* Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School, Worcester, Massachusetts
01655-0103; and Department of Biological Chemistry and Molecular Pharmacology and Dana Farber Cancer Institute, Harvard
Medical School, Boston, Massachusetts 02115
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Abstract |
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Members of the eukaryotic heat shock protein 70 family (Hsp70s) are regulated by protein cofactors that contain domains homologous to bacterial
DnaJ. Of the three DnaJ homologues in the yeast
rough endoplasmic reticulum (RER; Scj1p, Sec63p, and
Jem1p), Scj1p is most closely related to DnaJ, hence it
is a probable cofactor for Kar2p, the major Hsp70 in
the yeast RER. However, the physiological role of
Scj1p has remained obscure due to the lack of an obvious defect in Kar2p-mediated pathways in scj1 null mutants. Here, we show that the scj1 mutant is hypersensitive to tunicamycin or mutations that reduce N-linked
glycosylation of proteins. Although maturation of glycosylated carboxypeptidase Y occurs with wild-type kinetics in
scj1 cells, the transport rate for an unglycosylated mutant carboxypeptidase Y (CPY) is markedly
reduced. Loss of Scj1p induces the unfolded protein response pathway, and results in a cell wall defect when
combined with an oligosaccharyltransferase mutation.
The combined loss of both Scj1p and Jem1p exaggerates the sensitivity to hypoglycosylation stress, leads to
further induction of the unfolded protein response
pathway, and drastically delays maturation of an unglycosylated reporter protein in the RER. We propose
that the major role for Scj1p is to cooperate with Kar2p
to mediate maturation of proteins in the RER lumen.
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Introduction |
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KAR2P and Lhs1p (Cer1p/Ssi1p), members of the
heat shock protein 70 family (Hsp70s)1 perform
several diverse functions in the lumen of the rough
endoplasmic reticulum of Saccharomyces cerevisiae. Kar2p,
which is the yeast homologue of mammalian BiP, acts as a
chaperone to mediate protein folding in the ER lumen (te
Heesen and Aebi, 1994; Simons et al., 1995
). BiP/Kar2p
prevents inappropriate interactions between hydrophobic segments of nascent polypeptides via ATP hydrolysis-dependent cycles of peptide binding and release (Flynn et
al., 1989
; Gaut and Hendershot, 1993
). Reducing agents,
glycosylation inhibitors, and other agents that interfere
with protein folding in the RER induce the unfolded protein response (UPR) pathway leading to enhanced expression of Kar2p and other RER chaperones including protein disulfide isomerase (Kozutsumi et al., 1988
; Cox et al., 1993
). Mutations in the essential KAR2 gene were first isolated in a screen for yeast that have unilateral karyogamy
defects (Rose et al., 1989
). Although the precise role of
Kar2p in nuclear fusion during karyogamy is not fully understood, kar2 mutants are also defective in an in vitro assay that monitors homotypic fusion of ER derived vesicles
(Latterich and Schekman, 1994
). Class I kar2 mutants are
also defective in translocation of proteins across the endoplasmic reticulum (Vogel et al., 1990
). Loss of Lhs1p, a
nonessential and presumably less abundant RER Hsp70
protein, causes cytoplasmic accumulation of a subset of
yeast secretory precursors suggesting a partial overlap in
function between Kar2p and Lhs1p in nascent chain translocation (Baxter et al., 1996
; Craven et al., 1996
; Hamilton
and Flynn, 1996
). Disruption of the LHS1 gene reduces
the ability of yeast to refold heat-induced protein aggregates in the endoplasmic reticulum (Saris et al., 1997
).
Transcription of the LHS1 gene is induced by the UPR
pathway but not by elevated temperatures (Baxter et al.,
1996
; Craven et al., 1996
).
The ATP hydrolysis cycle of Hsp70 proteins is regulated
by cofactors that are members of the DnaJ protein family
(as reviewed by Bukau and Horwich, 1998). DnaJ proteins
interact with Hsp70 via a conserved ~80-residue J domain.
The yeast endoplasmic reticulum contains three members
of the DnaJ protein family (Sec63p, Scj1p and Jem1p) that
likely cooperate with Kar2p and Lhs1p (Sadler et al.,
1989
; Schlenstedt et al., 1995
; Nishikawa and Endo, 1997
). Sec63p, an integral component of the yeast translocation
complex (Deshaies et al., 1991
; Brodsky and Schekman,
1993
; Panzner et al., 1995
), has a J domain located within a
lumenally disposed loop (Sadler et al., 1989
). Synthetic interactions between kar2 mutants and sec63-1 provide genetic support for a direct interaction between Kar2p and
Sec63p (Scidmore et al., 1993
). An amino acid substitution
in the J domain of Sec63p (sec63-1 allele) confers a temperature sensitive defect in protein translocation due to a
reduced interaction with Kar2p (Brodsky and Schekman,
1993
) resulting in precursor stalling within the Sec61 translocation pore (Lyman and Schekman, 1995
). Hydrolysis
assays have shown that the J domain of wild-type Sec63p,
but not the sec63-1 mutant, stimulates ATP hydrolysis by
Kar2p (Corsi and Schekman, 1997
). Posttranslational translocation of proteins across the yeast RER is ATP dependent (Brodsky and Schekman, 1993
; Panzner et al., 1995
)
leading to the hypothesis that the interaction between
Kar2p and Sec63p promotes nascent chain transport
through the Sec61 complex by iterative cycles of nascent
chain binding and ATP hydrolysis-dependent release
(Brodsky and Schekman, 1993
; Lyman and Schekman,
1995
).
The roles of Scj1p and Jem1p in the RER are less well
defined. The growth rate of yeast is not altered by disruption of either the SCJ1 or the JEM1 gene (Schlenstedt et al.,
1995; Nishikawa and Endo, 1997
); simultaneous disruption
of both genes yields a strain that is inviable at 37°C (Nishikawa and Endo, 1997
). Synthesis of Jem1p and Scj1p,
unlike Sec63p, is induced by treatments that activate the
UPR pathway (Schlenstedt et al., 1995
; Nishikawa and
Endo, 1997
). The COOH-terminal J domain of Jem1p is
believed to interact with Kar2p, as the nuclear fusion defect that occurs when
jem1 strains are mated also occurs
when the J domain of Jem1p bears a point mutation identical to the sec63-1 allele that interferes with the Sec63p-Kar2p interaction (Nishikawa and Endo, 1997
). Several
lines of evidence suggest that the lumenal Scj1 protein can
interact with Kar2p in the ER via the conserved NH2-terminal J domain (Schlenstedt et al., 1995
). The J domain
from Scj1p, but not the J domains from DnaJ homologues
in the yeast cytoplasm (Sis1p) or mitochondria (Mdj1p),
can replace the essential J domain in Sec63p to promote
translocation of proteins across the RER (Schlenstedt et al.,
1995
). Synthetic genetic interactions between
scj1 and
certain kar2 alleles strongly suggest that Scj1p serves as a
cofactor for Kar2p (Schlenstedt et al., 1995
). Despite this genetic evidence for a functional interaction between Scj1p
and Kar2p, cells lacking Scj1p do not have detectable defects in protein translocation across the RER (Blumberg,
H., and P.A. Silver, unpublished results) or in nuclear
membrane fusion during mating (Nishikawa and Endo,
1997
). Consequently, a functional role for Scj1p in cellular
processes that require Kar2p remains to be elucidated.
The oligosaccharyltransferase (OST) catalyzes the transfer of a high-mannose oligosaccharide onto asparagine residues of nascent polypeptides entering the lumen of the
rough endoplasmic reticulum. Mutagenesis of the N-X-T/S
sites in acid phosphatase (AP) and carboxypeptidase Y
(CPY) has shown that hypoglycosylated proteins either
fail to fold and are retained in the ER (AP), or exit the ER
at reduced rates (CPY; Riederer and Hinnen, 1991; Winther et al., 1991
) indicating that N-linked oligosaccharides
are important for protein folding in yeast. Mutations in
each of the eight genes that encode the subunits of the
yeast OST result in reductions in the in vivo and in vitro
OST activity (for a review see Silberstein and Gilmore,
1996
). Isolation of the OST mutants indicates that significant underglycosylation of glycoproteins is tolerated by yeast with a functional UPR pathway (Cox et al., 1993
).
In an effort to obtain insight into the role of the Scj1
protein in the endoplasmic reticulum, yeast mutants were
sought that were inviable when combined with a scj1 mutation. The synthetic lethal screen yielded a mutant that
could be complemented by the OST1 gene. We found that
mild reductions in N-linked glycosylation are poorly tolerated in the absence of Scj1p, as a
ost3
scj1 mutant has a
severe growth defect at 37°C compared with
ost3 mutants that grow at wild-type rates. Loss of Scj1p induces
the UPR pathway, without yielding a detectable defect in
cell wall biosynthesis. However,
scj1 cells are hypersensitive to reducing agents and tunicamycin, and show a delay
in the transport of nonglycosylated CPY from the RER to
the Golgi. Jem1p and Scj1p appear to have partially overlapping functions as cofactors for Kar2p. Although the
mild reduction in growth rate of the
ost3
jem1 mutant at 37°C appears to argue against an important role for Jem1p
in protein folding, overexpression of Jem1p can suppress
the slow growth phenotype of the
ost3
scj1 mutant at
37°C. Our results suggest that Scj1p cooperates with Kar2p
to mediate folding of proteins under conditions of stress
induced by hypoglycosylation.
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Materials and Methods |
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Growth Media
Yeast were grown in YPDA medium (2% bacto-peptone, 1% yeast extract, 2% glucose, 0.003% adenine sulfate) to avoid accumulation of the
red pigment in ade2 cells, or in synthetic minimal media (0.67% yeast nitrogen base, 2% glucose) supplemented with adenine and the appropriate
amino acids or uracil. Growth media for drug sensitivity assays was prepared by adding aliquots from stock solutions of tunicamycin (10 mg/ml in
0.1 M NaOH), Calcofluor White (10 mg/ml in 1:1 ethanol-water), hygromycin B (10 mg/ml in water), or -mercaptoethanol to YPDA-2% agar
immediately before pouring the plates.
Isolation of Mutants Synthetically Lethal with scj1
The strain for the synthetic lethality screen was constructed in several
steps. PSY586 was obtained as a segregant from a cross of MS4 and MS20
(provided by Mark Rose, Princeton University, Princeton, NJ). The plasmid pPS753 was constructed to precisely disrupt the SCJ1 gene. Plasmid
pPS176, which contains the SCJ1 gene (Blumberg and Silver, 1991) as a
4-kb KpnI fragment in pBluescript, was digested with NsiI and BalI to excise the SCJ1 gene plus 100 bp of 5' and 29 bp of 3' flanking sequence. A
958-bp SmaI-PstI fragment from pJJ248 (Jones and Prakash, 1990
) containing the TRP1 gene was cloned into pPS176 digested with NsiI and BalI
to obtain pPS753. KpnI-digested pPS753 was used to transform PSY586 to
tryptophan prototrophy to obtain the strain designated PSY729. Southern
blotting confirmed the disruption of the SCJ1 gene in PSY729. PSY586
was crossed to CH1305 (Kranz and Holm, 1990
) to introduce the ade3 mutation; after sporulation and tetrad dissection white colonies (ade2ade3)
were selected to obtain PSY7-10D. The strains for the synthetic lethality
screen (PSY759 and PSY761) were segregants from the diploid obtained
by crossing PSY7-10D with PSY729. To construct the URA3 ADE3 SCJ1
plasmid (pPS720) for the colony sectoring assay, a 3,673-bp fragment containing the ADE3 gene was cloned into BamHI-SpeI digested pRS426, a
URA3 marked 2µ vector (Christianson et al., 1992
) to obtain the plasmid pPS719. A 1.7 kb fragment containing the SCJ1 gene was excised from
YEpSCJ1 (Schlenstedt et al., 1995
) by digestion with BamHI and SalI,
and ligated to BamHI-SalI digested pPS719. The strain PSY759 was transformed to uracil prototrophy with pPS720. The resulting strain was grown
to a density of 5 × 107 cells/ml at 30°C in synthetic minimal media lacking
uracil and mutagenized by UV-exposure to 50% survival as described
(Lawrence, 1991
). Nonsectoring mutants were selected and isolated by restreaking three times on YPD plates. Nonsectoring mutants did not grow
on plates containing 5-fluoro-orotic acid (5-FOA) unless they were first
transformed with a LEU2 marked SCJ1 plasmid, thereby showing that the
synthetic phenotype of the mutant strain is due to a requirement for the
SCJ1 gene, not the ADE3 or URA3 genes. Nonsectoring mutants were
transformed with a yeast genomic library on a 2µ LEU2-marked plasmid
(a gift from Richard Kolodner, Harvard Medical School, Boston, MA) to
identify genes that could suppress the synthetic lethal phenotype.
Strain Constructions
To minimize the effects of strain background, PSY771 was crossed with
RGY132, an -haploid derived by sporulation of YPH274 (Sikorski and
Hieter, 1989
). A temperature-sensitive, trp
his
lys
haploid (ost1-6
SCJ1) derived from the initial cross was backcrossed with RGY132 and
sporulated to obtain RGY140, and a nontemperature-sensitive, trp+ his
lys
haploid (OST1
scj1::TRP1) from the initial cross was backcrossed
to RGY132 and sporulated to obtain trp
(RGY141) and trp+ (RGY142)
haploids. RGY324 (derived from the diploid RGY302; Karaoglu et al.,
1995a
) was mated with RGY142. The resulting diploid was sporulated,
and asci dissected to obtain strains RGY143A-D that are the haploid
progeny from a tetratype ascus. To obtain RGY144, the SCJ1 gene was
disrupted by transforming RGY324 to uracil prototrophy with the KpnI-linearized URA3 marked plasmid p
SCJ1 (Blumberg and Silver, 1991
).
Correct integration of the plasmid into the SCJ1 gene was confirmed by PCR. The strain RGY145 was obtained by sporulation of the diploid obtained by crossing RGY144 with RGY131.
A one-step PCR-based gene disruption method (Wach et al., 1994;
Wach, 1996
) was used to disrupt the JEM1 gene to obtain RGY146. PCR
was used to generate a DNA fragment containing a HIS5 gene from S.
pombe flanked by 5' (nucleotides
37 to 5) and 3' (nucleotides 2015 to
2055) regions from the JEM1 gene (Wach, 1996
). The pFA6a-HIS3MX6 template for the PCR was kindly provided by Peter Philippsen (University of Basel, Switzerland). After transformation of RGY324 with the disruption cassette, histidine prototrophs were selected and integration of the
cassette into the JEM1 gene was confirmed by PCR. RGY146 was crossed
with RGY142 to obtain a diploid, which after sporulation and dissection
yielded RGY147 and RGY148.
Radiolabeling and Immunoprecipitation of Proteins
To obtain strains expressing unglycosylated CPY (ug-CPY), yeast were
transformed with the URA3 marked centromeric plasmid pJW373, that
bears a modified PRC1 gene that lacks all four N-glycosylation sites (Winther et al., 1991). The plasmid pJW373 was a generous gift from Jakob R. Winther (Carlsberg Laboratory, Denmark).
Yeast were grown at 25°C in synthetic minimal media supplemented
with the appropriate amino acids until mid-log phase (0.5-0.8 OD at 600 nm). Cells collected by centrifugation were resuspended at a density of 6 A600/ml of media and radiolabeled for 10 min at 25°C with 50 µCi of Tran-
35S-label (ICN Biomedicals Inc., Costa Mesa, CA) per A600 unit of cells. In
pulse-chase experiments, 100 µl of a fresh solution of unlabeled methionine and cysteine (each at 5 mg/ml) was added after 10 min to initiate the
chase incubation that continued at the same temperature. Aliquots of the
radiolabeled cultures were taken at the indicated times, added to an equal
volume of 20 mM sodium azide, frozen in liquid nitrogen and stored at
80°C. Rapid lysis of cells with glass beads and immunoprecipitation of
carboxypeptidase Y (CPY) was done as described (Rothblatt and Schekman, 1989
). Immunoprecipitation of Kar2p was performed as described
for CPY using 5 µl of antiserum against Kar2p (a gift from Peter Walter, UCSF) per 1.5 A600 of labeled yeast cells. Coimmunoprecipitation of unfolded protein substrates with Kar2p was performed as described (Simons
et al., 1995
). In brief, Kar2p was immunoprecipitated from radiolabeled
yeast cells that were lysed with glass beads in nondenaturing lysis buffer
(10 mM Tris-Cl [pH 8.0], 0.3M NaCl) containing 2% CHAPS and 30 U/ml
potato apyrase. The Kar2p immunoprecipitates were washed once with
nondenaturing lysis buffer containing 0.5% CHAPS and boiled in the
presence of 1% SDS. CPY was immunoprecipitated from denatured
Kar2p immunocomplexes as described (Simons et al., 1995
).
RNA Extraction and Northern Blot Analysis
Yeast cells were grown in YPDA at 25°C until mid-log phase. As indicated, tunicamycin was added 2 h before cells were collected at a concentration of 1 µg/ml. Whole-cell RNA was prepared using the hot phenol
procedure (Kohrer and Domdey, 1991). Samples of RNA (20 µg) were resolved by 1% agarose/formaldehyde gel electrophoresis and transferred
to Hybond-N+ membranes (Amersham Corp.) in 20× SSC for 20 h (Sambrook et al., 1989
). The membranes were prehybridized for 2 h and hybridized overnight in 35% formamide at 42°C as described (Sambrook et
al., 1989
) with probes specific for yeast KAR2 (nucleotides 1088 to 2376)
or JEM1 (nucleotides 156 to 1293) generated by PCR. Membranes were
washed twice with 1× SSC, 0.1% SDS at 55°C and exposed. The Hybond
membranes were stripped according to manufacturer's recommendations
before reprobing with a 1.5-kb EcoRI/ScaI fragment of the ACT1 gene.
pDH6, a construct bearing the ACT1 was provided by Allan Jacobson,
UMMC. Prehybridization, hybridization, and washes were performed
as described above for the KAR2 probe. Hybridization probes were
32P-labeled using the Oligolabeling Kit (Pharmacia Biotech, Inc., Piscataway, NJ). Radioactive bands were quantified with a PhosphorImager (Molecular Dynamics, Inc., Sunnyvale, CA) and visualized by autoradiography.
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Results |
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Identification of ost1-6 as a Temperature-sensitive Allele of the OST1 Gene
Previous studies have established that expression of Scj1p
is not essential for viability of yeast, yet the homology between Scj1p and the E. coli DnaJ protein strongly suggests
that Scj1p functions as a cofactor for Kar2p and/or Lhs1p,
DnaK homologues located within the yeast endoplasmic
reticulum. To further explore the role of Scj1p in the yeast
RER, the ade2ade3 colony-sectoring screen (Koshland et al.,
1985; Bender and Pringle, 1991
) was used to identify mutations that were incompatible with a null allele of the SCJ1
gene. Two
scj1ade2ade3 strains that differed with respect
to mating type were transformed with a plasmid bearing the URA3, ADE3 and SCJ1 genes. After UV mutagenesis,
yeast mutants were isolated that were unable to lose the
plasmid by their failure to form sectored red and white
colonies. Potential synthetic lethal strains were then tested
for the ability to grow on plates containing 5-FOA. From
among 45,000 colonies that were screened, a single mutant
strain (PSY771) was unable to lose the plasmid bearing the SCJ1 gene.
PSY771 was inviable at 37°C in the presence of the SCJ1
plasmid. Mating with a parental strain (PSY761) showed
that the synthetic lethal and temperature sensitive phenotypes were both recessive, and furthermore these two phenotypes cosegregated after sporulation and tetrad dissection. To identify the mutant gene responsible for the
synthetic lethal phenotype, PSY771 was transformed with a high copy yeast genomic plasmid bank. Plasmids that
were subsequently isolated from sectoring transformants
were found to contain the OST1 gene encoding the subunit of the yeast OST complex (Silberstein et al., 1995
).
Crosses between PSY771 and two previously characterized temperature sensitive ost1 mutants (RGY121 and
RGY122; Silberstein et al., 1995
) yielded diploids that
were temperature sensitive, indicating that PSY771 bears
a mutant allele of ost1 which we designate as ost1-6. Finally, we noted that PSY771 could lose the SCJ1 plasmid
at 25°C, provided that the growth media contained 2 M
glycerol, suggesting that the
scj1ost1-6 strain is osmotically fragile in standard growth media.
The initial ost1-6 strain was backcrossed several times
with RGY131 to minimize the effect of strain background
on the subsequent analysis, and to separate the scj1 and
ost1-6 mutations. In this new genetic background, the resulting ost1-6 strain displays a temperature-sensitive phenotype (Fig. 1 A). Previously isolated ost1 mutants have
reduced OST activity as detected by pleiotropic hypoglycosylation of N-linked glycoproteins synthesized in vivo at
both the permissive (25°C) and restrictive (37°C) temperatures (Silberstein et al., 1995
). Biosynthesis of the soluble
vacuolar glycoprotein CPY by wild-type,
scj1, ost1-6, and
ost1-6 cells complemented with pRS316-OST1 was analyzed to determine whether the ost1-6 mutant has reduced
OST activity at the permissive temperature. CPY is synthesized as a proenzyme that acquires four N-linked oligosaccharides upon translocation into the lumen of the endoplasmic reticulum. The 67-kD ER form of proCPY
(p1CPY) is transported to the Golgi complex, where the
core oligosaccharides are elongated by the addition of mannose residues to yield the 69-kD Golgi form of proCPY
(p2CPY). Upon transport to the vacuole, proteolytic removal of an 8-kD propeptide generates the mature 61-kD
vacuolar form of CPY. CPY was immunoprecipitated
from yeast cultures that were radiolabeled with 35S methionine for 1 h at 25°C (Fig. 1 B). As expected, the predominant form of CPY synthesized by the wild-type and
scj1 mutant has four N-linked oligosaccharides. In addition to fully glycosylated CPY, the ost1-6 mutant strain
synthesized hypoglycosylated variants of CPY that lack
between one and three oligosaccharides (Fig. 1 B,
1,
2,
and
3 forms of CPY). Notably, the synthetic lethal phenotype, the temperature sensitive growth defect (Fig. 1 A), and the glycosylation defect of the ost1-6 mutant (Fig. 1 B)
are corrected by expression of Ost1p from a centromeric
plasmid. From the preceding results, we conclude that the
temperature-sensitive phenotype of the ost1-6 allele is due
to reduced levels of N-glycosylation caused by a mutation
in OST1, the
subunit of the OST complex.
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A Synthetic Growth Defect Results when ost3 and
scj1 Are Combined
We postulated that the synthetic lethal phenotype produced by the combination of the scj1 and ost1-6 mutations was not caused by an exaggeration of the glycosylation defect inherent to ost1-6, as glycosylation of CPY in
the
scj1 strain is indistinguishable from that of the wild-type strain. However, given that Scj1p is proposed to cooperate with the ER chaperone Kar2p (Schlenstedt et al.,
1995
), it is formally possible that folding or oligomeric assembly of the mutant form of Ost1p is impaired in a
scj1
strain thereby causing an indirect reduction in OST activity. To address this possibility, the
scj1 strain was crossed
to a milder OST mutant caused by disruption of the nonessential OST3 gene encoding the 34-kD subunit of the
OST. We selected the
ost3 mutant for this analysis because the strain grows at a wild-type rate at 25, 30, and
37°C (Karaoglu et al., 1995a
). Moreover, immunoprecipitation experiments from strains that express an epitope-tagged OST subunit (Stt3p) have revealed that the integrity and stability of the OST complex is not compromised
in yeast cells that do not express Ost3p (Karaoglu et al.,
1997
). After sporulation of the
ost3
scj1 diploid, dissection of tetrads yielded four viable colonies at 25°C from
tetratype asci. As reported previously (Blumberg and Silver, 1991
; Karaoglu et al., 1995a
), the
ost3 and
scj1
mutants grow at wild-type rates on YPD plates incubated at 25°C or 37°C. In contrast, the
ost3
scj1 double
mutant has a severe growth defect at 37°C, but not at 25°C
(Fig. 2 A).
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CPY was immunoprecipitated from yeast cultures that
were pulse labeled at 25°C for 10 min and then chased for
0 or 15 min to determine whether the loss of Scj1p exaggerates the glycosylation defect of a ost3 mutant (Fig. 2
B). Nontranslocated prepro-CPY is not detected in the immunoprecipitates from
scj1 cells (Fig. 2 B), confirming
the previous observation that Scj1p does not have a role in
protein translocation across the ER (Blumberg, H., and
P.A. Silver, unpublished observations). The 67-kD p1
form of CPY was the predominant product after the 10-min pulse of wild-type or
scj1 cells. CPY immunoprecipitates from the
ost3 mutant contain p1CPY as well as
more rapidly migrating variants of p1CPY that lack one or
two oligosaccharides. Notably, the same glycoforms of
p1CPY were seen in the
ost3
scj1 mutant after the 10-min pulse label. After a 15 min chase, mature vacuolar
CPY is the major product detected in the wild-type and
mutant strains, together with residual p1CPY and p2CPY.
Additional polypeptides corresponding to CPY glycoforms lacking one, and in some cases, two core oligosaccharides are obtained from both the
ost3 and the
ost3
scj1 strains. Since the absence of Scj1p does not
cause a defect in N-linked glycosylation, the
ost3 and
ost3
scj1 strains synthesized similar CPY glycoforms. We conclude that the synthetic lethal phenotype of the
scj1ost1-6 mutant is not explained by a further reduction
in OST activity in the mutant strain, but instead indicates
that
scj1 cells do not tolerate hypoglycosylation of proteins.
As Scj1p is proposed to interact functionally with Kar2p, one would predict a synthetic growth defect for an ost1-6kar2 double mutant. To test this prediction, we crossed the ost1-6 mutant with the temperature-sensitive (ts) kar2-159 mutant. After sporulation and dissection of 27 asci at 25°C on YPD plates, we obtained 21 parental ditype tetrads (4 viable ts colonies), one nonparental ditype tetrad (2 viable non-ts colonies) and 5 tetratype tetrads (3 viable colonies, 2 of which were ts). This distribution of tetrads is consistent with the linkage of both KAR2 (YJL034W) and OST1 (YJL002C) to the centromere of chromosome X. Spores corresponding to the ost1-6kar2-159 double mutant germinated at 25°C and gave rise to microcolonies containing 2-4 cells, indicating a synthetic lethal interaction between the kar2-159 and ost1-6 mutations.
The Maturation of Unglycosylated CPY Is Delayed in
scj1 Strains
A reduction in N-linked glycosylation due to a mutation in
the OST would be predicted to interfere with the glycoprotein folding in cells that lack a component of the ER
chaperone machinery. Although the results obtained by
pulse-chase labeling of the ost3
scj1 mutant (e.g., Fig. 2
B) suggest a possible delay in ER-exit of the hypoglycosylated variants of CPY, the presence of multiple CPY glycoforms prevented accurate quantitation of CPY transport to the vacuole. To determine whether Scj1p is involved in
folding and ER exit of glycoproteins, we asked whether
processing and transport of CPY and a nonglycosylated
mutant form of CPY is altered in the
scj1 mutant. We
constructed wild-type and
scj1 mutant strains that have
the chromosomal CPY gene (PRC1) as well as a CEN
plasmid-borne CPY gene that contains point mutations in
each of the four consensus N-X-T/S sites for N-linked glycosylation. The unglycosylated CPY mutant has been used
as a reporter protein in previous studies since the expression, stability and activity of the CPY mutant are not affected by elimination of the glycosylation sites (Winther
et al., 1991
). However, transport of the unglycosylated CPY mutant from the RER to the vacuole occurs at a reduced rate in wild-type cells (Winther et al., 1991
), and is
further slowed in kar2 mutants (te Heesen and Aebi,
1994
).
CPY was immunoprecipitated from yeast cultures that
were pulse-labeled for 10 min at 25°C, and then chased for
the indicated times (Fig. 3 A). The ER and Golgi precursor forms of wild-type CPY (p1CPY, p2CPY) and of the
mutant CPY (pug-CPY) can be readily distinguished from
the mature vacuolar forms of wild-type CPY and mutant
ug-CPY by their different mobilities after PAGE in SDS.
After a 10-min pulse label, the ER and Golgi precursors
are the predominant forms of CPY and ug-CPY detected
in both yeast strains. Conversion of the precursors to the
more rapidly migrating mature forms during the chase period can be used to estimate the rate at which folded CPY
exits the ER. The effect of the Scj1 protein on transport
was quantified by determining the percentage of mature
CPY (Fig. 3 B, left) and mature ug-CPY (Fig. 3 B, right) during the 1-h chase incubation. During the chase period,
similar percentages of mature CPY were obtained from
the wild-type and scj1 strains, suggesting that Scj1p is not
required for a rate-limiting step in transport of fully glycosylated CPY from the ER to the vacuole (Fig. 3 B). Consistent with previous reports (Winther et al., 1991
), transport of unglycosylated CPY from the ER to the vacuole
was slightly delayed compared with transport of wild-type CPY (Fig. 3 B, compare solid bars in left and right panels).
In contrast to what was observed for wild-type CPY, the
scj1 yeast cells were found to transport unglycosylated
CPY to the vacuole 1.7-fold slower than the wild-type
strain (Fig. 3 B, right). Loss of Scj1p delays the folding of
hypoglycosylated forms of CPY, albeit to a lesser extent
than reported previously for several kar2 alleles (te Heesen and Aebi, 1994
).
|
The Folding Defect of scj1 Strains Is Aggravated in the
Absence of Jem1p
Expression of two of the ER DnaJ homologues (Scj1p and
Jem1p) is induced by tunicamycin (Schlenstedt et al., 1995;
Nishikawa and Endo, 1997
); hence the UPR pathway regulates expression of both proteins. A partial overlap of
function for Jem1p and Scj1p is suggested by the observation that the
scj1
jem1 double mutant is viable at 25°C
but not at 37°C (Nishikawa and Endo, 1997
). We constructed a series of strains lacking the nonessential JEM1 gene to determine whether Jem1p participates in processes that mediate folding of hypoglycosylated glycoproteins in the ER. Growth on YDP plates revealed a much
weaker growth defect for the
ost3
jem1 mutant than for
the
ost3
scj1 mutant at 37°C (not shown). However, the
ost3
scj1
jem1 triple mutants were inviable at 25°C
(data not shown). These results reinforce the hypothesis
that Jem1p and Scj1p perform related but nonidentical
functions in the RER.
The intracellular transport of glycosylated and unglycosylated CPY was analyzed in the wild-type, jem1 and
scj1
jem1 mutant strains to determine whether Jem1p is
also involved in protein folding (Fig. 4 A). The time course
for conversion of pug-CPY to mature unglycosylated CPY
was remarkably similar in the wild-type and
jem1 strains
(Fig. 4 B). However, the transport of unglycosylated CPY
to the vacuole was drastically delayed in the
scj1
jem1
mutant; after a 60-min chase period, <30% of the total ug-CPY precursor had been converted to the mature form
(Fig. 4 B). The 3.2-fold slower transport of ug-CPY in the
scj1
jem1 mutant relative to wild-type cells is similar to
that observed for several kar2 alleles (te Heesen and Aebi,
1994
), and is more severe than that observed for the
scj1
mutant (Fig. 3 B). Interestingly, the maturation of CPY
expressed from the chromosomal PRC1 gene was also delayed in the strain that lacks both DnaJ homologues, since
the ER precursor (p1CPY) can still be detected after a 60-min chase in the immunoprecipitates from the
scj1
jem1
strain (Fig. 4 A). This delay in transport of wild-type CPY
from the ER to the vacuole in the
scj1
jem1 mutant was
dependent upon the simultaneous expression of the unglycosylated CPY mutant (data not shown). Nontranslocated
precursor forms of Kar2p were not detected when Kar2p
was immunoprecipitated from pulse-labeled
scj1
jem1
cells (not shown), indicating that protein translocation is
not perturbed when two of the three DnaJ homologues are absent.
|
Next, we tested whether overexpression of Jem1p could
suppress the temperature sensitive growth defect of the
ost3
scj1 mutant. The double mutant was transformed
with URA3 marked high copy plasmids that either lack a
DNA insert, or contained the SCJ1 gene or the JEM1 gene
as inserts. As additional controls, the
scj1
jem1 mutant was transformed with the empty vector, or the JEM1 plasmid. As expected, all the strains grew at similar rates at
25°C (Fig. 4 C). The temperature sensitive lethal phenotype of the
scj1
jem1 mutant was rescued by the plasmid
that contained the JEM1 insert, but not by the empty vector. The severe growth defect of the
ost3
scj1 mutant
was also suppressed by high copy plasmids that contain either the SCJ1 gene or the JEM1 gene. Suppression of the
growth defects for the
scj1
jem1 and
ost3
scj1 strains
was plasmid dependent as neither strain grew at a wild-type rate at 37°C on minimal media plates that contain
5-FOA (not shown).
Prolonged Binding of Kar2p to Unglycosylated CPY in
the scj1 and
scj1
jem1 Mutants
Loss of the Scj1p selectively delays export of unglycosylated CPY from the ER. Presumably, the ability of Kar2p
to mediate pug-CPY folding is impaired when one or more
of the DnaJ homologues are not expressed. Based upon
previous studies using conditional kar2 mutants (te Heesen
et al., 1994; Simons et al., 1995), we would predict that the
unfolded pug-CPY remains associated with Kar2p for an
extended period in
scj1 and
scj1
jem1 cells. To test this
prediction, antibodies specific for Kar2p were used to immunoprecipitate the chaperone under nondenaturing conditions together with any unfolded protein substrates. A
subsequent immunoprecipitation under denaturing conditions using antibodies to CPY allowed an estimation of the
fraction of p1CPY and pug-CPY that were associated with
Kar2p either 0 or 60 min after pulse labeling (Fig. 5). Although a faint p1CPY band could be detected in the Kar2p
immunoprecipitates from the 0-min chase, quantitation of
the data revealed that <5% of the total p1CPY was associated with Kar2p in wild-type,
scj1 and
scj1
jem1 cells.
A greater fraction of pug-CPY was associated with Kar2p
in wild-type cells immediately after pulse labeling (Fig. 5 A
[8%] and B [13%]). After a 1-h chase, neither p1CPY nor
pug-CPY remained bound to Kar2p in wild-type cells, nor
did we observe artifactual coimmunoprecipitation of p2-CPY or mature CPY with Kar2p in 0 min or 60 min samples. Typically, a slightly greater fraction of the pug-CPY
was associated with Kar2p in the
scj1 (Fig. 5 A, 11%) and
scj1
jem1 (Fig. 5 B, 12%) mutants than in the wild-type
strain immediately after pulse labeling. In contrast to what
we observe in wild-type cells, complexes between Kar2p and pug-CPY persist in the
scj1 and
scj1
jem1 mutants.
Quantification of the data in Fig. 5 B indicated that 21%
of the remaining pug-CPY was bound to Kar2p in the
scj1
jem1 mutant after a 1-h chase. The native coimmunoprecipitation procedure may underestimate the fraction
of unfolded proteins that are bound to Kar2p, given that chaperone-substrate complexes may dissociate during the
time required for the immunoprecipitation.
|
ER DnaJ Proteins Help Protect Yeast from Stress-inducing Agents
The synthetic growth defect produced by the combination
of a reduction in OST activity and a null allele of Scj1p is
most readily explained by a protein folding defect that is
ultimately responsible for reduced exit of hypoglycosylated proteins from the ER. To determine whether loss of
Scj1p or Jem1p makes yeast cells more sensitive to the
stress caused by accumulation of unfolded proteins in the
ER, the growth of wild-type and mutant strains was compared on plates that contained sublethal concentrations of
tunicamycin or -mercaptoethanol (Fig. 6). All the strains analyzed grew at wild-type rates on YPDA plates incubated at 25°C. The
scj1 and the
ost3
scj1 mutant strains
showed reduced growth rates compared with the wild-type
strain when the YPDA plates contained tunicamycin at a
concentration of 1 µg/ml. A less-pronounced sensitivity to
tunicamycin, as detected by a slight reduction in colony
size, was observed for the
ost3
jem1 strain relative to the
ost3 strain. The
ost3 strain is slightly more resistant to
tunicamycin than the wild-type strain. Resistance to low
concentrations of tunicamycin is also a property of the ost1
and ost2 mutants; the degree of resistance is roughly proportional to the severity of the glycosylation defect (Silberstein and Gilmore, unpublished observation). The
ost3 and the
jem1 mutants were also less sensitive to
-mercaptoethanol than the wild-type strain or the other
mutants. Notably, RER proteins involved in disulfide bond
formation including protein disulfide isomerase and Ero1p
are regulated by the UPR pathway (Frand and Kaiser,
1998
). Whereas mutations in the OST result in increased,
rather than decreased, resistance to agents that promote
protein folding stress, we consider it unlikely that the OST
has an intrinsic role as a chaperone. In contrast, the
ost3
scj1 and
ost3
jem1 double mutant strains showed a
more pronounced growth defect than the wild-type and
scj1 strains in plates containing
-mercaptoethanol. Apparently, the combination of hypoglycosylation stress and
redox stress is not tolerated in cells that are deficient in
Scj1p or Jem1p.
|
Assembly of the yeast cell wall requires the biosynthesis
and transport of glycoproteins, GPI anchored proteins,
and -1,6 and
-1,3 glucans (Stratford, 1994
). Yeast with
mutations that block different stages in the synthesis of
N-linked oligosaccharides were found to exhibit increased
sensitivity to aminoglycoside antibiotics like hygromycin B
(Dean, 1995
) or compounds that interfere with cell wall assembly such as Calcofluor White (Ram et al., 1994
; Lussier
et al., 1997
). To determine whether the absence of Scj1p
results in a defect in cell wall biosynthesis, mutant and
wild-type strains were plated on YPD plates that had various concentrations of Calcofluor White or hygromycin B
(Fig. 6). As expected for an OST mutant, the
ost3 strain
was hypersensitive to hygromycin B and Calcofluor White.
It should be noted that yeast with more severe defects in
the OST (e.g., ost1-3 or ost1-4) show little or no growth on
plates that contain 10 µg/ml of hygromycin B (Karaoglu
et al., 1995b
). The
scj1 strain did not show a significant increase in sensitivity to either Calcofluor White or hygromycin B. In contrast, the loss of Scj1p aggravates the cell
wall defect of the
ost3 mutant as shown by a further decrease in colony size on plates that contain Calcofluor
White.
The UPR Pathway Is Induced in scj1,
ost3 and
jem1 Strains
The accumulation of unfolded proteins in the ER is a signal for increased synthesis of ER chaperones that are regulated by the UPR pathway (Kozutsumi et al., 1988). Increased transcription of the KAR2 gene (Normington et al.,
1989
; Rose et al., 1989
), the promoter of which contains a
22-bp UPR element (UPRE; Mori et al., 1992
), is indicative of an increased content of unfolded proteins in the
yeast ER. The SCJ1 and JEM1 genes contain UPRE (Blumberg and Silver, 1991
), and transcription of both
genes is induced by agents that cause protein folding stress
(Schlenstedt et al., 1995
; Nishikawa and Endo, 1997
).
Transcription of KAR2 and JEM1 mRNAs was quantified
by probing Northern blots of total RNA prepared from
yeast cultures grown at 25°C (Fig. 7, A and B). Incubation
of wild-type cells for 2 h in the presence of sufficient tunicamycin to completely block assembly of the dolichol-linked oligosaccharide donor for N-linked glycosylation
results in a 4.9-fold increase in KAR2 mRNA expression
and a 3.5-fold increase in JEM1 mRNA expression. Previous investigators have reported similar extents of KAR2
mRNA induction by tunicamycin treatment (Cox et al.,
1993
; Baxter et al., 1996
). Disruption of the OST3 gene or
the SCJ1 gene results in a 2- and a 2.7-fold increase in
KAR2 mRNA relative to the wild-type strain, respectively. The combined loss of both Ost3p and Scj1p results
in a 3.5-fold induction of KAR2 mRNA, consistent with
the view that loss of Scj1p aggravates the protein folding
stress caused by hypoglycosylation. In each strain, induction of JEM1 mRNA was slightly lower than induction of
KAR2 mRNA (Fig. 7 B). Similar results were obtained for
the
ost3,
ost3
scj1 and
scj1 strains when induction of
the UPR pathway was analyzed by comparing the incorporation of 35S methionine into Kar2p in the wild-type and
mutant strains (Fig. 7 C and not shown). Induction of
Kar2p was also evaluated in strains in which the JEM1
gene was disrupted (Fig. 7 C). We found that loss of Jem1p
has less effect on Kar2p expression than loss of Scj1p. As
observed for the
ost3
scj1 double mutant (Fig. 7 A), disruption of the JEM1 gene in the context of a
ost3 strain
or a
scj1 strain leads to a further increase in Kar2p expression (Fig. 7 C).
|
Redox Stress Aggravates the Protein Folding Defect in
the scj1
jem1 Mutant
The presence of a reducing agent (e.g., DTT) in yeast or
mammalian cell culture media arrests the folding of proteins that contain disulfide bonds in the ER (Braakman et al.,
1992; Simons et al., 1995
). The arrest in folding is reversible; dilution of the reducing agent restores the normal redox potential of the ER, at which point folding of disulfide
bond containing proteins resumes (Braakman et al., 1992
).
Studies in yeast have shown that unfolded p1CPY accumulates when yeast are pulse labeled after DTT treatment
(Jämsä et al., 1994
). After removal of DTT, the subsequent folding and transport of p1CPY to the Golgi is
blocked in yeast strains that have conditional kar2 mutations (Simons et al., 1995
).
To determine whether Scj1p and Jem1p were also required for the conformational maturation of DTT-reduced
p1CPY and pug-CPY, we compared transport of CPY and
ug-CPY to the vacuole in wild-type, scj1 and
scj1
jem1
mutant cells that were radiolabeled in the presence of
DTT. In agreement with previous reports (Jämsä et al.,
1994
; Simons et al., 1995
), only the reduced p1 precursor forms of CPY or pug-CPY accumulate when cells are
pulse labeled for 30 min in the presence of DTT (Fig. 8 A).
After dilution of the reducing agent, p1CPY and pug-CPY
are oxidized and fold into transport competent forms, as
judged by the disappearance of p1CPY and pug-CPY.
Transport of fully glycosylated CPY occurred with very
similar kinetics in wild-type and
scj1 mutant cells (Fig. 8
B, left). We conclude that Scj1p is not required for a rate-limiting step in the folding of DTT-reduced CPY in the
endoplasmic reticulum. Exit of p1CPY from the ER was
delayed, but not prevented, in the
scj1
jem1 mutant relative to the wild-type strain (Fig. 8 B, right). After dilution
of the reducing agent, transport of unglycosylated CPY to
the vacuole was delayed roughly twofold in the
scj1 mutant relative to the wild-type strain, just as we had observed in the absence of DTT treatment. The loss of both
DnaJ homologues had a much more profound effect upon
folding and ER-exit of pug-CPY. We did not detect conversion of pug-CPY to ug-CPY during the 90 minute incubation after dilution of the DTT in the
scj1
jem1 mutant
(Fig. 8 B, right), suggesting that the DTT-reduced unglycosylated CPY cannot be rescued when the folding machinery is compromised by loss of both Scj1p and Jem1p.
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Discussion |
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Here, we have analyzed the genetic interactions between mutations in genes encoding subunits of the OST and null alleles of the SCJ1 and JEM1 genes encoding homologues of the bacterial chaperone DnaJ. We observed that the protein folding stress induced by hypoglycosylation causes a synthetic growth defect in cells that do not express Scj1p. Cells that lack both Scj1p and Jem1p are extremely sensitive to hypoglycosylation. Our data provide the first direct evidence that Scj1p and Jem1p perform a role in protein folding in the lumen of the yeast ER.
Insight into the role of Scj1p as a regulatory cofactor for
Kar2p in the ER lumen is provided by considering the
ATP hydrolysis-dependent reaction cycle of their prokaryotic homologues DnaJ and DnaK. The affinity of the peptide binding domain of DnaK for substrate polypeptides is
controlled by the ATPase cycle of DnaK, which is in turn
regulated by DnaJ and GrpE which stimulate ATP hydrolysis and ribonucleotide exchange, respectively (for recent
reviews see Hartl, 1996; Bukau and Horwich, 1998
). The
ATP-bound conformation of DnaK binds and releases
polypeptide substrates rapidly, whereas the ADP-bound
conformation of DnaK binds peptides with higher affinity
due to a reduced rate of peptide dissociation. Eukaryotic DnaJ homologues have been shown to stimulate the intrinsic ATPase activity of Hsp70 proteins (Cyr et al., 1992
;
Braun et al., 1996
; Schumacker et al., 1996
); hence it is
likely that Scj1p stimulates the ATPase activity of Kar2p.
DnaJ is able to bind unfolded polypeptides and deliver
these substrates to the ATP-bound conformation of DnaK
(Szabo et al., 1994
). The substrate binding domain of DnaJ has been mapped to a zinc finger-like domain that is conserved in many (Scj1p, Mdj1p and Ydj1p), but not all
(Sec63p and Jem1p) eukaryotic DnaJ homologues (Szabo
et al., 1996
). The presence in Scj1p of the cysteine-rich zinc
finger-like domain argues strongly that Scj1p recognizes
unfolded polypeptides in the ER lumen, and initiates folding reactions by cooperation with Kar2p. After ATP hydrolysis and GrpE-mediated dissociation of ADP the substrate polypeptide dissociates from DnaK and proceeds
along a folding pathway or is instead rebound by DnaJ for
another cyclic interaction with DnaK
Genetic Interactions between OST Mutants and ER Chaperone Mutants
The ost1-6 allele that was identified in the synthetic lethality screen is phenotypically similar to other previously
described ost1 mutants (Silberstein et al., 1995). The mutation in Ost1p confers a temperature sensitive growth
phenotype even though nascent glycoproteins are hypoglycosylated at both the permissive and restrictive temperatures. As reported previously for other ost1 mutants
(Silberstein et al., 1995
), expression of Kar2p is induced in
ost1-6 cells at the permissive temperature (Silberstein, S., and R. Gilmore, unpublished observation). Given the role
for N-linked oligosaccharides in protein folding, a synthetic genetic interaction is anticipated for yeast strains
that have defects in an OST subunit and the Kar2 protein.
Indeed, a slow-growth phenotype is observed in each case
for double mutant strains that combine a wbp1 mutation (wbp1-1 or wbp1-2) with a kar2 mutation (kar2-1, kar-159,
or kar2-203; te Heesen and Aebi, 1994
). Here, we observed a synthetic lethal phenotype for the ost1-6kar2-159
double mutant.
The glycosylation defect caused by ost1-6 does not severely compromise growth of the mutant strain at 25°C.
However, loss of Scj1p was not tolerated in an ost1-6 background at any temperature. Unlike the kar2 mutants that
are defective in multiple RER-associated pathways, scj1
mutants have no previously reported defects in protein
translocation, nuclear membrane fusion during karyogamy, protein folding, or resistance to severe heat shock.
Consequently, several different explanations were considered for the inviability of the
scj1ost1-6 mutant. The
discovery that the
scj1ost1-6 mutant is viable on plates
that contain an osmotic stabilizing agent argues strongly
against the catastrophic loss of fully assembled OST complexes in the
scj1ost1-6 mutant. As
scj1 mutants are wild-type with respect to N-linked glycosylation of proteins, the lethal phenotype of the double mutant cannot be
easily explained by the additive effect of two lesions in oligosaccharide addition. Synthetic lethal interactions of the
latter type do occur when an OST mutant (e.g., wbp1-2) is
combined with an alg (asparagine linked glycosylation)
mutant that blocks assembly of the full-sized dolichol-linked oligosaccharide donor for glycosylation (Stagljar et al.,
1994
).
To investigate the effect of deleting Scj1p in a viable
strain with defects in N-glycosylation, we constructed the
ost3
scj1 mutant. The
ost3
scj1 mutant shows a synthetic growth defect, without showing a concomitant reduction in N-glycosylation of proteins, indicating that
Scj1p does not have a direct or indirect effect upon the
efficiency of the glycosylation reaction. The more restrictive phenotype of the
scj1ost1-6 mutant relative of the
ost3
scj1 mutant suggests that the requirement for Scj1p becomes greater as the severity of hypoglycosylation increases.
A Role for Scj1p in Protein Folding
For many proteins that enter the secretory pathway, the
addition of N-linked oligosaccharides is an important
modification that precedes the successful acquisition of
tertiary structure. Until proteins fold, they are not packaged into vesicles for intracellular transport, but are instead retained in the endoplasmic reticulum. Here, we observed that the rate of ER-export for a mutant form of
CPY that lacks N-linked oligosaccharides was reduced
twofold in cells that lack Scj1p. The reduced transport rate
for unglycosylated-CPY was in marked contrast to the
wild-type transport rate for fully-glycosylated CPY in cells
that lack Scj1p. Folding-deficient proteins are retained in
the ER via prolonged interactions with the Hsp70 chaperone BiP or Kar2p (De Silva et al., 1990). The slow maturation of unglycosylated CPY, but not fully glycosylated
CPY, was previously observed in kar2 mutants (te Heesen
and Aebi, 1994
). The unglycosylated precursor (pug-CPY)
was shown to be present in a complex with Kar2p mutant proteins that had lesions in the ATPase domain (i.e., kar2-159 and kar2-203; te Heesen and Aebi, 1994
). Here, we
observed a prolonged association between Kar2p and the
unglycosylated CPY in the endoplasmic reticulum. Our results indicate that in addition to Kar2p, efficient folding of
unglycosylated CPY requires the DnaJ homologue Scj1p. The major fate for soluble proteins that fail to fold in the
yeast RER is dislocation back to the cytosol for subsequent degradation by the proteasome (Hiller et al., 1996
).
As reported previously, the degradative pathway does not
appear to be taken by pug-CPY in wild-type cells (Winther et al., 1991
). Here, we did not observe a selective loss
of pug-CPY during the 1-h chase incubation in mutant
strains that lack Scj1p or Jem1p. An in vitro assay for the
proteasome-mediated degradation of unglycosylated pro-
-factor did not disclose an important role for Scj1p in
degradation of this substrate (McCracken and Brodsky,
1996
).
Delayed transport of folding-impaired hypoglycosylated
proteins to the cell wall provides the most likely explanation for the synthetic phenotypes that arise when scj1 is
combined with mutations in the OST. Support for the latter conclusion is provided by the observation that
scj1
cells are hypersensitive to tunicamycin. In the absence of
an OST mutation,
scj1 mutants lack an obvious cell wall
defect. However, the cell wall defect of the
ost3 mutant was greatly exaggerated in a yeast strain that lacks Scj1p.
Folding of DTT-reduced CPY has been found to require
Kar2p, as strains carrying kar2 alleles with mutations that
map in the ATPase domain (e.g., kar2-159) are unable to
form native disulfide bonds in p1CPY after removal of
DTT resulting in the formation of high molecular mass aggregates of Kar2p and unfolded p1CPY (Simons et al.,
1995
). We did not obtain evidence that Scj1p was required for CPY maturation after redox stress, suggesting that
DTT-reduced CPY may exist as a compact folding intermediate that rapidly acquires a native conformation after
removal of reductant without intervention of Scj1p. However, unglycosylated CPY did not fold correctly in the
scj1
jem1 mutant after redox stress, but instead was retained in the endoplasmic reticulum. Interestingly, we did
observe that
ost3
scj1 cells are hypersensitive to DTT,
suggesting that Scj1p does not act exclusively during glycoprotein folding, but instead has a broader role in the maturation of folding impaired proteins in the RER.
Survival of Yeast without Scj1p
How can we resolve the apparent paradox between the
nonessential status of the SCJ1 gene and the postulated
role for Scj1p as a regulatory cofactor for the essential
Kar2 protein during protein folding in the ER lumen. The
UPR pathway induces enhanced expression of RER chaperones and protein folding enzymes to permit survival under conditions that interfere with protein folding in the
RER. Interestingly, the UPR pathway is dispensable for cell viability under normal growth conditions (Cox et al.,
1993). One possible explanation for the nonessential nature of Scj1p would be that Scj1p only acts as a chaperone
when unfolded proteins accumulate in the RER. However, we found that strains lacking Scj1p express elevated
levels of Kar2p and Jem1p in the absence of other perturbants, indicating that loss of Scj1p causes protein folding
stress in the RER. We propose that Scj1p is the primary cochaperone for Kar2p in protein folding reactions under
normal physiological conditions.
A second explanation for the nonessential nature of
Scj1p is suggested by the presence of one essential and two
nonessential DnaJ homologues in the same cellular compartment raising the possibility that these proteins perform partially redundant functions. A complete functional
redundancy of Scj1p, Sec63p and Jem1p can be discounted for several reasons. Of the three DnaJ homologues, only
Sec63p has the correct localization as a component of the
large Sec complex (Deshaies et al., 1991; Panzner et al.,
1995
) to recruit Kar2p to the protein translocation channel
to interact with translocation substrates (Brodsky and
Schekman, 1993
; Corsi and Schekman, 1997
). Before this
report, a partial overlap of function for Scj1p and Jem1p
was suggested by the temperature sensitive growth phenotype of the
scj1
jem1 mutant and by the observation that
expression of both proteins is controlled by the UPR pathway (Schlenstedt et al., 1995
; Nishikawa and Endo, 1997
).
The wild-type growth phenotype of the scj1 mutant is
most readily explained by the induction of the UPR pathway leading to enhanced expression of RER chaperones
including Kar2p, Lhs1p and Jem1p. Kar2p binds nascent
polypeptides as they enter the ER lumen via the Sec61
channel (Sanders et al., 1992
), hence the initial interaction
between an unfolded protein and Kar2p is mediated by
Sec63p (Corsi and Schekman, 1997
). For wild-type proteins that fold rapidly, this initial Scj1p-independent interaction between Kar2p and the hydrophobic segments of
the nascent polypeptide may be sufficient to initiate productive folding in the RER lumen. In the absence of mutations or perturbants that interfere with protein folding in
the RER, enhanced expression of Kar2p and Jem1p may
compensate for loss of Scj1p. Conceivably, a higher lumenal concentration of Kar2p may result in Scj1p-independent binding of Kar2p to slowly folding proteins in the
RER lumen. Furthermore, suppression of the temperature-sensitive growth phenotype of the
ost3
scj1 strain
by the high copy JEM1 plasmid argues strongly that UPR-mediated induction of Jem1p expression will help compensate for loss of Scj1p.
Overlapping, but Nonidentical Roles for Jem1p and Scj1p
Induction of Kar2p expression was greater in the scj1
mutant than in the
jem1 mutant. Consistent with the relatively minor reduction in growth rate at 37°C for the
ost3
jem1 mutant, we observed that
jem1 yeast cells
are not hypersensitive to tunicamycin and are able to
transport unglycosylated CPY to the vacuole at a wild-type rate. We conclude that the viability of
scj1 cells cannot be adequately explained by a complete redundancy of
Scj1p and Jem1p. Nonetheless, several observations reported here suggest a role for Jem1p in protein folding
in the RER. In comparison to the
jem1 mutant, the
scj1
jem1 double mutant shows a further elevation in
Kar2p expression, and a more severe delay in transport of
ug-CPY to the vacuole. Furthermore, expression of a folding impaired protein (i.e., ug-CPY) in the
scj1
jem1 double mutant interferes with ER exit of fully-glycosylated
wild-type CPY suggesting that the protein folding capacity
of the ER has been exceeded. Surprisingly, expression of
Jem1p from a high copy vector largely alleviates the temperature sensitive growth defect of the
scj1
ost3 mutant.
Overexpression of Jem1p may be able to compensate for loss of Scj1p, even though Jem1p lacks the cysteine rich
domain that has been implicated in substrate recognition.
These data, together with the inviability of a
ost3
scj1
jem1 strain, are consistent with Jem1p acting as a second
regulatory chaperone for Kar2p-mediated protein folding
in the RER. Although Jem1p was initially believed to be a
type II integral membrane protein (Nishikawa and Endo,
1997
), a more recent study has established that the initiation codon for Jem1p was incorrectly assigned, and that
Jem1p is a peripheral membrane protein associated with the lumenal face of the yeast RER (Nishikawa and Endo,
1998
). Conceivably, the primary role of Jem1p as a chaperone may be to recruit Kar2p (or Lhs1p) to the membrane
to interact with unfolded membrane proteins. Consequently, a soluble substrate like unglycosylated proCPY
may not be the optimal substrate to reveal a folding delay caused by loss of Jem1p.
![]() |
Footnotes |
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Received for publication 9 June 1998 and in revised form 14 September 1998.
Address all correspondence to Reid Gilmore, Department of Biochemistry and Molecular Biology, University of Massachusetts Medical School,
55 Lake Avenue North, Worcester, MA 01655-0103. Tel.: (508) 856-5894. Fax: (508) 856-6231. E-mail: reid.gilmore{at}banyan.ummed.edu
Dr. Schlenstedt's present address is Medizinische Biochemie, Gebaeude 44, Universitaet des Saarlandes, D66421 Homberg, Germany.
We thank Mark Rose for yeast strains (kar2-159, MS4, and MS20) and for the 2µ JEM1 plasmid (pMR3270). We thank Jakob Winther for providing the plasmid pJW373 that encodes the unglycosylated CPY mutant and Peter Walter for providing the antibody to Kar2p. The authors wish to thank Peter Philippsen for providing pFA6a-HIS3MX6.
This work was supported by Public Health Services grants GM43768 (R. Gilmore) and GM47385 (P.A. Silver).
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Abbreviations used in this paper |
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5-FOA, 5-fluoro-orotic acid; AP, acid phosphatase; CPY, carboxypeptidase Y; Hsp70, heat shock protein 70 family; OST, oligosaccharyltransferase; ug-CPY, unglycosylated CPY; UPR, unfolded protein response.
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References |
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