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1 Division of Molecular Pharmacology and Pharmacogenomics, Department of Biochemistry and Molecular Biology, Kobe University Graduate School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, Japan
2 Laboratory of Molecular Pharmacogenomics, School of Pharmaceutical Sciences, Kinki University, Kowakae 3-4-1, Higashi-Osaka 577-8502, Japan
3 University of the Philippines Manila, Manila 1000, Philippines
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Abstract |
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/
) adaptor protein (AP) complexes that are involved in clathrin-mediated membrane trafficking. Here, we show that in Schizosaccharomyces pombe the deletion strains of each individual subunit of the AP-1 complex [Apl2 (β), Apl4 (), Apm1 (µ) and Aps1 ()] caused distinct phenotypes on growth sensitivity to temperature or drugs. We also show that the 
apm1 and apl2 mutants displayed similar but more severe phenotypes than those of aps1 or apl4 mutants. Furthermore, the apl2aps1 and apl2apl4 double mutants displayed synthetic growth defects, whereas the aps1apl4 and apl2apm1 double mutants did not. In pull-down assay, Apm1 binds Apl2 even in the absence of Aps1 and Apl4, and Apl4 binds Aps1 even in the absence of Apm1 and Apl2. Consistently, the deletion of any subunit generally caused the disassociation of the heterotetrameric complex from endosomes, although some subunits weakly localized to endosomes. In addition, the deletion of individual subunits caused similar endosomal accumulation of v-SNARE synaptobrevin Syb1. Altogether, results suggest that the four subunits are all essential for the heterotetrameric complex formation and for the AP-1 function in exit transport from endosomes. |
Introduction |
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Adaptor protein complexes are widely distributed among eukaryotes. Four AP complexes (AP-1, AP-2, AP-3 and AP-4) are found in humans, mice and Arabidopsis thaliana, whereas three AP complexes (AP-1, AP-2 and AP-3) are found in Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae (budding yeast) and Schizosaccharomyces pombe (fission yeast) (Boehm & Bonifacino 2001). Each AP complex, composed of four distinct subunits called adaptins, has two large adaptins (one each of /
/
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and β1-4 respectively), one medium adaptin (µ1-4) and one small adaptin (1-4) (Lewin et al. 1998).
Some valuable insights into the function of the AP complexes have been gathered from subcellular localization studies and from genetic analyses. In mammalian cells, studies have shown that AP-2 appears to localize almost exclusively to the plasma membrane, whereas AP-1, AP-3 and AP-4 localize to the intracellular compartments such as the trans-Golgi network (TGN) and/or endosomes (Boehm & Bonifacino 2001). In mouse, the involvement of µ1A-adaptin in the endosome-to-TGN transport has been reported, and ‘knock-outs’ of µ1A-adaptin or -adaptin of AP-1 caused embryonic lethality (Zizioli et al. 1999; Meyer et al. 2000). In budding yeast, the involvement of AP-1 in the transport of chitin synthase III from endosomes to the TGN has been reported (Valdivia et al. 2002); however, the deletion of the genes encoding adaptins of the AP-1 complex yielded no discernible phenotypes, except when combined with a temperature-sensitive allele of the clathrin heavy chain gene.
In our previous study in fission yeast, we identified a mutation in the apm1+ gene encoding the µ subunit of the AP-1 complex. The apm1 mutant cells showed chloride- and FK506-sensitive phenotypes and the massive accumulation of the exocytic v-SNARE Syb1 in the Golgi/endosomes (Kita et al. 2004).
In the present study, we report the isolation and characterization of a mutation in the aps1+ gene, encoding the subunit of the AP-1 complex, which showed valproic acid (VPA) sensitivity. In aps1-v2 mutants, the FK506- and temperature-sensitivities were not so severe compared with that of the apm1-1 mutants. Similarly, the apm1 and apl2 mutants displayed more severe phenotypes than those of aps1 or apl4 mutants. Furthermore, in pull-down assay, Apm1 specifically binds Apl2 even in the absence of Aps1 and Apl4, and Aps1 specifically binds Apl4 even in the absence of Apm1 and Apl2. Our results suggest that the four adaptin subunits are all essential for the function of the AP-1 complex in both endosome-to-Golgi and endosome-to-plasma membrane transport in fission yeast.
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Results |
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We have developed a genetic screen for mutants that are hypersensitive to VPA, and identified six complementation groups (vas1-6 for valproic acid-sensitive). The first gene identified in this screen was vas1+/vps45+ encoding a homolog of the human hVps45 and S. cerevisiae Vps45p. In the previous study, we showed that in wild-type cells VPA at low concentrations caused vacuolar fragmentation and inhibited the glycosylation and secretion of acid phosphatase, and we also showed that several membrane trafficking mutants were hypersensitive to VPA, suggesting that hypersensitivity of the mutants to VPA is related to the defects in membrane trafficking (Miyatake et al. 2007). Here, we describe the isolation and characterization of another complementation group, the vas2-1 mutant. As shown in Fig. 1a, the vas2-1 mutant cells grew equally well compared with that of wild-type cells at 27 °C on a yeast extract-peptone-dextrose (YPD) plate. The vas2-1 mutant cells, however, showed high temperature sensitivity and failed to grow on a YPD plate containing 7 mM VPA at 27 °C, whereas wild-type cells grew normally (Fig. 1a).
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subunit of the AP-1 complex
The vas2+ gene was cloned by complementation of the VPA sensitivity of the vas2-1 mutant cells (Fig. 1a, +7 mM VPA, +aps1+). The vas2+ gene also complemented the temperature sensitivity of the vas2-1 mutant cells (Fig. 1a, YPD 36 °C, +aps1+). Nucleotide sequencing of the cloned DNA fragment revealed that the vas2+ gene is identical to the aps1+ gene (SPAP27G11.06c), which encodes a protein of 162 amino acids that is highly similar to the human 1 subunit (52.53% identity) (Takatsu et al. 1998) and S. cerevisiae Aps1p (45.51% identity) (Phan et al. 1994) (Fig. 1b). Linkage analysis was performed (see Experimental procedures) and results indicated allelism between the vas2-1 mutation and the aps1+ gene. We therefore renamed vas2-1 as aps1-v2. Similar to aps1-v2 mutants, the aps1 cells also showed temperature and VPA sensitivities (Fig. 1a, aps1 + vector), (Fig. 1a, vas2-1 + vector).
To identify the mutation site in the aps1-v2 allele, the genomic DNA from the aps1-v2 mutant cells was isolated, and the full-length coding region of the aps1-v2 gene was sequenced. Sequence analysis revealed that the start codon ATG was mutated to ATA by a G-to-A transition (ATG
ATA) (data not shown).
Deletion mutants of individual adaptin subunit of the AP-1 complex displayed distinct phenotypes
We have previously isolated two mutant alleles namely apm1-1 and its1-1/apl2-i1, with mutations in the genes encoding the medium µ subunit and the large β subunit of the AP-1 complex respectively (Sugiura et al. 2002; Kita et al. 2004). The apm1-1 mutation resulted in chloride and FK506 sensitivities (Kita et al. 2004), and the its1-1/apl2-i1 mutation resulted in FK506 and temperature sensitivities (Sugiura et al. 2002). Here, we isolated a third mutant allele named aps1-v2, with mutation in the gene encoding the small subunit of the AP-1 complex that resulted in VPA sensitivity.
Because the three mutant alleles, namely apl2-i1, apm1-1 and aps1-v2, were isolated using varied genetic screens, we compared altogether the phenotypes of these mutant cells as follows. As regards FK506 sensitivity, the growth of apl2-i1 and apm1-1 mutant cells was completely inhibited in the presence of FK506 (Fig. 2a), whereas that of aps1-v2 mutant cells was only partially inhibited. As regards temperature sensitivity, the growth of apm1-1 and apl2-i1 mutant cells was very sensitive to high temperature, whereas that of aps1-v2 mutant cells was not very sensitive.
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), Apm1 (µ), and Aps1 ()], and compared the phenotypes of the deletion strains as follows. As regards FK506 sensitivity, the growth of apl2 and apm1 cells was completely inhibited in the presence of FK506 (Fig. 2b), whereas that of apl4 and aps1 cells was partially inhibited. As regards temperature sensitivity and VPA sensitivity, the apl2 and apm1 cells were more sensitive when exposed to 34 °C or to 5 mM VPA compared with those of apl4 and aps1 cells (Fig. 2b). Thus, aps1 and apl4 showed similar phenotypes, and apm1 and apl2 showed similar but more severe phenotypes than those of aps1 and apl4 mutants.
We then constructed the double knockout cells, and compared their phenotypes with those of the parental single knockout cells. The aps1apl4 double knockout cells showed phenotype indistinguishable from that of aps1 or apl4 single knockout cells, and the apl2apm1 double knockout cells showed phenotype indistinguishable from that of apl2 or apm1 single knockout cells (Fig. 2c). However, the apl2apl4 and apl2aps1 double knockout cells were more sensitive to high temperature compared with each of the single knockout cells or with the aps1apl4 and apl2apm1 double knockout cells (Fig. 2c).
Binding among the four subunits of the AP-1 complex
To further examine the relative importance of each AP-1 subunit, we investigated the complex formation among the four subunits. First, we examined the binding between Apm1 and the other adaptin subunits. For this, we expressed green fluorescent protein (GFP), Aps1-GFP, GFP-Apl2 or GFP-Apl4 in strains wherein Apm1-GST (glutathione S-transferase) was expressed from a chromosomally integrated fused gene. In the pull-down assay, Apm1-GST binds GFP-Apl2, but not GFP-Apl4 or Aps1-GFP (Fig. 3a). To test whether the binding of Apm1 and Apl2 is dependent on Apl4 or Aps1 subunit, we examined the association of Apl2 and Apm1 in aps1 or apl4 cells. As shown in Fig. 3b, Apm1 still binds Apl2, even in aps1 and apl4 cells. Second, we examined the binding between Apl4 and the other adaptin subunits. For this, we expressed GST, GST-Aps1, Apm1-GST or GST-Apl2 in strains wherein GFP-fused Apl4 was expressed from a chromosomally integrated fused gene. In the pull-down assay, GFP-Apl4 binds GST-Aps1 but not Apm1-GST or GST-Apl2 (Fig. 3c), and Apl4 still binds Aps1 even in apm1 and apl2 cells (Fig. 3d).
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In our previous study we reported that in wild-type cells, Apm1-GFP localized to the Golgi/endosomes based on the colocalization with FM4-64 (Kita et al. 2004). Hence, this prompted us to examine whether the other adaptin subunits namely Aps1, Apl2 or Apl4 also colocalized with FM4-64 at an early stage of endocytosis. As shown in Fig. 4, the dot-like fluorescence of Aps1, Apl2 and Apl4 colocalized with FM4-64-positive structures, suggesting that the four adaptin subunits of the AP-1 complex are all localized to the Golgi/endosomes.
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apm1 cells, it was noted that Aps1-GFP weakly localized to the Golgi/endosomes (Fig. 5a, arrows, apm1), while such specific fluorescence was hardly observed in apl2 or apl4 cells (Fig. 5a). With GFP-Apl2 in wild-type cells, Apl2 localized to the Golgi/endosomes, whereas in apl4, apm1 or aps1 cells Apl2 failed to localize to the Golgi/endosomes (Fig. 5b). With GFP-Apl4 in wild-type cells, Apl4 localized to the Golgi/endosomes in a pattern similar to that of Aps1, Apl2 or Apm1, although the nuclear fluorescence was less prominent when compared with the other adaptin subunits. In apm1 cells, GFP-Apl4 weakly localized to the Golgi/endosomes (Fig. 5c, arrows, apm1), while such specific fluorescence was hardly observed in aps1 or apl2 cells (Fig. 5c). With Apm1-GFP in wild-type cells, as shown here and as shown in our previous study (Kita et al. 2004), Apm1 localized to the Golgi/endosomes (Fig. 5d, arrows, wt). In aps1 cells, Apm1-GFP localized to the large structures reminiscent of the enlarged endosomes (Fig. 5d, arrows, aps1), and such specific fluorescence was not observed in apl2 or apl4 cells. Also, it was noted that Apm1 localized to the spindle pole body (SPB) in apl2 or apl4 cells (Fig. 5d, arrowheads).
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aps1 cells
Syb1 is a homologue of the budding yeast Snc1, a member of the v-SNARE synaptobrevin family proteins, that is mostly observed at the cell surface and that undergoes endocytosis and is transported from the endosome to the Golgi (Lewis et al. 2000). In our previous study, we reported that apm1 cells showed a massive accumulation of GFP-Syb1 in the Golgi/endosomes (Kita et al. 2004). This prompted us to examine the localization of GFP-Syb1 in aps1 cells. In wild-type cells, GFP-Syb1 was detected at the cell surface as well as in small punctate structures in the cytoplasm, and was enriched in the medial region and cell ends (Fig. 6a, wt, arrowheads). In aps1 cells, GFP-Syb1 failed to localize to the cell surface; instead GFP-Syb1 was observed as large dot-like structures with bright fluorescence in the cytoplasm (Fig. 6a, aps1, arrows). In deletion cells of other adaptin subunits, similar large dot-like accumulation of GFP-Syb1 was also observed (Fig. 6a).
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aps1 cells expressing GFP-Syb1 were loaded with FM4-64 dye and examined by fluorescence microscopy. In wild-type cells, most of the cytoplasmic GFP-Syb1 was seen as small punctate structures that colocalized with FM4-64 (Fig. 6b, wt). In aps1 cells, GFP-Syb1 also colocalized well with FM4-64, although it was observed as large dot-like structures (Fig. 6c, aps1). Krp1 mainly localized to the Golgi in wild-type cells
Krp1 is a fission yeast homologue of the budding yeast Kex2 and the human furin that resides in the Golgi and cycles between the Golgi and endosomes (Brickner & Fuller 1997). SPAC144.18 gene is the fission yeast homologue of the budding yeast VRG4, which encodes a Golgi GDP-mannose transporter that specifically resides throughout the Golgi complex (Dean et al. 1997), thus we named the gene as vrg4+.
To characterize the cytoplasmic large dot-like structures of GFP-Syb1, we investigated the colocalization of Krp1 and Vrg4 marker proteins with FM4-64. First, we examined the colocalization of Krp1 with Vrg4. In wild-type cells, most of Krp1-red fluorescent protein (RFP) colocalized with GFP-Vrg4 (Fig. 7a), indicating that Krp1 mainly localized to the Golgi apparatus. We then examined the colocalization of Krp1 with FM4-64. The bright FM4-64 staining at the growing end of the cell and at the cell equator appears to overlap with Krp1-GFP (Fig. 7b, arrow).
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aps1 cells
In aps1 cells, most of the small punctate structures of Vrg4 did not colocalize with FM4-64, although a small part of the large punctate structures of Vrg4 appeared to colocalize with FM4-64 (Fig. 7c). The result suggests that the large dot-like structures mainly represent endosomes, and in part, represent Golgi. In aps1 cells, moreover, Krp1-GFP colocalized well with FM4-64, and was observed as large dot-like structures (Fig. 7d), suggesting that Krp1 failed to localize to the Golgi and accumulated in the endosomes.
GFP-Syb1 colocalized well with FM4-64 in aps1 cells (Fig. 6c), therefore, the large dot-like structures in the cytoplasm whereby GFP-Syb1 accumulated are most likely to be enlarged endosomes, but not the Golgi.
Deletion of end4+ recovered the localization of GFP-Syb1 to the cell surface in aps1 cells
As described earlier, in aps1 cells GFP-Syb1 failed to localize to the cell surface and accumulated as large dot-like structures in the cytoplasm (Fig. 6a). To clarify the mechanism underlying these observations, we constructed double mutant cells combining the deletions of end4+ and aps1+ or apm1+ genes. End4 is a homologue of the budding yeast Sla2p/End4p that is required for endocytosis (Iwaki et al. 2004). In wild-type cells, GFP-Syb1 was detected at the cell surface as well as in small punctate structures in the cytoplasm, and was enriched in the medial region and cell ends (Fig. 6b). In end4 cells, however, GFP-Syb1 localized almost evenly all around the cell surface, and was dispersedly distributed as very small punctate structures in the cytoplasm (Fig. 8a, upper panel). These observations are consistent with the notion that End4 is essential for endocytosis. Notably in some end4 cells, GFP-Syb1 also polarized to the cell surface at the putative growing end (Fig. 8a, arrows). As shown in Fig. 8a in double mutant cells, namely aps1end4 and apm1end4, GFP-Syb1 clearly localized to the cell surface but the distribution of Syb1 to the cell surface was less pronounced than that in the end4 single mutant cells. Notably in the double mutant cells, the large dot-like structures of GFP-Syb1 were still observed (Fig. 8a, lower panel) but its size was significantly decreased compared with that in the aps1 single mutant cells (Fig. 6c).
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aps1 cells
The above results in aps1end4 and apm1end4 cells suggest that even in the absence of endocytosis GFP-Syb1 still accumulated in endosomes. To examine this possibility, we constructed an endocytosis-defective mutant Syb1V43AM46A, and named it Syb1AA. Extensive studies of the Syb1 homologue Vamp2 in animal cells and Snc1 in S. cerevisiae have defined these two residues as being specifically important for internalization from the plasma membrane (Grote et al. 1995; Lewis et al. 2000). In aps1 cells, the cell surface localization of Syb1AA was clearly observed (Fig. 8b, arrowheads), and the distribution of Syb1AA to the cell surface was less pronounced in aps1 cells than that in wild-type cells (Fig. 8b). Furthermore, in aps1 cells, the large dot-like structures of Syb1AA were still observed, and the fluorescence colocalized with FM4-64 (Fig. 8b, arrows). Together with the results obtained from the aps1end4 and apm1end4 double mutant cells, we reason that the accumulation of Syb1 in aps1 cells is not due to the endocytosed Syb1 alone, but that other pathways for endosomal accumulation of Syb1 may also be involved.
Deletion of vps45+ decreased the large dot-like structures of GFP-Syb1 in aps1 cells
To address the above hypothesis, we constructed aps1vps45 double mutants and observed the localization of GFP-Syb1. Vps45, a member of the Sec1 family, localizes to the endosomes and is involved in the step of membrane fusion (Cowles et al. 1994; Miyatake et al. 2007). As shown in Fig. 8c in aps1vps45 double mutant cells, the large dot-like structures of GFP-Syb1 were significantly decreased compared with that in aps1 cells. Strikingly, in aps1vps45 cells, the endocytosis-defective mutant Syb1AA localized to the cell surface and the large dot-like structure was barely observed (Fig. 8c, Syb1AA). These results suggest that Vps45 is required for Syb1 transport from the Golgi to endosomes.
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Discussion |
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subunit of the AP-1 complex. Here, we showed a detailed analysis of the deletion strains of each individual subunit and provided some insight into the function of the AP-1 complex in exit transport from endosomes. Deletion phenotypes, biochemical interaction and localization in the AP-1 complex
In the present study, our genetic data showed that apm1 and apl2 mutants displayed similar growth defect whereas aps1 and apl4 mutants displayed similar growth defect. Furthermore, the apl2aps1 and apl2apl4 double mutants displayed synthetic growth defects, whereas the apl2apm1 and aps1apl4 double mutants did not. Consistently, we confirmed that Apm1 (µ) binds Apl2 (β) even in the absence of Apl4 and Aps1, and that Apl4 () binds Aps1 () even in the absence of Apm1 and Apl2. The distinct phenotypes on growth sensitivity to temperature or drugs of the mutant alleles and the single deletion or double knockouts of the four subunits of AP-1 complex suggest a distinct role for each adaptin subunit for cell function.
There are reported studies based on the biochemical and yeast three-hybrid data suggesting that β/µ and / hemicomplexes are formed in mammalian cells (Janvier et al. 2003; Doray et al. 2007), and that µ adaptin recognizes the tyrosine-based motif (Ohno et al. 1995; Owen & Evans 1998; Owen et al. 2004) and that / hemicomplex recognizes the dileucine-based motif (Bonifacino & Traub 2003). However, there is no reported study of the biological effects of the β/µ or / hemicomplexes in vivo. Our genetic data are strikingly consistent with the hemicomplex formation, and it is interesting to speculate that the β/µ or / hemicomplexes may be formed in fission yeast, and that the β/µ and the / pairs may have different roles in the heterotetrameric complex, and deletion of each subunit may result in the complete loss-of-function of one hemicomplex, whereas the other hemicomplex may have function. Further studies are needed to demonstrate the formation and function of the hemicomplexes in fission yeast.
How Aps1 and Apl4 localized to the Golgi/endosomes in the absence of Apm1 can be explained by three possibilities. First, it is reported that Rab4 effector rabaptin-5 forms a ternary complex with the / hemicomplex of the AP-1 complex, and that Rab4-GTP acts as a scaffold for a rabaptin-5–adaptin complex on recycling endosomes (Deneka et al. 2003), thereby raising the possibility that Apl4/Aps1 binds to the membrane via other proteins that localize to Golgi/endosomes. Second, the membrane association may be achieved by binding to cargo proteins because the adaptins, Apl4/Aps1 and Apl2/Apm1, carry cargo-binding motifs. Third, in S. pombe the membrane association may be mediated by phosphoinositide PI4P or the small GTPase Arf1, similar to that in mammalian cells (Heldwein et al. 2004).
In the present study, it was also noted that among the four subunits of the AP-1 complex Apm1-GFP specifically localized to SPB in wild-type cells, and that the localization of Apm1 to SPB was not affected by the deletion of genes encoding Aps1, Apl2 or Apl4, although its localization to the Golgi/endosomes was abolished. Together with the results that Apm1 colocalizes and interacts with the SPB protein Sad1 at SPB by fluorescence microscopy and two-hybrid analysis (Kita et al. 2004), we reason that Apm1 may have an additional function related to SPB or cell separation that is independent of AP-1 function.
AP-1 complex plays roles in exit transport from endosomes
Recent studies have shown that the AP-1 complex plays a role in exit transport from the TGN to endosomes (Le Borgne & Hoflack 1998), endosomes to the TGN (Meyer et al. 2000), and TGN or endosomes to the plasma membrane (Folsch et al. 1999); however, it is currently unclear whether all of these roles are compatible. In addition, there is much ambiguity in the literature regarding the distinction between the Golgi and endosome compartments in the yeast study. By investigating where and how GFP-Syb1 accumulates in aps1 cells, we show that the AP-1 adaptor complex plays its role in both endosome-to-Golgi and endosome-to-plasma membrane transport in fission yeast.
In wild-type cells, most of Krp1-RFP colocalized with GFP-Vrg4, indicating that Krp1 mainly localizes to Golgi. In aps1 cells, both Krp1 and GFP-Syb1 accumulated as large dot-like intracellular structures that colocalized with FM4-64. On the basis of the absence of the Golgi marker Vrg4, these enlarged structures were judged to be endosomes. This can be explained by our hypothesis that the AP-1 complex plays a role in the transport of Krp1 and Syb1 from endosomes to the Golgi and the disruption of any adaptin subunit abolishes this function of the complex, thereby resulting in the accumulation of Syb1 and Krp1 in the endosomes.
In the present study, using an endocytic mutant strain end4 and an endocytosis signal-deficient mutant Syb1AA, we showed that Syb1 is transported from the Golgi to endosomes even in the absence of endocytosis. We also showed that in aps1 cells, the deletion of vps45+ decreased the accumulation of GFP-Syb1 and abolished the accumulation of GFP-Syb1AA in the endosomal compartment. These results suggest that the block of both endocytosis and Golgi-to-endosome transport is necessary for eliminating the endosomal Syb1 accumulation in aps1 cells.
As described above, Vps45 seems to play a role in the transport of Syb1 from the Golgi to endosomes. This suggests that in vps45 cells (Miyatake et al. 2007), the loss of cell surface GFP-Syb1 is caused by the decreased amount of endosomal Syb1 which in turn is transported to the plasma membrane and the AP-1 complex is involved in this transport. Consistently, in aps1 cells the cell surface fluorescence of GFP-Syb1AA was weaker than that in wild-type cells. Thus, the absence of GFP-Syb1 fluorescence from the plasma membrane in aps1 cells may be caused, at least in part, by a defect in the endosome-to-plasma membrane transport.
In S. cerevisiae, endocytosis-deficient Snc1V40AM43A shows a complete loss of polarized localization (Valdez-Taubas & Pelham 2003). In S. pombe, however, GFP-Syb1AA was still polarized to the cell tips. Furthermore, in some end4 cells although the polarity was not so evident compared with that in wild-type cells, GFP-Syb1 also polarized to the plasma membrane at the putative growing end. Taken together, these results suggest that the polarized localization of Syb1 in fission yeast is not only caused by endocytic mechanisms but may also be caused by the polarized secretion at the growing cell ends.
Altogether, our study indicates that four adaptin subunits are all essential for the function of the heterotetrameric AP-1 complex in the exit transport from endosomes. Also, it would be interesting to speculate that the / and β/µ hemicomplexes might be formed in fission yeast and might fulfill distinct functions. Further studies would be necessary in order to provide a definitive answer as to the formation and function of the hemicomplexes.
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Experimental procedures |
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Schizosaccharomyces pombe strains used in this study are listed in Table 1. The complete medium YPD and the Edinburgh minimal medium (EMM) have been described previously (Toda et al. 1996). Standard genetic and recombinant-DNA methods (Moreno et al. 1991) were used except where noted. FK506 was provided by Fujisawa Pharmaceutical Co. (Osaka, Japan). Gene disruptions are denoted by lower-case letters representing the disrupted gene followed by two colons and the wild-type gene marker used for disruption (for example, aps1::ura4+). Gene disruptions are abbreviated by the gene and preceded by (for example, aps1). Proteins are denoted by roman letter and only the first letter is capitalized (for example, Aps1).
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The vas2-1 mutant (KP1330) was isolated in a screen using nitrosoguanidine-mutagenized S. pombe cells as described previously (Zhang et al. 2000). To clone the vas2+ gene, vas2-1 mutant was transformed with an S. pombe genomic DNA library constructed in the vector pDB248. Leu+ transformants were replica-plated onto YPD plates containing VPA at 27 °C and the plasmid DNA was recovered from transformants that showed a plasmid-dependent rescue. These plasmids also complemented the VPA sensitivity of the vas2-1 mutant. By DNA sequencing, the suppressing plasmids were identified to contain the aps1+ gene (SPAP27G11.06c).
To investigate the relationship between the cloned aps1+ gene and the vas2-1 mutant, linkage analysis was performed as follows. The entire aps1+ gene was subcloned into the pUC-derived plasmid containing S. cerevisiae LEU2 gene and was integrated by homologous recombination into the genome of the wild-type strain HM123. The integrant was mated with the vas2-1 mutant. The resulting diploid was sporulated, and tetrads were dissected. A total of 30 tetrads were dissected. In all cases, only parental ditype tetrads were found, indicating allelism between the aps1+ gene and the vas2-1 mutation (data not shown).
Tagging of the aps1+, apl2+ (SPBC947.02), apl4+ (SPCP1E11.06) and vrg4+ (SPAC144.18) genes
The aps1+, apl2+ or vrg4+ gene was amplified by polymerase chain reaction (PCR) with the genomic DNA of wild-type cells as a template. For aps1+, the sense primer for GST-Aps1 was 5'-CGG GAT CCA TGT CTA TAA AGT TCT TTC TAC TC-3', and the antisense primer was 5'-CGG GAT CCT TAA CGC TTC TTC ACG CTG CCC AC-3'; and the sense primer for Aps1-GFP was 5'-CGG GAT CCC ATG TCT ATA AAG TTC TTT CTA C-3', and the antisense primer was 5'-CGG GAT CCG CGG CCG CCA CGC TTC TTC ACG CTG CCC AC-3'. For apl2+, the sense primer was 5'-CGG GAT CCA TGG TTC CAA AGT TGT TTC-3' and the antisense primer was 5'-CGG GAT CCT TAA AGC CCT AAC AGA TCA TC-3'. For apl4+, the sense primer was 5'- CCG GAA TTC GCG GCC GCA TGC AAA CAA CAC ATC CAA AG-3' and the antisense primer was 5'- CCG GAA TTC GCG GCC GCT ATT GTA AAA GGT CAG ATG GC-3'. For vrg4+, the sense primer was 5'- CGC GGA TCC ATG GAT AAT CAT ATG CTA AAC CG-3' and the antisense primer was 5'-CGC GGA TCC TTA AGA CTT TGA CAG ACT ATC GCG C-3'. The amplified product containing aps1+, apl2+ or vrg4+ was digested with BamHI, and the resulting fragment was subcloned into BlueScriptSK (+) (Stratagene, La Jolla, CA, USA). The amplified product containing apl4+ was digested with EcoRI, and the resulting fragment was subcloned into BlueScriptSK (+) (Stratagene).
For ectopic expression of proteins, we used the thiamine-repressible nmt1 promoter (Maundrell 1993). Expression was repressed by the addition of 4 µM thiamine to EMM. To express GFP-Aps1, Aps1-GFP, GFP-Apl2, GFP-Apl4 or GFP-Vrg4, the complete open reading frame (ORF) of aps1+, apl2+, apl4+ or vrg4+ was ligated to the C- or N-terminus of the GFP carrying the S65T mutation (Heim et al. 1995). The expression of pREP1-Aps1-GFP, pREP1-GFP-Apl2 and pREP1-GFP-Apl4 complemented the phenotypes of the aps1, apl2 and apl4 cells respectively (data not shown). To obtain the chromosome-borne GFP, the fused genes were subcloned into the vector containing the ura4+ marker under the control of the nmt1 promoter, and was integrated into the chromosome at the ura4+ gene locus of KP1248 as described (Cheng et al. 2002; Kita et al. 2004).
Gene deletion
A one-step gene disruption by homologous recombination was performed (Rothstein 1983). For aps1::ura4+ disruption construct, the genomic DNA including aps1+ was amplified using the sense primer 5'-CGG AAT TCA ATA CTG AAT ATC ATC TAT G-3' and the antisense primer 5'-CGG GAT CCC TTT CAT TTA TTG TTT AGT C-3'. The BamHI/EcoRI fragment containing aps1+ was subcloned into the BamHI/EcoRI site of pGEM-7Zf. A PstI/SmaI fragment containing ura4+ was then inserted into the PstI/HincII site of the previous construct.
For apl2::ura4+ disruption construct, the ORF of apl2+ was amplified using the sense primer 5'-CGG GAT CCA TGG TTC CAA AGT TGT TTC-3' and the antisense primer 5'-CGG GAT CCT TAA AGC CCT AAC AGA TCA TC-3'. The BamHI fragment containing apl2+ was subcloned into the BamHI site of BlueScriptSK (+), in which SmaI and HincII were ligated together to delete both EcoRI and PstI sites. An EcoRI/PstI fragment containing ura4+ was then inserted into the EcoRI/PstI site of the previous construct.
For apl4::ura4+ disruption construct, genomic DNA including apl4+ (SPCP1E11.06) was amplified using the sense primer 5'-CCG CTC GAG ATG CAA ACA ACA CAT CCA AAG-3' and the antisense primer 5'-CCG CTC GAG GCG GCC GCC TTG TAA AAG GTC AGA TGG C-3'. The XhoI/NotI fragment containing apl4+ was subcloned into the XhoI/NotI site of BlueScriptSK (+). A BamHI fragment containing ura4+ was then inserted into the BamHI/BglII site of the previous construct.
The fragment containing the disrupted aps1+, apl2+ or apl4+ gene was transformed into diploid cells. Stable integrants were selected on medium lacking uracil. The disruption of the gene was checked using genomic Southern hybridization (data not shown).
Site-directed mutagenesis
Syb1AA (V43A and M46A) was generated using the Quick Change mutagenesis kit (Stratagene). In the PCR reaction, the mutant primers 5'-GCA GCA GAT TGA CGA TAC TGC AGG GAT TGC GCG TGA AAA CAT TTC TAA GG-3' and 5'- CCT TAG AAA TGT TTT CAC GCG CAA TCC CTG CAG TAT CGT CAA TCT GCT GC-3' were used to change Val43 (GTG) and Met46 (ATG) into Ala (GCA and GCG respectively).
Microscopy and miscellaneous methods
Methods in light microscopy, such as fluorescence microscopy which was used to observe the localization of GFP- or RFP-tagged proteins, were performed as described (Kita et al. 2004). FM4-64 labeling was performed as described previously (Kita et al. 2004). Krp1-RFP was expressed as described (He et al. 2006). Cell extract preparation and immunoblot analysis were performed as described previously (Sio et al. 2005). Microscopic analysis was performed using a microscope (Axioskop 2 plus; Carl Zeiss, Inc., West Germany) equipped with a Plan-Fluar 100x 1.45 oil objective (Carl Zeiss, Inc.). Samples were observed at room temperature. Photographs were taken using the SPOT 2 digital camera in combination with the Spot32 software version 2.1.2 (Diagnostic Instruments, Sterling Heights, MI). CorelDRAW graphics suite version 11.0 (Corel Corporation, Ottawa, Canada) was used for preparation of the figures.
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Acknowledgements |
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Footnotes |
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* mayan{at}med.kobe-u.ac.jp |
References |
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Received: 23 March 2009
Accepted: 21 May 2009
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