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1 Department of Chemistry, Graduate School of Science, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
2 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, 5-1-5 Kashiwanoha, Kashiwa 277-8562, Japan
3 The Institute for Advanced Research, Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
4 The Core Research for Evolutional Science and Technology (CREST), Japan Science Technology Corporation (JST), Nagoya University, Chikusa-ku, Nagoya 464-8602, Japan
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Abstract |
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and sec71 mutant zygotes using electron microscope with the freeze-substituted fixation method. Three-dimensional reconstitution of nuclear structures from electron microscope serial sections revealed that Jem1p facilitates nuclear inner-membrane fusion and spindle pole body (SPB) fusion while Sec71p facilitates nuclear outer-membrane fusion. Two haploid SPBs that failed to fuse could duplicate, and mitotic nuclear division of the unfused haploid nuclei started in jem1 and sec71 mutant zygotes. This observation suggests that nuclear inner-membrane fusion is required for SPB fusion, but not for SPB duplication in the first mitotic cell division. |
Introduction |
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) mate to produce diploid cells. Haploid cells that respond to mating pheromones first exit the mitotic cell cycle to produce a mating-proficient cell with a mating-specific projection. Contact between the partner cells leads to cell fusion and subsequent nuclear fusion, resulting in formation of a diploid zygote. The process of nuclear fusion is called karyogamy, the final step of the mating pathway in yeast (for review, see Rose 1996). Karyogamy consists of two consecutive steps, nuclear congression and nuclear membrane fusion. The onset of nuclear congression requires emanation of cytoplasmic microtubules from the spindle pole bodies (SPBs) immediately after the cell fusion. The SPB is the sole microtubule organizing center in yeast and apparently consists of three major layers, the outer plaque on the cytoplasmic side, the inner plaque on the nucleoplasmic side and the central plaque embedded in the nuclear envelope (Jaspersen & Winey 2004). The microtubule from the SPBs interconnects the two nuclei, which allows the nuclei to approach each other. When the two nuclei are in close contact, the two SPBs become closely apposed.
In the second step of karyogamy, the outer and the inner membranes of the two haploid nuclei start to fuse, and the two SPBs also fuse each other to produce a single SPB of diploid cells. The SPB fusion apparently starts from the satellite on the half bridge, which is a specialized region of the nuclear envelope connected to the core SPB (Jaspersen & Winey 2004). The newly formed diploid SPB initially exhibits a characteristic V-shaped structure, but later reverts to a planar form seen in vegetative cells (Byers & Goetsch 1975).
Yeast mutants defective in karyogamy (Kar–) have been isolated and characterized (Rose 1996). Zygotes of the mutants defective in the nuclear congression have two haploid nuclei that are positioned far apart in the cells (Kurihara et al. 1994). Mutants defective in the nuclear membrane fusion produce zygotes in which two haploid nuclei are juxtaposed but do not fuse (Kurihara et al. 1994; Nishikawa & Endo 1997; Brizzio et al. 1999). Both genetic and biochemical analyses of karyogamy mutants led to identification of the components involved in karyogamy. For example, BiP/Kar2p, an Hsp70 molecular chaperone in the endoplasmic reticulum (ER) (Rose et al. 1989), and its partner J proteins, Jem1/Kar8p (Nishikawa & Endo 1997) and Sec63p (Ng & Walter 1996) were found to facilitate nuclear fusion in karyogamy. Jem1p is an ER J-protein peripherally associated with the lumenal face of the ER membrane, and has partly overlapped functions with another ER lumenal J-protein, Scj1p, in protein folding/assembly and ER-associated degradation of aberrant proteins (Nishikawa et al. 2001). Sec63p forms a complex in the ER membrane with Sec71/Kar7p and Sec72p and mediates protein import into the ER (Brodsky & Schekman 1993). The Sec63p complex likely affects the nuclear membrane fusion by assisting import or assembly of Kar5p, a component of SPB involved in nuclear membrane fusion during karyogamy (Brizzio et al. 1999). Identification of Nep98/Mps3p, a component of the half bridge of the SPB, as a Jem1p-interacting protein suggested that Jem1p functions in nuclear membrane fusion by regulating assembly of Nep98p in the SPB (Nishikawa et al. 2003).
Electron microscopic (EM) examination of permanganate-fixed zygotes revealed two distinct phenotypic classes in nuclear fusion mutants with respect to the ultrastructures of nuclear organization (Brizzio et al. 1999). In kar8 mutant zygotes, two haploid nuclei make a direct contact through membranous bridges that span the gap between the two nuclei. Serial EM sections indicate that the bridge contains a significant lumen that traverses as much as 400 nm. In contrast, the bridges observed in kar2, kar5 and kar7/sec71 zygotes has no apparent lumen and are observed only within a single section of 70 nm (Kurihara et al. 1994). kar5kar8 double mutant zygotes contain bridges that are similar to what is observed in kar5 mutant zygotes, suggesting that Jem1/Kar8p functions after Kar5p.
In the present work, we analyzed nuclear structures in zygotes of nuclear fusion mutants, jem1, sec71 and jem1sec71 by EM using the freeze-substituted fixation method. This method has an advantage over the conventional permanganate-fixation method used in the previous studies (Kurihara et al. 1994; Brizzio et al. 1999) in that it gives minimal effects on intact proteinaceous structures such as the SPB, nuclear pores and microtubules. On the basis of the EM pictures and the three-dimensional organization of the nuclear structures reconstituted from serial EM sections we will discuss the roles of Jem1p in the nuclear fusion process.
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Results |
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We performed EM analyses of nuclear membranes and SPBs in zygotes of jem1, sec71 and jem1sec71 mutants by using the freeze-substituted fixation method. Haploid mutant cells of the opposite mating types were mated for 2 h at 30 °C, and zygotes were fixed and prepared for EM analyses. Figure 1A shows an electron micrograph of a thin section of the jem1 mutant zygote, and Fig. 1B–D show magnifications from serial sections. Bridges connecting two juxtaposed haploid nuclei (indicated by small allows in Fig. 1D) were observed. The bridges were continuous to the outer nuclear membrane of the haploid nuclei and the lumen was clearly observed. EM analyses of 42 jem1 mutant zygotes by serial sections showed that nuclear fusion can take place at multiple fusion sites; while a single bridge was observed in 11 mutant zygotes, two, three and four bridges were also found in 23, 5 and 3 mutant zygotes, respectively. On the other hand, fusion of the inner nuclear membrane was not observed in any sections of the jem1 mutant zygotes analyzed.
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mutant zygotes (Fig. 2A–D, Supporting Information Movie S1). The two haploid nuclei were connected by tubular and cisternal elements, and the cisternal elements sometimes spanned even as long as 10 sections (60 nm thickness/section; Fig. 2D).
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mutant zygotes, sec71 mutant zygotes showed different nuclear structures (Fig. 3; Fig. 3B–E show magnifications from serial sections). Although the outer membranes of the two haploid nuclei were in close contact at the half bridge region (Fig. 3D, small arrows), we could not observe a bridge containing a lumen between the two juxtaposed nuclei. Connection of the two nuclei was observed only in a single EM section and gaps were found between the two membranes in the neighboring sections (Fig. 3B,C,E). No bridge containing a lumen was observed in any sections of the 32 analyzed mutant zygotes with two apposed nuclei. Three-dimensional reconstitution of the nuclear structure of a sec71 mutant zygote showed that two haploid nuclei are in close contact at one small region (Fig. 3C,D).
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sec71 double mutant showed similar nuclear structure organization to that observed in sec71 mutant zygotes (Fig. 4; Fig. 4B–E show magnifications from serial sections). Two haploid nuclei were in close contact at a small region near the SPBs, and no bridge with a lumen connecting the two nuclei was observed in any sections of the 37 mutant zygotes examined. These results are consistent with the previous report by Brizzio et al. (1999) that Sec71/Kar7p and Kar5p function before Jem1/Kar8p in karyogamy.
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Defects in membrane fusion of karyogamy mutants can be analyzed by the in vitro homotypic ER/nuclear membrane fusion assay developed by Latterich & Schekman (1994). This assay measures intermixing of ER luminal contents, which is expected to reflect the ability of the ER/nuclear membrane to fuse from the cytosolic side. Briefly, the acceptor membrane carries wild-type glucosidase I encoded by the CWH41 gene (Romero et al. 1997), which trims terminal glucosyl residues from core glycosylated proteins in the ER, and the donor membrane carries a 35S-labeled substrate for glucosidase. An in vitro synthesized precursor protein, prepro--factor was translocated into the donor membrane prepared from the cwh41 mutant, to serve as a substrate for the membrane fusion assay. Membrane fusion was assessed by measuring the percent of carbohydrate trimming of the imported ER form of -factor during incubation with the acceptor membrane prepared from wild-type (CWH41) cells. As a control, fusion between wild-type membranes exhibited 15%–20% trimming at 30 and 37 °C (Fig. 5, wt). Membranes from the jem1 mutant did not show defects in membrane fusion as well (Fig. 5, jem1), which is different from the previous report by Kurihara et al. (1994). On the other hand, membranes from the sec71 mutant showed temperature sensitive defects in membrane fusion (Fig. 5, sec71). Our results of the homotypic ER membrane fusion assay for wild-type, jem1 and sec71 cells fit well with the EM observation that the outer nuclear membranes can fuse with each other in jem1 mutant zygotes, but not in sec71 mutant zygotes. We thus conclude that Jem1p is not required for the ER/nuclear membrane fusion from the cytosolic side.
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EM ultrastructural analyses with the freeze-substituted fixation method enabled us to analyze intact organizations of proteinaceous structures such as SPBs and microtubules. Our results show that the jem1 mutant is also defective in the SPB fusion; each haploid nucleus in jem1 mutant zygotes contained SPBs (Fig. 1B,C, arrows). On the other hand, unfused nuclei had an ability to enter the mitotic nuclear division cycle. As shown in the three-dimensional reconstituted images of the nuclear structures (Fig. 2A,B), we sometimes observed unfused but duplicated SPBs and spindles in each nucleus (Fig. 1B,C, arrows). Nuclear microtubules emanating from the inner plaque of the SPB were often observed in jem1 mutant zygotes (Fig. 1B,C, arrowheads). These results suggest that the mitotic nuclear division cycle can start without completion of the nuclear membrane fusion and SPB fusion.
We further examined the nuclear fusion and division in zygotes by analyzing the spindle morphology. To visualize spindles in zygotes using fluorescence microscope, haploid cells expressing the GFP-Tub3p (-tubulin) fusion protein were mated. Spindle morphology was examined by GFP fluorescence and nuclei were visualized by DAPI staining. On the basis of those analyses, zygotes were classified into five different types with respect to the nuclear and spindle organization (Fig. 6). Type 1 and type 2 zygotes are those for Kar cells in which karyogamy proceeds normally. In type 1 zygotes, mitotic nuclear division was not complete, so that single nucleus with a short spindle was observed (Fig. 6A–C). On the other hand, in type 2 zygotes, mitotic nuclear division was completed, so that two nuclei connected by a nuclear spindle were observed (Fig. 6D–F). The other three types of zygotes correspond to Kar– zygotes. Type 3 zygotes have two haploid nuclei that are in close contact but do not fuse. The SPB of each nucleus was observed around the region where the two nuclei were in contact, and nuclear microtubules emanated from the SPBs to the other side of the nuclei (Fig. 6G–I). Type 4 zygotes also contained two haploid nuclei in close contact, but only short spindles were observed in each nucleus, indicating that SPB duplication had already occurred (Fig. 6J–L). In type 5 zygotes, the first mitotic division of at least one of the unfused haploid nuclei was completed, and these zygotes contained three or four nuclei. Mitotic division of the two haploid nuclei in single mutant zygotes was not synchronized, so that we often observed zygotes containing three nuclei, that is, one nucleus containing a short spindle and two nuclei connected by a spindle. We also observed zygotes containing four nuclei and two spindles connecting each pair of the nuclei in Kar– zygotes (Fig. 6M–O).
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, sec71 and jem1sec71 cells of the opposite mating types expressing the GFP-Tub3p fusion protein were mated at 30 °C for 2 h or 4 h, and the ratios of zygote types in each cross were analyzed (Table 1). Wild-type crosses resulted in zygotes, 96% of which exhibited the Kar phenotype and were classified into type 1 (77%) zygotes and type 2 (19%) zygotes after 2 h of mating. Prolonged (4 h) incubation increased the fraction of type 2 zygotes to 33%, indicating progression of mitotic nuclear division in those zygotes. On the other hand, when jem1 cells of the opposite mating types were mated for 2 h, all zygotes exhibited Kar– phenotypes and were classified into type 3 (50%), type 4 (37%) and type 5 (13%) zygotes. After 4 h of mating, the fraction of type 3 zygotes decreased to 4%, whereas type 4 and type 5 zygotes became 32% and 59%, respectively. These results indicate that, in jem1 zygotes, haploid nuclei were still capable of proceeding to enter the mitotic division cycle when they failed in nuclear fusion. The unfused haploid nuclei appear to retain their abilities to divide even after the first mitotic division, as we often observed cells containing more than two nuclei (Fig. 6K,L). Progression of mitotic nuclear division was also observed for sec71 zygotes, although it was slightly delayed. Two hour of mating of sec71 cells resulted in type 3 (77%) and type 4 (20%) zygotes, but type 5 zygotes were not observed at this time point. Four hours of mating resulted in decrease in the fraction of type 3 zygotes to 11%, but instead the fractions of type 4 and type 5 zygotes increased to 46% and 25%, respectively. Crosses of jem1sec71 cells gave essentially the same results as were obtained for the crosses of sec71 mutant cells.
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Discussion |
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, sec71 and jem1sec71 mutant zygotes using electron microscope. The EM pictures showed that bridges were formed between apposed nuclei in jem1 mutant zygotes, suggesting the fusion of the outer, but not the inner, nuclear membranes (Fig. 1). In parallel with this observation, the in vitro ER membrane fusion assay revealed that ER/nuclear membranes prepared from jem1 mutant cells are proficient for homotypic membrane fusion. This result does not agree with the previous observation (Kurihara et al. 1994) that ER/nuclear membranes prepared from the kar8-1333 mutant were defective in homotypic membrane fusion. The reason for the discrepancy is not clear at the moment. In contrast to jem1 zygotes, EM analyses showed that both sec71 mutant zygotes and jem1sec71 double mutant zygotes were defective in the outer nuclear membrane fusion, and ER/nuclear membranes prepared from sec71 mutant cells were defective in homotypic membrane fusion in vitro. As reported by Rose and colleagues (Kurihara et al. 1994; Brizzio et al. 1999) previously, the fusion defect of sec71 membranes was observed only when membranes were incubated at 37 °C but not at 30 °C. In contrast in the present study, the sec71 mutant zygotes showed the nuclear fusion defect at 30 °C. Probably, there is a step requiring Sec71p in outer-membrane fusion, which could be bypassed in the in vitro fusion assay using isolated membranes. Taken together, we concluded that Jem1p functions after the outer nuclear membrane fusion i.e. downstream of Sec71p in karyogamy. This sequence of the nuclear membrane fusion events seems to be conserved in karyogamy of the other yeast species as suggested by the EM analyses of the fission yeast, Schizosaccharomyces pombe, zygotes (Hirata & Tanaka 1982).
While Melloy et al. (2007) reported a single nuclear bridge between the two haploid nuclei in a wild-type zygote, we observed multiple bridges in jem1 mutant zygotes (Fig. 1). In wild-type zygotes, two haploid nuclei are in close contact at their SPBs, and nuclear fusion starts at the region near the SPB and completes within minutes (Melloy et al. 2007). In the case of jem1 mutant zygotes, nuclear fusion was arrested after the outer membrane fusion over a prolonged period. Probably, outer-membrane fusion can take place at any region of the nuclei that is not restricted to the region close to the SPBs, and this could result in multiple nuclear bridges observed in jem1 mutant zygotes.
Melloy et al. (2007) reported that nuclear fusion during yeast mating occurs by three consecutive fusions of the outer nuclear membrane, the inner nuclear membrane and the SPB. Because jem1 mutant cells are defective in the fusion of the inner nuclear membranes, Jem1p most likely functions in the inner nuclear membrane fusion. Owing to the advantage of the freeze-substituted fixation method, we were able to analyze by EM the structures of intact SPB and microtubules in zygotes, which were hardly retained by the permanganate fixation used in the previous studies (Kurihara et al. 1994; Brizzio et al. 1999), and found that jem1 mutant cells are defective in the SPB fusion during mating. Our results provide genetic evidence that completion of nuclear inner-membrane fusion is required for the SPB fusion. Another possibility is that Jem1p also functions in the SPB fusion. We previously identified Nep98/Mps3p, an essential nuclear membrane protein localized in the half bridge of the SPB as a Jem1p interacting protein. The temperature-sensitive nep98-7 mutant showed defects in both nuclear congression and nuclear fusion, indicating that Nep98p is involved in the karyogamy pathway (Nishikawa et al. 2003). Jem1p likely interacts with Nep98p at the half bridge where nuclear membrane fusion starts, thereby regulating the SPB fusion in karyogamy. Alternatively, it is possible that the inner nuclear membrane fusion requires the fusion activity of SPB. Jem1p may function in inner-membrane fusion through regulating the SPB fusion. Furthermore analyses will reveal whether Jem1p-Nep98p interaction also regulates the inner nuclear membrane or not.
We observed here that the zygotes could enter the first mitotic cell cycle without completion of the nuclear membrane fusion. The SPB duplication and mitotic nuclear division proceeded in half the jem1 zygotes that failed in nuclear fusion after 2 h of mating. Approximately 90% of the mutant zygotes contained nuclei in the mitotic division cycle after 4 h, suggesting that the efficiency in nuclear division in jem1 zygotes is similar to that in wild-type zygotes. At the initial stage of mating, haploid cells respond to mating pheromones, rendering their cell division cycle arrested in the G1 phase. The observed early onset of the mitotic division cycle in jem1 zygotes means that cells were released from the G1 arrest at some point during mating, and that completion of the nuclear fusion event is not essential for initiation of the mitotic nuclear division cycle. In contrast to jem1 zygotes, only 20% of the sec71 zygotes contained nuclei with duplicated SPBs and approximately 80% of the mutant zygotes contained nuclei in the process of nuclear fusion. Failure in the outer nuclear membrane fusion may cause delay in the onset of mitotic nuclear division cycle in sec71 zygotes. Nevertheless, release from the G1 arrest does not require completion of nuclear fusion in mating, so that division of the haploid nuclei results in production of growing cells with haploid nuclei that look like cytoductants in the crosses of Kar– mutants. It should be noted that we often observed asynchronous division of two haploid nuclei in Kar– zygotes. This is in striking contrast to what was observed in vertebrate cells. In vertebrate cells, two independent spindles in the common cytoplasm elongate in the similar timing (Rieder et al. 1997). Because nuclear envelope breakdown does not occur during the yeast mitosis, asynchronous elongation of two spindles in Kar– zygotes may suggest that the anaphase onset is regulated by factors in the nucleoplasm, not in the cytosol. Identification and analysis of machineries directly involved in the nuclear membrane fusion during mating will reveal the mechanisms that regulate the sequential events of nuclear fusion and division in the yeast mating process.
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Experimental procedures |
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Yeast strains used in this study are SEY6210 (MAT
ura3 leu2 trp1 his3 lys2 suc2), SEY6211 (MATa ura3 leu2 trp1 his3 ade2 suc2) (Robinson et al. 1988), SNY1028 (MAT
jem1::LEU2 ura3 leu2 trp1 his3 lys2 suc2), SNY1029 (MATa jem1::LEU2 ura3 leu2 trp1 his3 lys2 suc2) (Nishikawa & Endo 1997). The SEC71 gene was cloned by PCR using primers 5'-GCGCTCGAGCGTCAAAA GTTTTAGGAAC-3' and 5'-GCGGAGCTCTTAAGTAGTGA GCAAGAAG-3', and the amplified DNA fragment was introduced into the XhoI and SacI sites of pBluescriptII SK(+) to give pBSSEC71. The sec71::LEU2 allele was constructed by replacing the 470-bp MunI/XbaI fragment of pBSSEC71 with the 2-kb SalI/XbaI fragment of pJJ283 (Jones & Prakash 1990) containing the LEU2 gene. The constructed null allele was introduced into SEY6210 by the one-step gene disruption method (Rose et al. 1990) to produce SNY1039 (MAT
sec71::LEU2 ura3 leu2 trp1 his3 lys2 suc2). SNY1040-7C (MATa sec71::LEU2 ura3 leu2 trp1 his3 lys2 suc2) was constructed by crossing SNY1039 and SEY6211. The jem1::LEU2 sec71::LEU2 double mutants used in this study are SNY1053-10D (MATa jem1::LEU2 sec71:: LEU2 ura3 leu2 trp1 his3 suc2) and SNY1053-17D (MAT
jem1::LEU2 sec71::LEU2 ura3 leu2 trp1 his3 suc2), which were constructed by crossing SNY1029 and SNY1039.
Yeast strains used for in vitro membrane fusion assay were constructed as follows. A plasmid, pBSPEP4, was constructed by cloning the 1.9-kb XhoI/SacI fragment containing the PEP4 gene of the genomic clone (a gift from Dr Y. Wada) into the XhoI and SacI sites of pBluescriptII SK(+). The pep4::TRP1 allele was constructed by replacing the 850-bp EcoRI/EcoRV fragment of pBSPEP4 with the 820-bp EcoRI/StuI fragment of pJJ280 (Jones & Prakash 1990) containing the TRP1 gene. The CWH41 gene was cloned by PCR using primers 5'-GCGGAGCTCTACTTC AGAGGCATTCAG-3' and 5'-CGCCTCGAGTGATAGTCG TAGTTGG-3', and the amplified DNA fragment was introduced into the XhoI and SacI sites of pBluescriptII SK(+) to give pBSCWH41. The cwh41::HIS3 allele was constructed by replacing the 1.1-kb XbaI/BamHI fragment of pBSCWH41 with the 1.3-kb XhoI/BamHI fragment of pJJ217(Jones & Prakash 1990) containing the HIS3 gene. Null alleles were introduced into yeast cells by the one-step gene disruption method (Rose et al. 1990). Multiple mutants were generated by the second round of transformation. Genotypes of the constructed strains are SNY1062 (MAT
pep4::TRP1 ura3 leu2 trp1 his3 lys2 suc2), SNY1063 (MAT
pep4::TRP1 jem1::LEU2 ura3 leu2 trp1 his3 lys2 suc2), SNY1064 (MAT pep4::TRP1 sec71::LEU2 ura3 leu2 trp1 his3 lys2 suc2), SNY1066 (MAT
pep4::TRP1 cwh41:: HIS3 ura3 leu2 trp1 his3 lys2 suc2), SNY1067 (MAT pep4:: TRP1 cwh41::HIS3 jem1::LEU2 ura3 leu2 trp1 his3 lys2 suc2) and SNY1068 (MAT
pep4::TRP1 cwh41::HIS3 sec71::LEU2 ura3 leu2 trp1 his3 lys2 suc2).
A plasmid for expression of GFP-Tub3p from the CUP1 promoter was constructed as follows. A DNA fragment containing the TUB3 ORF was amplified by PCR using primers 5'-GCGCT CGAGTGAGAGAGGTCATTAGTA-3' and 5'-TATCCGCGG TTAGAACTCCTCAGCGTA-3'. The amplified DNA fragment was digested with XhoI and SacII and introduced into the XhoI and SacII sites of pTYSC157 (T. Yoshihisa, personal communication) to give YCpUC-GFP-TUB3. Standard recombinant techniques were used with Escherichia coli strain TG1 (supE hsd5 thi
(lac-proAB) F'[traD36 proAB+
lacIq lacZM15]).
Yeast cells were grown in YPD medium (1% yeast extract, 2% polypeptone and 2% glucose) or SCD medium (0.67% yeast nitrogen base without amino acids, 0.5% casamino acids and 2% glucose) supplemented with 20 µg/mL each of adenine and tryptophan. Quantitative mating assays were performed as described previously (Nishikawa & Endo 1997). Yeast transformation was performed as described previously (Keszenman-Pereyra & Hieda 1988).
Microscopic analysis
The synchronyzed mating reaction was performed as described by Azpiroz & Butow (1993). For fluorescence microscopy, cells on the membrane filters were suspended in sterile water and fixed as reported (Nishikawa et al. 1994). The fixed cells were suspended in 1 M sorbitol, 0.1 M Hepes–NaOH, pH 7.5, 5 mM sodium azide, 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) and observed using an inverted fluorescence microscope (IX70; Olympus) equipped with fluorescein or DAPI filter sets. Cell images were taken by using a cooled CCD camera system (MicroMAX; Princeton Research Instruments) with IPLab image processing software (Scanalytics Inc.). For electron microscopy, cells on the membrane filters were scraped off with one-hole copper grids and fixed by the freeze-substituted fixation method (Sun et al. 1992). The sections were examined and photographed on a JEOL2010 transmission electron microscope or a Hitachi H-7600 transmission electron microscope at 100 kV. Preparation of serial thin sections and computer-aided three-dimensional reconstructions were performed as reported (Sun et al. 1992) except that TRI 3D software (Ratoc System Engineering Co., Ltd, Tokyo, Japan) was used. The three-dimensional images of jem1, sec71 and jem1sec71 mutant zygotes were constructed based on 45, 26 and 22 serial sections, respectively.
In vitro ER fusion assay
Reagents for the in vitro homotypic ER membrane fusion assay were prepared as described by Latterich & Schekman (1994). Microsomal membranes were prepared from yeast cells grown at 30 °C (wild type and jem1 strains) or at 23 °C (sec71 strains) in YPD. We used null mutants of the CWH41 gene, which encodes glucosidase I (Romero et al. (1997), as glucosidase I-negative strains instead of the gls1-1 mutant strains used in the original paper. Following strains were used in this assay; SNY1062 (CWH41), SNY1066 (cwh41), SNY1063 (jem1
CWH41), SNY1067 (jem1
cwh41), SNY1064 (sec71
CWH41), SNY1068 (sec71
cwh41). Fusion assay reactions consist of 37.5 µg of cwh41 membranes containing untrimmed gpF, 37.5 µg of CWH41 membranes and an ATP regeneration system, in a total volume of 25 µL. Fusion was allowed to proceed for 1 h either at 30 °C or at 37 °C as indicated. Chilled reactions were treated with trypsin, and then trypsin inhibitor to degrade gpF that was not membrane enclosed. Reactions were terminated by the addition of 15 µL of 3X Laemmli sampling buffer with β-mercaptoethanol at final concentration of 2%, and the proteins were analyzed by SDS-PAGE. The percentage of glucose trimming was quantified by radioimaging using a Storm 860 image analyzer (GE Healthcare).
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Acknowledgements |
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Footnotes |
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* Correspondence: shuh{at}biochem.chem.nagoya-u.ac.jp
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References |
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Accepted: 25 August 2008
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