containing proteins, Vps5p and Vps17p, as potential targets of PI 3-P in sporulation. Vps5p and Vps17p migrated toward the forespore membrane in a pik3+-dependent manner. Mutations in the PX domains abolished this migration, suggesting that PX domain-PI 3-P interaction is important. We have shown the similarity of vps5 or vps17 cells to pik3 and cells, suggesting a relationship between Pik3p and Vps5p or Vps17p. The accumulation of the condensed forespore membrane like bodies positive in Spo3-GFP suggests that some membrane component may be missing because of the problem in vesicle transport in pik3, vps5, or vps17 cells (Fig. 7A). Accumulation of vesicle-like structures in the ascus supports this idea. Furthermore, over-expression of Vps5p and Vps17p in pik3 cells partly rescued the spore formation, resulting in some viable spores. Because it is often seen that over-expression of the proteins can partially suppress the defects of the absence of the upstream protein, it is likely that Vps5p and Vps17p function downstream of Pik3p. However, we did not observe the elongated forespore membrane without enclosure of nuclei or multilamellar structure, reflecting failure of closing the forespore membrane. Likewise, forespore membranes of pik3 cells, whose defect was partly suppressed by over-expression of Vps5p or Vps17p, still showed a multilamellar structure (data not shown). These results suggest that there may be additional downsteam factors of PI 3-kinase responsible for the other phenotypes of pik3 cells. Indeed, we have some evidence that Vps27p, which is another putative PtdIns(3)P binding protein, is involved in a different step in the sporulation of S. pombe in a PtdIns 3-kinase-dependent manner (unpublished data). Taken together, Vps5p and Vps17p may be probable targets of PtdIns(3)P, especially in the onset of the forespore membrane formation.
How do Vps5p and Vps17p contribute to the forespore membrane formation? We have shown that PtdIns(3)P is present on the forespore membrane (Onishi et al. 2003). Because endosomes are also positive in PtdIns(3)P, it is reasonable to argue that there may be some similarity in the function of these membranes. Vps5p and Vps17p have been shown to be involved in recycling of the proteins from endosomes to the Golgi apparatus as members of a complex called ‘retromer’ in S. cerevisiae in a PtdIns(3)P dependent manner (Seaman et al., 1998; Pfeffer 2001). We found that loss of the vps29+ or the vps35+ gene, whose homologues in S. cerevisiae are the other members of the retromer complex, also leads to incapability to form proper forespore membranes (data not shown). It is interesting to speculate that a retromer-like complex is functioning in vesicle transport in the forespore membrane formation. It is possible that there is membrane trafficking between the Golgi apparatus and the forespore membrane in sporulation, which is similar to the retromer-mediated one between Golgi and endosome in vegetative growth.
The growth of vps5 or vps17 cells was much slower than that of wild-type cells. This was more obvious in diploid than in haploid cells; the reason for this is not known. This slow growth of the cells is reasonable because the retromer-like complex may be functional in cells in vegetative growth. It is likely that the yeast cells divert the complex functioning in vegetative growth for formation of the forespore membrane when the cells move on to sporulation.
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Experimental procedures |
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The yeast strains used in this study are listed in Table 1. They are the derivatives of those originally described by Leupold (1950). YPD (complete medium) and SD (synthetic medium) were used for growing S. pombe strains (Iino & Yamamoto 1985). SSA (Egel & Egel-Mitani 1974) was used for induction of mating and sporulation.
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Expression of Spo3p and Psy1p fused with green fluorescence protein (GFP) was done by pAL(spo3-GFP) and pREP81(GFP-psy1), respectively, as described before (Nakamura et al. 2001). Construction of pGFP(vps5) and pGFP(vps17), which are the expression vectors for Vps5p and Vps17p fused with GFP, was done by inserting the coding sequences of the proteins between the BglII and SalI sites of pREP1sss, which was produced by inserting a SalI linker at the SmaI site of pGFT1 (Watanabe et al. 1997). Over-expression of the proteins was done by expression vectors based on pREP1sssal, pART1, or pART2. pART2 was produced by substitution of the LEU2 gene of pART1 with the S. pombe his2+ gene. The coding sequences of Vps5p or Vps17p were inserted at the BamHI site of pART2. Plasmid for coexpression of Vps5p and Vps17p (pREP(Vps5, Vps17)) were produced by insertion of the two genes tandemly between the BamHI and SalI sites of pREP1sss.
Cloning and disruption of the genes for PX-domain-containing proteins
The genes for the PX-domain-containing proteins were cloned into pT7blue T/A cloning vector (Novagen) after PCR with the primers listed in Fig. 1. These genes were named after the genes in S. cerevisiae. By use of the resulting plasmids, the plasmids for gene disruption were produced as shown in Fig. 1. The S. pombe strain This17 was transformed with the plasmids digested with appropriate restriction enzymes by the standard LiAc method (Okazaki et al. 1990) and was grown on SD plates. DNA was extracted from the resulting clones, and the gene disruption was confirmed by PCR reaction with appropriate primers.
Production of mutants of Vps5p and Vps17p
This was done by the methods described by Sawano & Miyawaki (2000). The primers used for Vps5pRR and Vps17pRR mutants were 3'-gttagcaatgtgactgtaTCTGCaGCatataatgattttgc and 3'-gttgtacaaaaatgttcAaCAaactcacgcagaattCaaaaaatt, respectively (the nucleotides that do not match the original genes are shown in capital letters).
Detection of the secreted CPY
This was performed using cells grown on nitrocellulose filters overnight (Black & Pelham 2000). Detection of the secreted CPY was carried out with rabbit polyclonal antibody against S. pombe Cpy1p as described before (Tabuchi et al. 1997).
Fluorescence microscopy
Cells were fixed with paraformaldehyde by the method of Hagan and Hyams (Hagan & Hyams 1988). The nuclear chromatin region was stained with DAPI at 1 µg/mL. For visualization of the vacuolar membrane, cells were harvested at 1 OD600 and then labelled with vital vacuolar dye FM4-64 [N-(3-triethylammoniumpropyl)-4-(p-diethylaminophenyl) pyridinium dibromido] as previously described (Vida & Emr 1995). The cells were observed under a fluorescence microscope IX70 (Olympus) or confocal laser microscope FLUOVIEW FV300 (Olympus).
Electron microscopy
Cells were mounted on a copper grid to form a thin layer and were immersed in liquid propane cooled with liquid nitrogen. The frozen cells were transferred to 2% OsO4 in aqueous acetone and kept at –80 °C for 48 h in a solid CO2-acetone bath. The samples were then gradually warmed by being kept at –35 °C for 2 h, at 4 °C for 2 h, and at room temperature for 2 h. After washing three times with anhydrous acetone, the samples were infiltrated with Spurr's resin at increasing concentrations (final conc. 100%) in anhydrous acetone. Next, a polymerization reaction was carried out in capsules at 50 °C for 5 h and 60 °C for 50 h. Thin sections were cut on a Reichest Ultracut S and stained with uranyl acetate and lead citrate. The sections were viewed on electron microscopes (JEOL 100CX at 80 kV and JOEL2010 at 100 kV).
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Acknowledgements |
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Footnotes |
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These authors contributed equally to this work. 
* Correspondence: E-mail: ayfukui{at}mail.ecc.u-tokyo.ac.jp |
References |
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Received: 12 December 2003
Accepted: 18 March 2004
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