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© The Rockefeller University Press,
0021-9525/1998//247 $5.00
The Journal of Cell Biology, Volume 140, Number 2,
, 1998 247-258
Article |
A Novel Fission Yeast Gene, tht1+, Is Required for the Fusion of Nuclear Envelopes during Karyogamy
Kansai Advanced Research Center, Communications Research Laboratory, Kobe 651-24, Japan
We have isolated a fission yeast karyogamy mutant, tht1, in which nuclear congression and the association of two spindle pole bodies occurs but the subsequent fusion of nuclear envelopes is blocked. The tht1 mutation does not prevent meiosis, so cells execute meiosis with two unfused nuclei, leading to the production of aberrant asci. The tht1+ gene was cloned and sequenced. Predicted amino acid sequence has no significant homology to previously known proteins but strongly suggests that it is a type I membrane protein. The tht1+ gene is dispensable for vegetative growth and expressed only in conjugating cells. Tht1p is a glycoprotein susceptible to endoglycosilase H digestion. Site- directed mutagenesis showed that the N-glycosylation site, as well as the COOH-terminal region of Tht1p, is essential for its function. A protease protection assay indicated that the COOH terminus is cytoplasmic. Immunocytological analysis using a HA-tagged Tht1p suggested that the protein is localized in nuclear envelopes and in the ER during karyogamy and that its levels are reduced in cells containing fused nuclei.
Abbreviations used in this paper: DAPI, 4,6-diamidino-2-phenylindole; EDTA, ethylenediaminitetraacetate; endoH, endoglycosilase H; FISH, fluorescent in situ hybridization; GFP, green fluorescent protein; NSF, N-ethylmaleimide-sensitive factor; ORF, open reading frame; SPB, spindle pole body.
Address correspondence to Osami Niwa, Kazusa DNA Research Institute, 1532-3 Yana, Kisarazu, Chiba 292, Japan. Tel: +81 438 52 3923; FAX: +81 438 52 3924; E-mail: niwa{at}kazusa.or.jp
KARYOGAMY, or nuclear fusion, is a process in which two haploid nuclei fuse to produce a diploid nucleus in yeast. Genetic and cytological analyses in Saccharomyces cerevisiae showed that karyogamy consists of at least two distinct processes: one is the congression of two nuclei and the other is the fusion of nuclear envelopes of juxtaposed nuclei (Rose, 1991; Kurihara et al., 1994). The mixing of chromosomes and nuclear matrices brought by the two nuclei must follow envelope fusion and results in the formation of a diploid nucleus, however, this aspect of karyogamy has received little attention to date. Genes identified in S. cerevisiae as required for the nuclear congression include KAR1, KAR3, CIK1, BIK1, and TUB2, which are all components of spindle pole body (SPB)1– microtubular system (Rose, 1991). These factors are also shown to be either essential or important for vegetative growth. KAR4 is involved in the expression of KAR3 and CIK1 and therefore also required for the nuclear congression (Kurihara et al., 1996). On the other hand, a class of endoplasmic reticulum proteins, such as Kar2p (BiP), Sec63p, Sec71p, Sec72p, and Jem1p (Normington et al., 1989; Rose et al., 1989; Ng and Walter, 1996; Nishikawa and Endo, 1997), were all shown to be required for the fusion of nuclear envelopes. BiP is know to be a ubiquitous ER lumenal protein which is a member of stress-inducible chaperones. BiP functions in modulating protein folding and protein translocation (Gething and Sambrook, 1992). Sec63p, Sec71p and Sec72p form a membrane-bound complex required for protein translocation (Deshaies et al., 1991; Green et al., 1992; Ng and Walter, 1996), and Kar2p has been shown to interact with Sec63p (Scidmore et al., 1993; Brodsky et al., 1995). However, it remains to be elucidated how these ER proteins are involved in the nuclear envelope fusion.
We have been interested in the karyogamy in Schizosaccharomyces pombe, because cytological studies have proven it to be an excellent model system to study the regulation of nuclear organization. Very dynamic yet genetically regulated rearrangement of chromosomes, as well as morphological changes in nuclear shape, can be observed upon entry into and during karyogamy and subsequent meiosis in the fission yeast. In vegetatively proliferating cells, centromeres are either located near the SPB as a cluster or linked to the SPB with microtubules. Thus, the SPB take a major role in the positioning and movement of chromosomes (Funabiki et al., 1993). In sharp contrast to the situation in the mitotic cell cycle, during karyogamy and meiotic prophase telomeres form a single cluster near the SPB and dominate in the chromosomal movement (Chikashige et al., 1994, 1997). The SPB appears to play a vital role not only in these chromosomal events but also in the movement of nucleus, that is, during both congression of two haploid nuclei and the oscillatory movement of a meiotic prophase nucleus along the long axis of the zygote, the SPB leads the nuclear movement (Chikashige et al., 1994). The nucleus in the meiotic prophase is also known to have characteristically elongated and ever changing morphology, often referred to as a "horse tail" nucleus (Robinow, 1977).
We have recently isolated a fission yeast mutant, kms1 (Shimanuki et al., 1997), which does not form the smoothly elongated horse tail nucleus in meiotic prophase but produces an aberrantly shaped nucleus in which the telomeres often fail to form a single cluster. Instead they tend to be distributed in multiple clusters. The kms1 mutant is also impaired in karyogamy and in the meiotic chromosome segregation and sporulation. These mutant phenotypes are most likely correlated with the failure of SPB function. In fact, we recently found that the kms1+ gene product may function as an indispensable component of the SPB when the telomere cluster associates with it (Shimanuki, M., unpublished observation). The observations of the kms1 phenotype further emphasized the importance of SPB function and chromosomal organization in sexual nuclear processes in fission yeast. In an effort to identify more factors involved in such a process, we previously made a collection of mutants that were defective in diploid formation after conjugation (Tange and Niwa, 1995). In this study we have isolated and characterized a mutant that is defective in the tht1 gene. Results described in this report indicate that the nuclear congression and the association of two SPBs takes place but that the subsequent fusion of nuclear envelopes is blocked in the mutant. The tht1+ gene encodes a novel glycoprotein transiently produced during conjugation. It is the first gene identified in the fission yeast that may be specifically required for the fusion of nuclear membranes during karyogamy.
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Materials and Methods |
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Mutant Isolation
We have made a mutant collection based on the genetic background of h90 mei1-102 leu1 tsh1, aiming a class of mutants defective in the diploid formation after conjugation (Tange and Niwa, 1995). Individual strains were examined for the nuclear morphology in zygotes by 4,6-diamidino-2-phenylindole (DAPI) staining. Strain DF4-3 found in this screening was subjected to detailed analyses in this study.
Determination of Ploidy
Homothallic yeast strains were incubated on a MEA medium at 30°C overnight to induce zygote formation. Individual zygotes were transferred onto fresh YEA medium using a micromanipulator and incubated at 30°C for several days. Colonies produced from the separated zygotes were examined for their ploidy by the flow cytometric analysis as described in Tange and Niwa (1995).
Plasmids
The multicopy plasmid pKD10 (Shimanuki et al., 1997) was used for subcloning and complementation test. Fragments inserted into the vector are shown in Fig. 6 A. For the overexpression experiments, pREP1, pREP42 (gifts from Dr. K. Maundrell, Glaxo Institute for Molecular Biology, Geneva, Switzerland), and pAS248 (Toda et al., 1991) were used. pREP1 and pREP42 carry the nmt1-promoter (Maundrell, 1993), which is inducible by thiamine depletion. A DNA fragment covering the tht1+-ORF was produced by PCR and inserted at the NdeI–BamHI site of pREP1 and pREP42 to make pNT45 and pNT97, respectively. pAS248 carries a constitutively active adh promoter. The BamHI–PstI fragment from pNT37 was blunt ended and inserted into the SmaI site in pAS248 to generate pNT79. The HA-tag sequence (AYPYDVPDYAGYPYDVPDYAMGYPYDVPDYA, repeated HA-epitopes are underlined) was amplified from a plasmid pHA41 (a gift from I. Hagan, University of Manchester, Manchester, UK) and inserted at the BsmI, HpaI, StuI, and NcoI sites in pNT28 to make pNT72, pNT73, pNT74, and pNT75, respectively. pNT72, pNT73, and pNT74 could not complement the tht1-1 mutation, whereas pNT75 could. A control plasmid, pNT77, containing the HA-tag sequence was constructed by replacing the NspV fragment of pNT28 with the HA-tag sequence. In this construct the HA-tag is fused to the NH2-terminal 38 amino acids of tht1+. The plasmid pEB9 carries the BiP gene tagged with the myc epitope (Pidoux and Armstrong, 1992). The plasmid D817, which was employed for staining the nuclear envelope in this study, had been isolated from a GFP-fusion genomic library based on a multicopy vector with the LUE2 selection marker. Sequence determination of the D817 clone showed that the 276 amino terminal residues of the 678–amino acid cytochrome P-450 reductase (Miles, 1992) were fused with GFP in this plasmid (Ding, D.-Q., and Y. Hiraoka, manuscript in preparation). GFP-tagged Kms1p was used to stain the SPB, which was expressed from pGK77, a pREP1-based plasmid. The fusion protein produced under the repressive condition was shown to localize to the SPB. Details about the localization of Kms1p will be published elsewhere.
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One Step Gene Disruption
The 2.9-kb SnaBI fragment of the tht1+ gene was inserted in the SmaI site of Bluescript II (Stratagene, La Jolla, CA), and the ura4+ gene sequence was substituted with the 1.7-kb NspV fragment in the insert to generate pNT31. This substitution nearly completely disrupts the tht1+ gene, leaving only 38 amino acid residues at its NH2 terminus. The KpnI–SacI fragment of pNT31 was excised and transformed into a diploid cell to obtain stable transformants, for which Southern blot hybridization was performed to verify the correct integration. For the disruption of the 740-bp open reading frame (ORF), the 2.2-kb NspV fragment bearing the ORF was inserted into the EcoRV site in Bluescript II (pNT60). The EcoT14I– EcoRV fragment of pNT60 was replaced with the ura4+ sequence to completely remove the ORF sequence and then the XhoI–SpeI fragment was cut out of this plasmid for transformation.
Northern Blot Hybridization
Total RNA was extracted according to Alfa et al. (1993). Poly(A)+ mRNA was purified by using the oligotex-dT30 (super; Takeda Pharmaceutical Co., Osaka, Japan). RNA probes were used for hybridization. Each probe was transcribed from the T7 promoter using the DIG RNA labeling kit (Boehringer Mannheim Corp., Indianapolis, IN) with the following template plasmid DNAs. For probe 1 (see Fig. 8) the XhoI–RsaI fragment (from nucleotide –1202 to –189, numbering is from the initiation codon of the tht1+ gene) was ligated with the XhoI–EcoRV site of Bluescript II to make pNT46. XhoI-digested pNT46 DNA was used as the template. For probe 2 the HindIII–EcoRV fragment (from nucleotide –1271 to +670) was cloned into the HindIII–SmaI site of pKD10 (pNT25). pNT25 was digested at the SalI site (located at –39). For probes 3, 4, and 5 the EcoRV fragment (+670 to +1588), the MunI fragment (+2030 to +2482; blunt ended), and the DdeI fragment (+2781 to +3333; blunt ended) were inserted at the EcoRV site of Bluescript II in an appropriate orientation to make pNT42, pNT58, and pNT59, and then digested with HindIII, HindIII, and XhoI, respectively, before use. Another plasmid, pNT47, was made by ligating the RsaI–HindIII fragment (+2356 to +2922) with the HincII and HindIII sites in Bluescript II followed by digestion with XhoI. This probe was used for the analysis of the disruptant of the 740-bp ORF (see text). Hybridization and washing was conducted using the standard conditions recommended by Boehringer Mannheim Corp., with the exception of probe 4, which was hybridized in a high SDS buffer without formamide to reduce the stringency of hybridization (Church and Gilbert, 1984). Band detection was performed with the DIG luminescent detection system (Boehringer Mannheim Corp.).
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Preparation of Cell Extracts and Western Immunoblotting
Cell extracts for Western immunoblotting were prepared according to Moreno et al. (1991) and Funabiki et al. (1996) with modifications. 3 x 107 cells were harvested and washed once with ice-cold STOP buffer (150 mM NaCl, 50 mM NaF, 10 mM ethylenediaminitetraacetate [EDTA], 1 mM NaN3, pH 8.0). Cells were resuspended in 150 µl of disruption buffer (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 10% glycerol, 30 mM NaCl, 1 mM DTT, 1 mM PMSF, 2 µg/ml pepstatin A, 10µM E-64) and disrupted with 0.3g glass beads (425–600 µm; Sigma Chemical Co., St. Louis, MO) by vortexing four times each for 20 s. 200 µl of disruption buffer was added to the disrupted cell suspension and centrifuged at 1,500 g for 5 min. Supernatant fraction was then centrifuged at 57,000 g for 20 min. Resultant supernatant was referred as the soluble fraction and the pellet was suspended in 60 µl of disruption buffer and referred as the insoluble fraction. To each fraction, one-fourth volume of loading buffer (0.25 M Tris-HCl, pH 6.8, 4% SDS, 40% glycerol, 10% mercaptoethanol, 40 mg/ml bromophenol blue) was added and boiled for 5 min and then chilled on ice. In some cases this boiling step was omitted. Before loading on a gel, samples were centrifuged at 10,000 g for 5 min to remove insoluble materials. Alternatively, cell extracts were prepared according to the alkaline lysis method (Silve et al., 1991) with a slight modification. 1.5 x 107 cells were washed once in 0.5 ml of ice–cold STOP buffer and resuspended in a minimum amount of STOP buffer. 0.5 ml of 1.85 M NaOH 7.5% mercaptoethanol was added to the cell suspension and incubated for 10 min on ice. 0.5 ml of 50% TCA was mixed and kept on ice for 10 min, followed by a centrifugation at 15,000 g for 10 min. The pellet was rinsed with 0.5 ml of 1 M Tris-OH and suspended in 50 µl of disruption buffer plus 17 µl of loading buffer. This mixture was kept on ice for 20 min and then centrifuged at 10,000 g for 5 min to remove insoluble materials. Proteins were run on 12.5% PAGE and transferred to a nitrocellulose membrane. Protein detection was performed by a chemiluminescence detection system (ECL; Amersham Corp., Arlington Heights, IL) with horse radish peroxidase-conjugated protein A (Amersham Corp.).
Protease Protection Assay
Methods described by Baker et al. (1990) and Garnier et al. (1996) were modified as follows. 5 x 109 conjugating cells were harvested after 5 h incubation in MSM medium, washed once with 50 mM Tris-HCl, pH 7.5, 50 mM EDTA and once again with the same buffer containing 1.2 M sorbitol (TES). Cells were digested at 30°C for 45 min with 0.5 mg/ml Zymolyase 100T (Seikagaku America, Inc., Rockville, MD) and 0.2 mg/ml lysing enzymes (Sigma Chemical Co.) in TES at a cell density of 5 x 108 cells/ml. The digested cells were washed once with solution A (50 mM Tris-HCl, pH 7.5, 10 mM EDTA, 30 mM NaCl, 0.5% 2-mercaptoethanol, 1 mM PMSF, 2 µg/ml pepstatin A, 10 µM E-64) containing 1.2 M sorbitol and suspended in 4 ml of lysis buffer (solution A containing 0.1 M sorbitol). The suspension was homogenized by a Dounce homogenizer (10 strokes), followed by centrifugation at 3,000 g for 10 min. The supernatant was centrifuged at 12,000 g for 10 min. The resultant pellet was suspended in 0.4 ml of lysis buffer, layered onto two tubes of 2.0-ml sucrose step gradient (1.0 ml each of 1.5 M sucrose and 1.2 M sucrose in lysis buffer), and then centrifuged at 100,000 g for 1 h. The microsomes at the 1.2/1.5 M interface were collected and washed once with lysis buffer and once with reaction buffer (lysis buffer without protease inhibitors), before being suspended in 1.0 ml of reaction buffer. 100-µl aliquots were incubated with TPCK-treated trypsin (0-5 µg/ml; Sigma Chemical Co.) at 25°C for 10 min. Reaction was terminated by adding 25 µl of 50% TCA. After 15 min incubation on ice, the pellet was collected (15,000 g for 10 min) and dissolved in 50 µl of solution A. Where indicated, Triton X-100 was added at a final concentration of 0.1% and the resulting solution was incubated at 25°C for 5 min before the addition of trypsin.
Overexpression of the tht1+ Gene
For the overexpression of the tht1+ gene in vegetative cells, HM123 (h– leu1) was transformed either with pNT45 or with pNT79. Transformants carrying pNT45 were transferred from EMM2 with thiamine to the thiamine-depleted medium at the cell density of 3 x 105 cells/ml at 30°C to induce the expression of the tht1+ gene.
Digestion with Endoglycosilase H
Cell extracts prepared by the alkaline lysis method were digested with endoglycosilase H (endoH; New England Biolabs Inc., Beverly, MA) according to the manufacturer's instructions.
Site-directed Mutagenesis
The Quikchange site-directed mutagenesis kit (Stratagene) was used to introduce desired mutations in pNT28. For each mutant gene the entire nucleotide sequence of the ORF was determined to confirm that only the desired changes had been introduced.
Fluorescent Staining
For indirect immunofluorescent staining, we followed the procedure described in Chappell and Warren (1989) but Novozyme was omitted from the digestion mixture. In this method, 4% formaldehyde was used for fixing cells but glutaraldehyde was not included. In some experiments, including GFP-DAPI double staining, the method described in Shimanuki et al. (1997) was followed. But for double staining, cell wall digestion was performed only briefly and the Triton X-100 treatment was omitted. Mouse monoclonal anti–HA antibody (12CA5; Boehringer Mannheim Corp.) was used in combination with a goat anti–mouse Cy3-conjugated antibody (The Jackson Laboratories, Bar Harbor, ME). We also used a goat anti–rabbit Cy3-conjugated antibody (Jackson Laboratories). For immunolocalization of the tagged BiP, mouse monoclonal anti–c-myc antibody (9E10; Sigma) was used (Pidoux and Armstrong, 1992). For membrane staining 0.25 or 0.125 µg/ml of DiOC6(3) (3,3'-dihexyloxacarboctanine iodide; LAMBDA, Graz, Austria) was added (Terasaki, 1994). Fluorescent in situ hybridization (FISH) was performed using a telomere specific probe cos212 according to Shimanuki et al. (1997). Microscopic observation was carried out with the Delta Vision system (Applied Precision Inc., Issaquah, WA). Coloring in all figures was arbitrary.
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85% were found to form haploid colonies. Under the same condition, tht1+ zygotes produced diploid colonies in >90% of the cases.
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1.8 kb that was interrupted with two introns. The presence of these introns has been confirmed by a PCR based cDNA analysis. We found that a cosmid sequence that had been previously deposited in the EMBL/ GenBank/DDBJ database (available under accession number Z50112) contained the tht1+ sequence. This cosmid (c13C5) had been mapped to the distal portion of the left arm of chromosome I (Hoheisel et al., 1993). We then made a complete loss-of-function mutant allele at the tht1 locus by a one-step gene disruption method (Materials and Methods). Strains carrying the disruptant allele were viable and did not show any defective phenotype in vegetative growth, but, as anticipated, were as defective in karyogamy as the original tht1-1 mutant. The predicted amino acid sequence of Tht1p along with a hydropathy plot is shown in Fig. 6. The calculated mol wt of the 577–amino acid protein is 66.9 kD. Computational searches have failed to find out any known proteins with significant sequence homology, indicating that Tht1p represents a novel class of proteins. There are three potential transmembrane segments in the COOH-terminal half of the protein, with positively charged amino acid residues enriched between segments 1 and 2 and near the COOH-terminal side of segment 3 (Fig. 6). There is also a typical signal peptide sequence at the NH2 terminus. These features of the protein may indicate that the tht1+ gene product is a type I membrane protein, with the COOH-terminal portion being in the cytoplasm (High and Dobberstein, 1992).
The Expression of tht1+ Gene Is Confined to Conjugating Cells
Northern blot hybridization analysis was performed using probes specific for the tht1+ gene (Fig. 7). About 3 h after the induction of conjugation by nitrogen starvation, two kinds of poly(A)+ transcripts of 1.8 and 3.8 kb appeared and the amount of these transcripts peaked at 5 h and then decreased (Fig. 7 A). The timing of the induction was roughly coincident with the appearance of copulating cells. Nitrogen starvation and the presence of two different mating type genes were not sufficient for the induction, since neither heterothallic haploid cells nor diploid cells heterozygous for the mating type gene could produce the transcripts after nitrogen starvation (Fig. 7 B). These results indicated that tht1 gene is only expressed in conjugating cells.
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50 bp upstream from the initiation codon) and that the 1.8-kb mRNA terminates 100 bp downstream from the termination codon. These results indicated that the tht1-ORF is transcribed with two different mRNAs. Since it was possible that the 740-bp ORF has some functional relation to the tht1 gene, we disrupted this ORF by a ura4+ substitution method (Materials and Methods). The disruptant was viable and displayed no karyogamy related phenotype. In this disruptant, the 0.8-kb mRNA was no longer transcribed and the 3.8-kb mRNA became shorter. The amount of the 1.8-kb mRNA in the disruptant was comparable to its level in wild-type controls (data not shown).
Identification of the tht1+ Gene Product
We prepared extracts from cells carrying a multicopy plasmid with the tht1+ gene (pNT28) and performed a Western immunoblot analysis using anti–Tht1p antibodies (1-3-2). We found a 67-kD protein specifically expressed in conjugating cells (Fig. 9 A). The temporal expression profile was very similar to the mRNA accumulation profiles detected by Northern hybridization analysis. Moreover, the protein levels were very low in strains without the plasmid or with the vector alone (Fig. 9 A), suggesting that the 67-kD band represented the tht1+ gene product. This protein partitioned to the insoluble fraction (Fig. 9 B). It could be barely solubilized with 1% Triton X-100 or with 2 M NaCl (data not shown). The same 67-kD insoluble protein was produced when we forced gene expression in mitotic cells by using the adh-promoter (see Fig. 9 C).
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As noted above, it was predicted from the amino acid sequence that the tht1+ gene product is a type I membrane protein, with its COOH terminus in the cytoplasm. To test this prediction, we prepared microsomes from conjugating cells expressing HA-tagged or wild-type Tht1p. One of the tagged proteins, Tht1-NcoI-HA, bears the HA epitope near the COOH terminus, while the other one, Tht1-StuI-HA, contained it on the NH2-terminal side of the presumed membrane-spanning domain. When the microsome samples were incubated with trypsin and subjected to Western immunoblot analysis using anti–HA antibodies, the band intensity of Tht1-NcoI-HA fusion decreased as the amount of trypsin increased (Fig. 11 A, lanes 1– 5). However, when Tht1-StuI-HA was examined there was a shift to a band that migrated with a mobility equivalent to a reduction in size of 3 kD and there was only a partial decrease in band intensity. (Fig. 11 A, lanes 11– 15), indicating that the HA sequence was more resistant to trypsin in Tht1-StuI-HA than in Tht1-NcoI-HA. The 55-kD band that appeared in the preparation of Tht1-StuI-HA was also resistant to trypsin digestion. This band probably corresponded to a polypeptide truncated near the COOH-terminal end of the third transmembrane segment. When trypsin digestion was performed in the presence of Triton X-100, which can permeabilize microsomes, the bands from Tht1-StuI-HA were no longer resistant to trypsin (Fig. 11 A, lanes 16–20). We then used anti–Tht1p antibody 1-3-2, which was raised against an segment from the NH2-terminal part of Tht1p, to probe the tryptic digests. Upon digestion of Tht1-NcoI-HA by trypsin, as the intensity of the 76-kD band decreased, a lower 64-kD band became more intense (Fig. 11 B, lanes 1– 3). The same 64-kD band was produced from Tht1p in the absence of Triton X-100 (Fig. 11 B, lanes 13– 15). In the presence of the detergent, this 64-kD band was not stable in either Tht1-NcoI-HA or Tht1p (Fig. 11 B, lanes 4–6 and 16–18). These results were all consistent with the notion that the COOH terminus of Tht1p is in the cytoplasm and the NH2-terminal portion is in the lumen so that it is protected from attack by trypsin in these assays.
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Aberrant Meiotic Divisions from Unfused Nuclei
In fission yeast meiosis, like in mitosis, the spindle is formed between separated SPBs in the nucleus while keeping nuclear envelope intact (Hirata and Tanaka, 1982). Before the SPB proceeds into the duplication/separation cycle in meiosis, the two haploid SPBs must fuse to make a single unified SPB in karyogamy. A couple of pieces of evidence shown in this study suggest that the SPBs have completed this binding process before the tht1 arrest point. First, in almost all of the cases, only one SPB signal could be observed between two juxtaposed nuclei using the SPB-specific probes, anti–Sad1 antibodies and GFP-fused Kms1p. A second piece of evidence is provided by the coordinated movement of the twin horse tail nuclei, where only a single SPB seems to lead the movement. However, we have not ruled out the possibility that the binding of the SPBs is incomplete in the mutant. Rather, we think it could be a considerable possibility, because there could be strong tension produced by such unusual configuration of unfused and bulky nuclei against the SPB. In any case, there must be a topological problem in the formation of the spindle in a mutant situation such as that seen in tht1 where one unified SPB or two separating SPBs have to be stuck to the two unfused nuclear envelopes. In fact, we could find very few normal spindles and DAPI-stained images of separating nuclei in the mutant zygotes (data not shown). Although we could not strictly rule out the possibility that meiotic division is prohibited from the twin horse tail state, we feel that it is unlikely judging from the efficiency of sporulation. Moreover, the tws1 mutation that blocks meiosis II (Nakaseko et al., 1984), when combined with the tht1 mutation, reduced the number of spores (data not shown), indicating that even in the aberrant meiosis/ sporulation in the tht1 mutant, meiosis II, and therefore meiosis I as well, does take place. In this regard we thought it interesting that a considerable number of mutant asci contained a large nucleus that was not encapsulated with the spore wall (Fig. 5, arrows). Since it is known that spore wall formation initiates from a modified SPB (Tanaka and Hirata, 1982), we first speculated that this type of nucleus might have arisen from an event where the SPB had been stripped off from one of the unfused nuclei. However, to our surprise, such nuclei in fact bore two or three anti–Sad1 stains (data not shown). This phenotype was very unusual and we have currently no plausible explanation for it. Nevertheless, it seems apparent that the SPB-spindle cycle became catastrophic in the mutant zygotic meiosis. It may be also worth considering here that like the "twin meiosis" described by Gutz (1967) where two unfused nuclei go into meiosis separately, the two haploid nuclei might undergo independent meiosis and sporulation (Iino and Yamamoto, 1985; Nurse 1985) in the tht1 mutant, thereby producing aberrant asci. However, if this were the case a large fraction of the mutant asci should contain eight spores, but only very few asci actually contained eight spores. This argument further supports the notion that the SPBs have unified in the tht1 mutant.
The Role of Tht1p in Karyogamy
During conjugation in the tht1 mutant, the juxtaposition of two nuclei is not followed by the fusion of nuclear envelopes. Mutants showing similar defective phenotype have been isolated in S. cerevisiae, those include kar2, kar5, kar7, kar8, sec63, sec71, sec72, and jem1 (Kurihara et al., 1994; Ng and Walter, 1996; Nishikawa and Endo, 1997). For some of these genes their products have been identified, but very little is known about how these genes are involved in nuclear fusion. For instance, Sec63p, Sec71p, Sec72p, and also Kar2p (BiP) are involved as a complex in protein translocation across the ER membrane (Deshaies et al., 1991; Green et al., 1992; Brodsky et al., 1995; Ng and Walter, 1996), but it has been shown that the activity required for protein translocation is not directly required for the fusion of nuclear envelopes (Ng and Walter, 1996).
The ER is contiguous with the nuclear membrane and so it has not been determined from which site(s) membrane fusion starts in karyogamy. However, it was demonstrated that nuclear fusion is roughly paralleled with the homotypic fusion of ER membranes (Latterich and Schekman, 1994). Mutants defective in the fusion of nuclear envelopes were also defective in the ER membrane fusion both in vivo and in vitro (Kurihara et al., 1994; Latterich and Schekman, 1994). In this respect it is interesting that Cdc48p, an ER protein with homology to Sec18p, has been shown to participate in the fusion of ER membranes (Latterich et al., 1995). Sec18p is a yeast homologue of N-ethylmaleimide–sensitive factor (NSF), which together with Sec17p (a homologue of soluble NSF attachment protein 
[-SNAP]) is required for vesicular docking. Thus, it appears that Cdc48p is a component of fusion/docking machinery, mainly functioning in the fusion of ER membranes. Whether Cdc48p is required for nuclear fusion is yet to be examined experimentally. Furthermore, it has been shown that some novel proteins which are tightly bound to ER membrane are required for the membrane fusion and that Kar2p may participate in the activation of these proteins (Latterich and Schekman, 1994).
At the present we can not speculate as to the function of Tht1p from its amino acid sequence because of the lack of homology to known proteins. We cannot strictly rule out the possibility that the absence of the fusion of nuclear envelopes is a secondary effect of incomplete SPB binding, although it is unlikely as discussed above. However, several lines of evidence described in this study strongly suggest that Tht1p is a novel ER protein. The primary amino acid sequence indicates that it is a type I membrane protein. Tht1p is actually modified with N-linked glycosylation, and partitions to the insoluble fraction. Finally, immunofluorescence microscopy localized the protein in nuclear envelope and some portion of cytoplasmic membranous structures. In vegetative cells containing overproduced Tht1p it could localize to ER. Taking this fact into consideration together with the observation that the tht1+ gene is transiently expressed only in conjugating cells, we suggest that Tht1p may be directly and specifically involved in the fusion of nuclear envelopes and the ER membranes during karyogamy. One of the key issues in further studies for elucidating the role of Tht1p in karyogamy may be the role of the COOH-terminal end of Tht1p, because this end of the protein was shown to reside in the cytoplasm and could therefore be interacting with some other factors or perhaps with itself to perform an essential function to achieve the membrane fusion. Searches for such factors are in progress and may help to reveal a novel class of molecules involved in membrane fusion events. Another important issue addressed in this study is the temporal expression of the gene. Particularly interesting is the rapid disappearance of Tht1p from the membranes after the completion of karyogamy. Elucidation of mechanism involved in such elaborate regulation should be an important step toward understanding the membrane dynamics in the cell.
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Acknowledgments |
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This work was supported by Grant-in-Aid for Scientific Research on Priority Areas (to Y. Hiraoka and O. Niwa) and by the Kazusa DNA Research Institute Foundation.
Submitted: 14 August 1997
Revised: 17 November 1997
Dr. Horio present address is School of Medicine, Tokushima University, 3-18-15 Kuramoto-cho, Tokushima, Tokushima 770, Japan.
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References |
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