(Received for publication, December 18, 1996, and in revised form, March 23, 1997)
From the Department of Chemistry, Faculty of Science, Nagoya University, Chikusa-ku, Nagoya 464-01, Japan
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 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 ER1 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.
Standard recombinant
techniques (14) were performed using Escherichia coli strain
TG1 (supE hsd5 thi
(lac-proAB)
F
[tra
36 proAB+
lacIq lacZ
M15]). Yeast strains SEY6210
(MAT
ura3 leu2 trp1 his3 lys2 suc2) and
SEY6211 (MATa ura3 leu2 trp1 his3 ade2 suc2) (15) were used in the construction of
jem1 and
scj1 strains. SEY621D was constructed by mating SEY6210
with SEY6211. Yeast cells were grown according to standard methods
(16). Quantitative mating assay was performed as described previously
(16).
The JEM1 gene
was cloned by PCR from yeast genomic DNA using the primers based on the
sequence deposited in the data base: 73A
(5-GCGGAGCTCTGCAGACGTGAACTATTAC-3
) and 73B
(5
-GCGCTCGAGGTGCTGGCTTTGCAATAA-3
). 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 oligonucleotide-directed mutagenesis (Sculptor
in vitro mutagenesis system, Amersham Corp.) using an
oligonucleotide (5
-CCAAAAAATACCAACCAGACAAAATAAAG-3
).
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,
YEpjem1, 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+ 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 (5-GCGCTCGAGTGATTACTACGCCTACCG-3
) and SCJB (5
-GCGAGCTCGAAGATGTCTGAAAT-3
). The amplified 1.5-kb fragment was
digested with XhoI and SacI and subcloned into a
polylinker site of pBluescript IISK+ (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+ 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.
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(ab)2 fragment and the rhodamine-conjugated sheep anti-mouse IgG antibody F(ab
)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 µg/ml 4
,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.
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 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 TemperatureTo
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+ 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+ Trp+ 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
self-crossed, 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.
|
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 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).
We are grateful to Drs. Y. Ohya and Y. Wada for plasmids, to Dr. S. D. Emr for strains, and to Dr. M. Nakai for the anti-BiP antiserum.