The Yeast JEM1p Is a DnaJ-like Protein of the Endoplasmic Reticulum Membrane Required for Nuclear Fusion*

DnaJ-like proteins are functional partners for Hsp70 molecular chaperones. Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitope-tagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of theER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The Δjem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the Δjem1 Δscj1 double mutant or defects in karyogamy of the Δjem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.

DnaJ-like proteins are functional partners for Hsp70 molecular chaperones. Complete nucleotide sequencing of yeast chromosome X has revealed that an open reading frame YJL073w encodes a novel member of the DnaJ-like protein family. The open reading frame represents a protein of 692 amino acids with a J-domain and one putative membrane-spanning segment. An epitopetagged version of the protein was anchored in the endoplasmic reticulum (ER) membrane and its J-domain faced the ER lumen. We therefore propose to designate this gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is not essential for cell growth, but double disruption of the JEM1 gene and the SCJ1 gene, which encodes another DnaJ-like protein in the ER lumen, causes growth arrest at elevated temperature. The ⌬jem1 mutant is defective in nuclear fusion, karyogamy, during mating. A mutant JEM1p carrying a mutation in the highly conserved His-Pro-Asp sequence in the J-domain could not complement either temperature-sensitive growth of the ⌬jem1 ⌬scj1 double mutant or defects in karyogamy of the ⌬jem1 mutant. JEM1p likely assists the functions of BiP, Hsp70 in the ER, including karyogamy.
DnaJ-like proteins, homologs of the bacterial chaperone protein DnaJ, mediate various cellular processes in cooperation with the members of another class of chaperone protein family, Hsp70 (1). DnaJ-like proteins have a well conserved "J-domain," which is responsible for their interactions with Hsp70. In eukaryotic cells, DnaJ-like proteins and members of the Hsp70 family are localized in various cellular compartments. The ER 1 of yeast, Saccharomyces cerevisiae, has two DnaJ-like proteins, SEC63p (2) and SCJ1p (3), and two Hsp70, BiP/ KAR2p (4,5) and LHS1p/SSI1p (6,7). BiP, LHS1p, and SEC63p facilitate post-translational translocation of proteins across the ER membrane (6,8,9), and BiP mediates protein folding in the ER (10). The yeast BiP and SEC63p are also involved in nuclear membrane fusion during mating (4,11,12). The function of SCJ1p is not clear, but a genetic interaction between the SCJ1 and KAR2 genes was reported (3).
Complete nucleotide sequencing of yeast chromosome X (13) has revealed that an open reading frame, designated YJL073w, on this chromosome encodes a novel member of the DnaJ-like protein family. In the present study, we have examined localization and function of this new DnaJ-like protein. We found that this protein is the third DnaJ-like protein in the ER; it is localized in the ER membrane in such transmembrane topology as the J-domain faces the ER lumen. We therefore propose to designate the gene JEM1 (DnaJ-like protein of the ER membrane) and to designate its gene product JEM1p. The JEM1 gene is nonessential for growth, but double disruption of the JEM1 gene and the SCJ1 gene causes growth arrest at elevated temperature. We also found that the ⌬jem1 null mutant is defective in nuclear membrane fusion.
Plasmids and Strain Constructions-The JEM1 gene was cloned by PCR from yeast genomic DNA using the primers based on the sequence deposited in the data base: 73A (5Ј-GCGGAGCTCTGCAGACGTGAAC-TATTAC-3Ј) and 73B (5Ј-GCGCTCGAGGTGCTGGCTTTGCAATAA-3Ј). The amplified 2.4-kb fragment was digested with SacI and XhoI and introduced into a yeast multi-copy plasmid pYO326 (17) to generate pSNJ1. The 3HA epitope tag, three tandem repeats of the influenza virus hemagglutinin (HA) epitope (YPYDVPDYA), was introduced at the C terminus of JEM1p at the DNA level by oligonucleotide-directed mutagenesis (18). The chimeric gene encoding JEM1p tagged with the 3HA epitope was subcloned into pYO326 (17) and a yeast single copy plasmid pRS316 (19) to generate pSNJ2 and pSNJ3, respectively. A H613Q mutation of the JEM1 gene was performed by oligonucleotidedirected mutagenesis (Sculptor in vitro mutagenesis system, Amersham Corp.) using an oligonucleotide (5Ј-CCAAAAAATACCAACCAGA-CAAAATAAAG-3Ј).
A null allele of JEM1 was constructed by replacing the 1.2-kb BamHI/SalI restriction fragment of pSNJ1 with a 2-kb BamHI/SalI fragment of pJJ282 containing the yeast LEU2 gene (20). The resulting plasmid, YEp⌬jem1, was digested with XhoI and SacI, and the 3.2-kb fragment was isolated and transformed into SEY6210, SEY6211, and SEY621D. Yeast transformation was performed by the lithium thiocyanate method (21). Leu ϩ transformants were selected, and the presence of the ⌬jem1 allele was confirmed by PCR using the primers 73A and 73B. The ⌬jem1 strains derived from SEY6210, SEY6211, and SEY621D were named SNY1028, SNY1029, and SNY1024, respectively.
The SCJ1 gene was cloned from yeast genomic DNA by PCR using the primers SCJA (5Ј-GCGCTCGAGTGATTACTACGCCTACCG-3Ј) and SCJB (5Ј-GCGAGCTCGAAGATGTCTGAAAT-3Ј). The amplified 1.5-kb fragment was digested with XhoI and SacI and subcloned into a polylinker site of pBluescript IISK ϩ (Stratagene) to generate pBSSCJ1. The SCJ1 gene was disrupted by inserting a 0.9-kb EcoRI/PstI restriction fragment from pJJ281 containing the TRP1 gene (20) into the EcoRI/PstI sites of pBSSCJ1. The resulting plasmid was named pBS⌬SCJ. The 2.0-kb XhoI and SacI fragment of pBS⌬SCJ was purified and transformed into SEY6210 and SEY6211, and Trp ϩ transformants were selected. Disruption of the SCJ1 gene was confirmed by PCR using the primers SCJA and SCJB. The ⌬jem1 strains derived from SEY6210 and SEY6211 were named SNY1025 and SNY1027, respectively.
Fluorescence Microscopy-Double label immunofluorescent staining of yeast cells was performed as described previously (22). The 12CA5 mouse monoclonal antibody, the fluorescein isothiocyanate-conjugated sheep anti-mouse IgG antibody F(abЈ) 2 fragment and the rhodamineconjugated sheep anti-mouse IgG antibody F(abЈ) 2 fragment were purchased from Boehringer Mannheim Yamanouchi (Tokyo, Japan). The rabbit anti-BiP antiserum was prepared against the MalE-KAR2 fusion protein. To analyze nuclear fusion during mating, cells of different mating types were mated as described previously (23). Cells were fixed in 3:1 (v/v) methanol-acetic acid at 4°C for 2 h, washed with distilled water, and resuspended in 1 g/ml 4Ј,6-diamidino-2-phenylindole (DAPI). Cells were viewed on an Olympus BH-2 epifluorescent microscope (Olympus, Tokyo) with filter sets suitable for DAPI, fluorescein, or rhodamine and photographed with T-MAX 400 film (Eastman Kodak Co., Rochester, NY) developed at ASA1600.

JEM1p Is a Yeast DnaJ-like Protein Anchored in the ER Membrane, and Its J-domain Faces the ER Lumen-JEM1
(YJL073) on yeast chromosome X encodes a DnaJ-like protein, which is 692 amino acids long, with a calculated molecular weight of 80,380. The predicted amino acid sequence of JEM1p revealed that JEM1p contains a possible J-domain near the C terminus (Fig. 1A), which shows 47% identity to that of E. coli DnaJ. JEM1p lacks a G/F-rich region and a cysteine-rich region, which are less conserved among DnaJ-like proteins (1).
We analyzed the subcellular location of JEM1p by indirect immunofluorescence microscopy. For this purpose, three tandem repeats of the HA epitope tag for recognition by the monoclonal antibody 12CA5 (24) were attached to the C terminus of JEM1p at the DNA level. This epitope-tagged version of JEM1p was expressed from a multicopy plasmid, and the cells were fixed, permeabilized, and stained with the 12CA5 antibody. Staining with the 12CA5 antibody showed perinuclear staining with several extensions in the cytoplasm (Fig. 1B, panel a). This staining is typical for yeast ER proteins, and nearly identical staining was observed with the anti-BiP antibodies (Fig.  1B, panel b). These results indicate that JEM1p resides exclusively in the ER.
Immunoblotting of cell extracts prepared from the strain expressing the 3HA-tagged JEM1p with the 12CA5 antibody showed a diffuse or doublet band with the apparent molecular mass of 73-79 kDa (Fig. 2, A and B), which was not detected in the extracts of cells that did not express the epitope-tagged JEM1p (not shown). This band shifted to a single sharp band of 71 kDa after treatment of the extracts with endoglycosidase H (not shown), suggesting that the 3HA-tagged JEM1p contained N-linked oligosaccharide chains and that the observed diffuse or doublet band reflected heterogeneity in glycosylation.
Because JEM1p has a stretch of uncharged hydrophobic amino acids between residues 50 and 70 near the N terminus (Fig. 1A), which is sufficiently long to function as a membrane anchor, we tested whether JEM1p is an integral membrane protein in the ER. Whole cell homogenates from the strain expressing the 3HA-tagged JEM1p were treated with 1 M NaCl, 2 M urea, 1% Triton X-100, 0.1 M sodium carbonate, pH 11.5, or 1% sodium deoxycholic acid, and extractability of JEM1p from membranes was analyzed ( Fig. 2A). Because JEM1p was resistant to extraction with 1 M NaCl, 2 M urea, or 0.1 M sodium carbonate, by which soluble and peripheral membrane proteins are extracted, JEM1p is indeed an integral membrane protein.
Interestingly, JEM1p was extracted from the ER membrane with 1% deoxycholic acid, an ionic detergent, but not with 1% Triton X-100, a nonionic detergent ( Fig. 2A). JEM1p may be interacting with other membrane proteins in the ER membrane.
The orientation of JEM1p in the ER membrane was examined by digestion with a proteolytic enzyme. Cell homogenates from the strain expressing the 3HA-tagged JEM1p were treated with trypsin in the absence or the presence of Triton X-100, and the intactness of the 3HA epitope tag of JEM1p was probed with the 12CA5 antibody. Although the 3HA-tagged JEM1p was not digested by trypsin in the absence of detergent, the 3HA epitope tag became susceptible to trypsin digestion when the ER membrane was lysed with Triton X-100 (Fig. 2B). Because the 3HA tag was introduced at the C terminus of Cells of SEY6210/pSNJ2 were grown in SCD medium (minimal medium containing 2% (w/v) glucose and 0.5% (w/v) casamino acids) lacking uracil at 30°C and analyzed by double label immunofluorescence microscopy using the 12CA5 monoclonal antibody and anti-BiP polyclonal antibodies. Panels a, b, and c show the same field of the fluorescent images stained with the 12CA5 antibody, anti-BiP antibodies, and DAPI, respectively. Bar, 2 m.

FIG. 2. JEM1p is an integral membrane protein with its Jdomain facing the ER lumen.
A, cell homogenates (29) prepared from the strain SEY6210/pSNJ2 were incubated in one of the following solutions on ice for 30 min and subsequently centrifuged at 315,000 ϫ g for 30 min. Buffer, buffer (10 mM Hepes-KOH, pH 7.4, 300 mM mannitol, 100 mM KCl, and 1 mM EGTA) alone; NaCl, buffer containing 1 M NaCl; Urea, buffer containing 2 M urea; Triton, buffer containing 1% Triton X-100; Na 2 CO 3 , buffer containing 0.1 M Na 2 CO 3 , pH 11.5; NaDOC, buffer containing 1% sodium deoxycholic acid. Pellets (P) and supernatants (S) derived from the same amount of the homogenates (Total) were subjected to SDS-polyacrylamide gel electrophoresis followed by immunoblotting with the 12CA5 antibody to detect the 3HAtagged JEM1p. B, cell homogenates prepared from the strain SEY6210/ pSNJ2 were incubated with 0.5 mg/ml trypsin in the absence (Ϫ Triton X-100) or the presence (ϩ Triton X-100) of 0.1% Triton X-100 on ice for the times indicated. The digestion was stopped by the addition of soybean trypsin inhibitor to 1 mg/ml, and the proteins were analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with the 12CA5 antibody.
JEM1p, the C-terminal part including the J-domain is obviously exposed on the lumenal side of the ER. This transmembrane topology of JEM1p is consistent with the fact that potential sites for N-linked glycosylation are present only in the C-terminal domain (Fig. 1) and that JEM1p is glycosylated. The presence of the J-domain in the ER lumen suggests that JEM1p is a partner protein for BiP and/or LHS1p, Hsp70s of the ER lumen.
Yeast Cells Lacking JEM1p Alone Show Normal Growth, but Those Lacking Both JEM1p and SCJ1p Cannot Grow at High Temperature-To assess the roles of JEM1p in vivo, we have constructed a null allele of the JEM1 gene. A JEM1/jem1::LEU2 heterozygous diploid was constructed and subjected to sporulation. Among 25 tetrads dissected, 18 produced 4 viable spores, and 7 produced 3 viable spores. The Leu ϩ phenotype was segregated 2:2 in all four viable asci. The ⌬jem1 mutant strain grew as well as wild-type strains at all temperatures tested between 14 and 37°C (not shown).
The yeast SCJ1 gene encodes a soluble DnaJ-like protein in the ER lumen (3). Disruption of the SCJ1 gene was not lethal for yeast cells. The jem1::LEU2 strain and the scj1::TRP1 strain of opposite mating types were crossed, and the resulting heterozygous diploid was sporulated and dissected. We obtained Leu ϩ Trp ϩ spores that contain disrupted alleles of both genes. The ⌬jem1 ⌬scj1 double disrupted strains grew as well as wild-type strains at 14, 23, and 30°C, but they did not grow at 37°C (Fig. 3A). A low copy number plasmid containing the fusion gene for the 3HA-tagged JEM1p rescued the temperature-sensitive growth defect of the ⌬jem1 ⌬scj1 double mutant (Fig. 3B). Therefore, the growth defect resulted from disruption of both JEM1 and SCJ1 genes. The genetic interactions between the JEM1 and the SCJ1 genes suggest that their gene products are involved in a common pathway of cellular processes, which remains to be revealed.
Disruption of the JEM1 Gene Causes a Defect in Karyogamy-In the sexual phase of the yeast, haploid cells of opposite mating types (MATa and MAT␣) mate each other to form diploid cells. The mating cells form projections, and the cells fuse where the two mating cells come in close contact. After cell fusion, the nuclei from both haploid cells fuse to produce a diploid nucleus. This step is called karyogamy (25). Analyses of the yeast mutants defective in karyogamy showed that this step can be divided into two steps; nuclear congression and nuclear fusion (26). Haploid nuclei move and align during the nuclear congression step, and then the two nuclei fuse. In zygotes of mutants defective in the nuclear fusion step, nuclei become closely juxtaposed but do not fuse (26). Although the ⌬jem1 mutant strain grows as well as wild-type strains at all temperatures tested, it is defective in karyogamy.
Cells of opposite mating types were mated, and nuclei of zygotes were stained with DAPI. A wild-type zygote possessed a single nucleus (Fig. 4a). In the ⌬scj1 mutant zygotes, we also observed a single nucleus (Fig. 4b), indicating that SCJ1p is not required for karyogamy. On the other hand, zygotes from the MATa ⌬jem1 and the MAT␣ ⌬jem1 cross contained two nuclei (Fig. 4c). The nuclei of the zygotes were in close proximity but did not fuse; 79% of ⌬jem1 zygotes contained two or more nuclei that did not fuse (Table I). We sometimes observed mitochondria migrating into the first bud of the zygote, indicating that the cell fusion occurred normally in the mutant. When selfcrossed, the ⌬jem1 mutant exhibited significant reduction in diploid formation. These phenotypes are characteristic of a class of karyogamy mutants that are defective in the fusion of the nuclear membrane (26). Crosses between ⌬jem1 cells and wild-type cells of opposite mating types efficiently produced diploid (Table I). Therefore, the karyogamy defect of the ⌬jem1 mutation is bilateral. We concluded that the loss of JEM1p function causes a defect in the step of nuclear membrane fusion in karyogamy.
The J-domain Is Required for the JEM1p Functions-Members of the DnaJ-like protein family contain a highly conserved His-Pro-Asp sequence in the J-domain, which appears to play a critical role in interactions with Hsp70 (27,28). We attempted to test the role of the J-domain in the functions of JEM1p by   FIG. 3. Simultaneous disruption of the JEM1 and the SCJ1 genes confers temperature-sensitive growth on yeast cells. A, haploid strains SNY1024 -15A (⌬jem1) and SNY1025 (⌬scj1) were mated, and the resulting diploid strain was sporulated and dissected. Four haploid segregants (SNY1026 -7A, SNY1026 -7B, SNY1026 -7C, and SNY1026 -7D) of the same ascus were grown on YPD plates at 23°C (left) for 3 days or at 37°C (right) for 2 days. wt, wild type. B, the ⌬jem1 ⌬scj1 double mutant strain (SNY1025-7A) harboring a single copy plasmid containing a fusion gene for 3HA-tagged JEM1p (pSNJ3, JEM1), 3HA-tagged JEM1(H613Q)p (pSNJ3-H613Q, jem1H613Q), or the vector alone (pRS316, vector) was grown on SCD lacking uracil at 23°C for 3 days (left) or at 37°C (right) for 2 days.
FIG. 4. The ⌬jem1 mutant is defective in karyogamy. Cells of opposite mating types were mated on YPD (pH 4.5, adjusted with 0.1 M sodium citrate) plates for 3 h at 23°C. The cells were then fixed and stained with 1 g/ml DAPI to visualize nuclei. a, a wild-type zygote has a single fused nucleus (SEY6210 ϫ SEY6211). b, a ⌬scj1 mutant zygote also contains a single fused nucleus (SNY1025 ϫ SNY1027). c, a ⌬jem1 mutant zygote has two juxtaposed nuclei (SNY1028 ϫ SNY1029). Bar, 2 m. introducing an H613Q mutation in its His-Pro-Asp sequence. When ⌬jem1 mutant cells harboring the HA-tagged jem1 H613Q mutant gene were self-crossed, 75% of the resulting zygotes showed karyogamy defects (not shown), and diploid formation was impaired (not shown). This demonstrates that the J-domain of JEM1p is essential for karyogamy. Interestingly, the jem1 H613Q mutant gene failed to complement the temperature-sensitive growth of the ⌬jem1 ⌬scj1 double mutant (Fig. 3B). Because karyogamy is not required for normal cell growth and disruption of the SCJ1 gene alone exhibits no obvious phenotype in karyogamy, the J-domain of JEM1p, together with SCJ1p, is involved in a process that is distinct from karyogamy. In this context, it is to be noted that treatment of yeast cells with tunicamycin, which leads to accumulation of malfolded proteins in the ER and triggers the unfolded protein response, led to an increased level of JEM1 mRNA (not shown).