* Institute of Biotechnology, University of Helsinki, Helsinki, Finland; and School of Biological Sciences, University of
Manchester, Manchester M13 9PT, United Kingdom
Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We
have recently shown that a marker protein in the ER of
Saccharomyces cerevisiae, denatured by exposure of
cells to 50°C after preconditioning at 37°C, was reactivated by an ATP-dependent machinery, when the cells
were returned to physiological temperature 24°C. Here
we show that refolding of the marker enzyme
Hsp150-
-lactamase, inactivated and aggregated by
the 50°C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of
Lhs1p, Hsp150
-
-lactamase failed to be solubilized
and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was
somehow associated with heat-denatured Hsp150
-
-lactamase, whereas no association with native marker
protein molecules could be detected. Similar findings
were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no
significant role in folding or secretion of newly synthesized Hsp150
-
-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins
may have specific features as compared to chaperones
assisting de novo folding. After preconditioning and
50°C treatment, cells lacking Lhs1p remained capable
of protein synthesis and secretion for several hours at
24°C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER,
refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental
heat-resistant survival machinery needed for recovery
of yeast cells from severe heat stress.
When Saccharomyces cerevisiae cells are shifted
from physiological temperature (24°C) to 50°C,
they die. However, if preconditioned at the temperature where heat shock genes are activated, 37°C, they
survive a subsequent thermal insult at 50°C and form colonies at 24°C. All organisms initiate a protective response
to heat stress at characteristic temperatures in a similar
way (1, 24, 28). Though the primary lesions causing cell
death from thermal insult are not known, denaturation of
proteins in various subcellular locations can be lethal, unless it is reversible (28, 30, 32, 34). Heat shock proteins,
which are upregulated during the preconditioning period,
have been assumed to function in the acquisition of thermotolerance (28, 30). However, little is known of how they
protect or repair heat-damaged target proteins, though a
wealth of information is available on their roles in folding
of newly synthesized proteins under physiological conditions (12). Moreover, the Hsp26, Hsp70, and Hsp90 classes
of heat shock proteins do not significantly affect thermotolerance in yeast (3, 31, 50). In contrast, Hsp104, an ATPbinding member of the Hsp100 (Clp) family of stress proteins, is essential for acquisition of thermotolerance in S. cerevisiae (35). In its absence, cytoplasmic protein aggregates generated by thermal insult to the cells at 50°C fail to
be solubilized, and heat-inactivated mRNA splicing is not
reactivated, resulting in cell death (29, 47).
The ER is the folding compartment for proteins destined to secretory organelles and to the exterior of the cell
(14). When a newly synthesized polypeptide chain emerges
in the ER lumen, it adopts a conformation dictated by its
amino acid sequence. Folding is thought to be assisted by
chaperones like the Hsp70 family member BiP/Kar2p, apparently by prevention of incorrect associations between
nonnative chains (10). We showed recently that a fusion
protein with Yeast Strains and Media
The following yeast strains were grown at 24°C in shake flasks overnight
to early logarithmic phase in YPD medium or synthetic complete (SC)1
medium lacking histidine (H645, H647, H656, and H660): H393 (Mat Metabolic Labeling and Immunoprecipitation
Metabolic labeling of cells (108/200 µl) was with 20 µCi of [35S]methionine/cysteine (1,000 Ci/mmol; Amersham International, Buckinghamshire, UK) in SC medium lacking methionine and cysteine. The labelings
were terminated with NaN3, and the culture medium and cell lysate samples were immunoprecipitated as described (19). Briefly, cells were lysed
mechanically with glass beads in NET buffer (0.05 M Tris-HCl, pH 8.0, containing 0.4 M NaCl, 5 mM EDTA, 1% NP-40, and 100 U/ml of aprotinin) in the presence of 2 mM PMSF. The lysates were boiled and precleared for 1 h at 4°C with protein A-Sepharose (Pharmacia LKB Biotechnology, Inc., Piscataway, NJ). The samples were immunoprecipitated with
anti- Coimmunoprecipitation and Western Analysis
Cells were lysed with glass beads in the presence of protease inhibitors as
above, but in the presence of Triton X-100 and absence of SDS and boiling. The lysates were precleared with preimmune serum (1:100) and protein A-Sepharose and incubated overnight with anti- Sucrose Gradient Centrifugation
Cells were lysed under nondenaturing conditions (see above) and loaded
on 10-40% (wt/vol) linear sucrose gradients and subjected to velocity sedimentation centrifugation (44). Fractions of 0.5 ml were collected from top
to bottom. The pellet was dissolved in 10 µl of 20% SDS and diluted in
0.5 ml of NET buffer. The fractions were immunoprecipitated under nondenaturing conditions.
Other Methods and Materials
For determination of Effect of Lhs1p on Reactivation of Heat-denatured
Hsp150 Our assay to study heat-denaturation and refolding of ERlocated proteins in living S. cerevisiae cells is presented at
the top of Fig. 1. Cells grown at 24°C are incubated in liquid medium for an h at 37°C. The marker protein
Hsp150
To study whether Lhs1p has a role in refolding of proteins
denatured by thermal insult to living cells, Hsp150 Aggregation and Solubilization of Heat-denatured
Hsp150 Next, we studied whether the thermal treatments caused
aggregation of Hsp150
Next we assayed the
In the absence of the LHS1 gene (strain H621, lhs1
Coimmunoprecipitation of Lhs1p with Heat-denatured
Hsp150 Coimmunoprecipitation experiments were performed to
study whether Lhs1p was associated with Hsp150
When a 50°C-treated sample, like the one in Fig. 5 A,
lane 2, was fractionated in a sucrose gradient before immunoprecipitation with anti- To study whether Lhs1p could be found in association
even with an authentic yeast glycoprotein, we repeated the
above coimmunoprecipitation experiment using anti-CPY
antiserum instead of anti-
Stability of Heat-denatured Marker Protein
To study the stability of heat-aggregated Hsp150
Role of Lhs1p in De Novo Folding and Secretion of
Hsp150 Lhs1p is required for translocation into the ER of a subset
of proteins (2, 6). Thus, we studied the role of Lhs1p in translocation, folding, and secretion of de novo-synthesized Hsp150
Proteins that normally acquire disulfide bonds are retained in the yeast ER if cotranslocational disulfide formation is prevented by incubating cells with a reducing agent
like DTT, though the secretory machinery remains functional. When oxidizing conditions are reestablished by removing DTT, the sulfhydryls are oxidized, and the proteins
undergo conformational maturation resulting in resumption of secretion (19, 44). To study whether Lhs1p was involved in conformational maturation of newly synthesized proteins, H602 (lhs1
The role of Lhs1p in conformational maturation of proCPY was then studied. Both in strains H602 (lhs1 Acquisition of Thermotolerance in the Absence
of Lhs1p
Next we studied whether Lhs1p affected the ability of
yeast cells to acquire thermotolerance. Strain H540 (lhs1
Though only 10% of lhs1
The open reading frame of the LHS1 gene (YKL 073) was
discovered in the Yeast Genome Sequencing Project. The
translated amino acid sequence begins with a potential NH2terminal signal peptide and terminates with an ER-retrieval
signal HDEL. The ER-located Hsp70 cognate BiP/Kar2p
has 25% identical and 50% similar amino acids with Lhs1p
(33). LHS1 encodes a glycoprotein of 113-119 kD, is located in an ER-like compartment, and is required for translocation of a subset of proteins (2, 6). The LHS1 gene and Lhs1p are also called SSI1 and Ssi1p, respectively (2). We show here that Lhs1p is involved in conformational repair and stability of heat-damaged protein in the yeast ER.
In the absence of a functional LHS1 gene, Hsp150 According to coimmunoprecipitation experiments, Lhs1p
was associated with heat-denatured Hsp150 Though heat-damaged aggregated Hsp150 In the presence of Lhs1p, Hsp150 Lhs1p thus appears to be involved both in translocation
and in conformational repair of heat-damaged protein.
Yeast BiP/Kar2p also has been assigned two functions. It is
generally needed for translocation (36, 48) and for conformational maturation of newly synthesized pro-CPY (44)
and Hsp150 Chaperone-dependent refolding of heat-denatured proteins has been shown before to occur in vivo in the cytosol
of yeast and mammalian cells, and in E. coli. Heat-denatured firefly luciferase was reactivated in S. cerevisiae in an
Hsp104-dependent manner (29). In E. coli, refolding of In the absence of Lhs1p, the ability of the yeast cells to
form colonies after thermal insult was reduced 10-fold,
whereas in the absence of Hsp104, thermotolerance is reduced 1,000-fold (35). Thermal insult did not result in
abrupt death of the lhs1-lactamase activity, Hsp150
-
-lactamase, was inactivated in the yeast ER upon shift of the cells to
50°C after preconditioning at 37°C. When the cells were
returned to 24°C, the enzyme was reactivated in an ATPdependent way, even in the absence of de novo protein
synthesis (21). In these studies the marker enzyme was accumulated in the ER of the temperature-sensitive secretion mutants sec23 or sec18 before the thermal insult at 50°C. When the marker enzyme was preaccumulated in
the Golgi in sec7 or sec14 mutants, it failed to be reactivated, demonstrating that the refolding apparatus was
confined to the ER (21). We show here that conformational repair of heat-damaged protein in the ER involves a
novel member of the Hsp70 family, Lhs1p. Lhs1p was
needed for reactivation, solubilization, and stabilization against proteolysis of the marker enzyme Hsp150
-
-lactamase, which was denatured and aggregated by thermal insult
to living cells. Lhs1p appeared to have no significant role in
folding and secretion of newly synthesized Hsp150
-
-lactamase molecules.
Materials and Methods
sec18-1 ura3-52 trp1-289 leu2-3,112 URA3::HSP150
-
-lactamase [43]),
H454 (Mata ura3-1 his3-11,15 leu2-3,112 trp1-2 ade2-1 can1-100 hsp104::
LEU2 [35]), H486 (Mata sec23-1 leu2-3,11 ura3-52 URA3::HSP150
-
-lactamase [12]), H503 (RCY127 Mata sec18-1 trp1-1 ade2-1 his3 ura3-52 lhs1::
TRP1[6]), H540 (RCY104 Mata ura3-52 ade2-1 trp1-1 lys2 his3 lhs1::TRP1
[6]), H541 (TR2 Mata ura3-52 ade2-1 trp1-1 lys2 his3 [27]), H602 (Mata ura3-52 ade2-1 trp1-1 lys2 his3 lhs1::TRP1 URA3::HSP150
-
-lactamase), H604 (Mata ura3-52 ade2-1 trp1-1 lys2 his3 URA3::HSP150
-
-lactamase), H621 (Mata sec18-1 ura3-52 ade2-1 trp1-1 his3 lhs1::TRP1 URA3::
HSP150
-
-lactamase), H645 (Mata sec18-1 ura3-52 ade2-1 trp1-1 his3
lhs1::TRP1 URA3::HSP150
-
-lactamase [pRC42]), H647 (Mata sec18-1
ura3-52 ade2-1 trp1-1 his3 lhs1::TRP1 URA3::HSP150
-
-lactamase
[pRC51]), H656 (Mata ura3-52 ade2-1 trp1-1 lys2 his3 lhs1::TRP1 [pRC51]),
and H660 (Mata sec18-1 trp1-1 ade2-1 his3 ura3-52 lhs1::TRP1 [pRC51]).
Strains H602, H604, and H621 were created by integrating the HSP150
-
-lactamase gene in plasmid pKTH4544 (43), into the genome of strains
H540, H541, and H503, respectively. Centromeric plasmid pRC42 containing the LHS1 gene (6) was introduced into strain H621, creating strain
H645, and centromeric plasmid pRC51 containing the c-myc-LHS1 gene
(6) was introduced into strains H621, H540, and H503 to create strains
H647, H656, and H660, respectively. Transformations were with the lithium acetate method (15). Strain H534 (Mat
sec18-1 ade2-1 his3-11,15
trp1 hsp104::LEU2 URA3::HSP150
-
-lactamase) was created by mating
strains H393 and H454 and dissecting the spores of the resulting diploid.
The 4-kb BamHI/SacI fragment containing the LHS1 gene was removed
from pRC40 (6) and ligated into BamHI/SacI-digested pRS313 (CEN
HIS3 [42]), to create pRC42. A 4.1-kb BamHI/SacI fragment containing a
modified copy of the LHS1 gene encoding Lhs1p tagged with the c-myc
epitope was removed from pRC45 (6) and ligated into pRS313 (CEN
HIS3) to create pRC51.
-lactamase antiserum (1:100) or anti-carboxypeptidase Y (CPY)
antiserum (1:100) and protein A-Sepharose for 2 h at 4°C. After washing
with diluted NET buffer (1:1), wash buffer (0.1 M Tris-HCl, pH 7.5, containing 0.2 M NaCl, 2 M urea, and 0.5% Tween-20), and with 0.1% SDS,
the precipitates were analyzed by SDS-PAGE.
-lactamase antiserum (1:100), anti-CPY antiserum (1:100), or preimmune serum (1:100) at
4°C, followed by a 3-h incubation with protein A-Sepharose at 4°C. The
precipitates were washed twice with NET buffer containing 10 mM CaCl2
and lacking EDTA, and twice with the same buffer diluted 1:1 and subjected to SDS-PAGE. Proteins were blotted from gel onto nitrocellulose
membrane (Hybond-C Extra; Amersham International) and immunostained with anti-
-lactamase antiserum (1:6,000) or anti-CPY antiserum
(1:1,000) and alkaline-phosphatase-conjugated anti-rabbit antibody (1:7,500;
Promega Corp., Madison, WI), or with monoclonal anti-c-myc antibody
(1:1,000; Chemicon International, Temecula, CA) and alkaline phosphatase-
conjugated anti-mouse antibody (1:3,000; Promega Corp.).
-lactamase activity, duplicate cell samples (25 × 106/0.5 ml of YPD or SC medium) were incubated at indicated temperatures. After termination of the incubation with NaN3, the cells were separated from the media, and cell-associated and secreted
-lactamase activity was assayed using nitrocefin as a substrate as described (43). Invertase
synthesis was induced by incubating cells (5 × 107/0.5 ml) in YPD medium
containing 0.1% glucose, and intracellular and cell wall-bound invertase
activity of duplicate samples was measured as described in (19). Thermotolerance was assayed by incubating duplicate cell samples (3 × 106/ml) in
YPD medium (except strain H647 in SC medium lacking histidine) in
Wassermann tubes successively at 37 and 50°C (35). After cooling on ice,
the cells were diluted and plated for 3-4 d at 24°C. Cycloheximide (CHX),
DTT, and NaN3 were from Sigma Chemical Co. (St. Louis, MO) and used
in final concentrations of 100 µg/ml, 20 mM, and 10 mM, respectively.
BSA and porcine thyroglobulin were from Sigma Chemical Co. SDSPAGE was in 8% reducing gels.
Results
-
-lactamase In Vivo
-
-lactamase is expressed under the heat-activated HSP150 promoter in a temperature-sensitive mutant (sec18), where ER-derived transport vesicles are unable to fuse with Golgi membranes at 37°C. The 37°C
incubation thus preconditions the cells to survive a subsequent thermal insult at 50°C, upregulates the synthesis of
the marker protein, and causes its retention in the preGolgi compartment. The cells are then incubated for 20 min at 50°C, resulting in inactivation of the
-lactamase fusion protein, and shifted to 24°C to monitor reactivation of
the marker enzyme. In tens of experiments, 20-70% of the
original
-lactamase activity was resumed in 3-6 h at 24°C in the presence of CHX (21). The variation in the reactivation yields evidently reflects the vulnerability of the cells to
extreme temperature. Hsp150
-
-lactamase consists of
Escherichia coli
-lactamase fused to the COOH terminus
of the Hsp150
-carrier, an NH2-terminal 321-amino acid
fragment of the yeast secretory glycoprotein Hsp150 (20).
The carrier is needed for translocation and to promote
proper folding of foreign proteins that by themselves are
not secretion-competent in yeast (25, 43).
Fig. 1.
Dependence of reactivation of heat-denatured Hsp150-
-lactamase on Lhs1p. (Top) Scheme of the renaturation assay.
After growth at 24°C, cells were incubated for 60-70 min at 37°C
(preconditioning), thereafter for 20 min at 50°C (thermal insult),
and then for 6 h at 24°C (recovery). Strains (A) H621 (lhs1
sec18), (B) H393 (sec18), and (C) H645 (lhs1
LHS1+ sec18)
were preincubated for 10 min at 37°C, pelleted, resuspended in
prewarmed medium, and incubated at 37°C for 1 h and then at 50°C for 20 min. CHX (closed circles) or NaN3 (open circles) was added, and the cells were incubated for the indicated times at 24°C. The
-lactamase activity of lysed cells was determined and plotted against time. The absence (lhs1
) and presence (WT) of
the LHS1 gene is indicated. The designation wild-type (WT) refers only to the LHS1 gene.
[View Larger Version of this Image (14K GIF file)]
-
-lactamase was expressed in S. cerevisiae sec18 cells lacking a
functional LHS1 gene because of targeted gene disruption
(strain H621, lhs1
sec18). When H621 cells were incubated at 37°C,
-lactamase activity accumulated inside of
the cells (Fig. 1 A), to a somewhat lower level than in wildtype cells (Fig. 1 B). Hsp150
-
-lactamase adopts an enzymatically active conformation upon translocation into
the ER and concomitant disulfide formation. The ER form
of Hsp150
-
-lactamase is primary O-glycosylated at many
serines and threonines of the carrier portion and migrates
in SDS-PAGE like a protein of ~110 kD, whereas the mature protein with extended O-glycans migrates like a 145-kD
protein (21, 43). Shift of the cells from 37 to 50°C abolished the
-lactamase activity. When CHX was added to
inhibit protein synthesis and the cells were shifted to 24°C for recovery, very little of the initial
-lactamase activity was resumed in 6 h (Fig. 1 A, closed circles). Parallel cells, which received NaN3 at the time of shift to 24°C, served as
a negative control: no reactivation was observed in the absence of ATP (open circles). H393 cells, which harbored
the wild-type genomic LHS1 gene and the sec18 mutation,
served as a positive control: 38% of the initial
-lactamase
activity, which had accumulated in the ER before inactivation at 50°C, was recovered in 6 h (Fig. 1 B, closed circles),
and NaN3 inhibited the reactivation (open circles). We
returned one copy of the wild-type LHS1 gene on a centromeric plasmid to strain H621, creating strain H645 (lhs1
LHS1+ sec18). Reactivation of heat-denatured Hsp150
-
-lactamase was rescued to the level observed in the wildtype strain H393, again in an ATP-dependent fashion (Fig.
1 C). In none of the strains was
-lactamase activity found
in the culture medium. These experiments show that
Lhs1p did not protect the marker protein from thermal inactivation but was involved in reactivation of the heatdenatured marker protein.
-
-lactamase
-
-lactamase. H393 cells (sec18)
were 35S-labeled for 1 h at 37°C (Fig. 2 A), a parallel cell
sample was incubated thereafter for 20 min at 50°C (Fig.
2 B), and a third sample was allowed to recover for 6 h at
24°C in the presence of CHX (Fig. 2 C). A fourth sample
received NaN3 for the time of recovery (Fig. 2 D). The
cells were lysed under nondenaturing conditions and subjected to velocity sedimentation centrifugation in sucrose
gradients. The fractions plus the pellet material (lane P)
were subjected to immunoprecipitation with anti-
-lactamase antiserum and SDS-PAGE analysis. After labeling
at 37°C, Hsp150
-
-lactamase was detected in the top
fractions, indicating that it was soluble (Fig. 2 A). When
immunoprecipitated from the gradient fractions, the protein migrated in SDS-PAGE as a doublet of ~95 and 110 kD. Thermal insult to the cells caused Hsp150
-
-lactamase
to form aggregates since it was detected across the whole
gradient and in the pellet fraction (Fig. 2 B). After the recovery period at 24°C, it sedimented again mainly in the
top fractions, showing that it was largely solubilized. Solubilization required metabolic energy, since it was inhibited
in the presence of NaN3 (Fig. 2 D). Since the sec18-1 mutation blocks fusion of ER-derived vesicles with the Golgi
membranes, at least part of our marker protein may have
resided in the vesicles in addition to the ER proper. Thus,
we repeated the aggregation assay described above using sec23-1 cells (H486), where the marker is blocked quantitatively in the ER proper. Similar results were obtained as
shown in Fig. 2, A-D, for the sec18-1 mutant. We have
shown before that resumption of enzymatic activity of
heat-inactivated Hsp150
-
-lactamase occurred equally
efficiently in the sec23-1 and sec18-1 mutants (21).
Fig. 2.
Reversible heat-induced aggregation of Hsp150-
-lactamase. (A) Strain H393 (sec18) was labeled, after a preincubation for 10 min at 37°C, with [35S]methionine/cysteine for 1 h at
37°C. (B) Parallel cells were incubated for 20 min at 50°C after
the labeling, or incubated thereafter for 6 h at 24°C in the presence of CHX (C) or NaN3 (D). The cells were lysed under nondenaturing conditions and centrifuged in linear 10-40% sucrose
gradients. 10 fractions from top to bottom plus the pellet (lane P)
were collected and immunoprecipitated with anti-
-lactamase,
followed by SDS-PAGE and fluorography. Molecular mass
markers of 97 and 69 kD are indicated on the left of each panel,
and the sedimentation of bovine serum albumin (4.6 S, 66 kD)
and thyroglobulin (19.3 S, 660 kD) are indicated at the bottom.
[View Larger Version of this Image (50K GIF file)]
-lactamase activity after fractionation. After incubation of H393 cells at 37°C,
-lactamase
activity coincided with 35S-Hsp150
-
-lactamase in fractions 1-3 of the gradient (Fig. 3 A). After the thermal insult, no activity could be detected in the fractions (Fig. 3 B),
whereas after 6 h at 24°C in the presence of CHX, ~22%
of the original
-lactamase activity reappeared in the top
fractions (Fig. 3 C). We conclude that our marker protein was soluble when accumulated in the ER, aggregated by
thermal insult to the living cells, and resolubilized in an
ATP-dependent fashion, even in the absence of de novo
protein synthesis, during recovery of the cells at 24°C.
Fig. 3.
-Lactamase activity of the gradient fractions. Strain
H393 (sec18) was incubated at 37°C (A), then at 50°C (B), and
thereafter for 6 h at 24°C with CHX (C). The cells were lysed and
subjected to sucrose gradient centrifugation as described in Fig. 2,
A-C. The fractions were assayed for
-lactamase activity.
[View Larger Version of this Image (16K GIF file)]
sec18), 35S-Hsp150
-
-lactamase labeled at 37°C was soluble and migrated in SDS-PAGE again as a doublet of 95 and 110 kD (Fig. 4 A). After the 50°C treatment, it was
found in aggregates (Fig. 4 B), and after the recovery period it was barely detectable (Fig. 4 C). After the thermal
insult and the recovery period, very little of the marker
protein could be immunoprecipitated. Exposure time for
B and C was five times longer than for A. Fractions 1-3
contained
-lactamase activity when the cells had been incubated at 37°C, whereas after thermal insult and recovery, no activity could be detected in the gradients (not
shown). In the rescue strain H645 (lhs1
LHS1+ sec18),
ATP-dependent solubilization of the Hsp150
-
-lactamase- containing aggregates occurred similarly as in strain H393
(see Fig. 2; not shown). Thus, heat-denatured Hsp150
-
lactamase remained in aggregates and was susceptible for
proteolysis in the absence of Lhs1p. Degradation in this
experiment occurred mainly by cellular proteases after cell
lysis since less degradation occurred in vivo, as shown below.
We conclude that thermal insult caused aggregation of ERlocated Hsp150
-
-lactamase, whether Lhs1p was present
or not. In the absence of Lhs1p, the Hsp150
-
-lactamase-containing aggregates failed to be solubilized and
were susceptible to proteolytic degradation in vitro.
Fig. 4.
Aggregation and degradation of Hsp150-
-lactamase
in the absence of Lhs1p. The same experiments as described in
the legend of Fig. 2, A-C, were performed using strain H621
(lhs1
sec18). B and C were exposed five times longer than A. The sedimentation of BSA (4.6 S) and thyroglobulin (19.3 S) is
indicated at the bottom.
[View Larger Version of this Image (79K GIF file)]
-
-lactamase and Pro-CPY
-
-lactamase. H647 cells (lhs1
c-myc-LHS1+ sec18) were incubated successively for 1 h at 37°C (Fig. 5, A and B, lanes 1),
20 min at 50°C (lanes 2), and with CHX at 24°C for 3 h
(lanes 3) or 6 h (lanes 4). The cells were lysed under nondenaturing conditions and divided in two. One lysate set
was immunoprecipitated with anti-
-lactamase antiserum
(Fig. 5, A and B, lanes 1-4) and the other with preimmune
serum (lanes 6-9). Both sets were divided in two and subjected to SDS-PAGE and Western analysis using anti-
c-myc antibody (Fig. 5 A) or anti-
-lactamase antiserum
(Fig. 5 B). After incubation of the cells at 37°C, no precipitation of c-myc-Lhs1p with anti-
-lactamase antiserum
could be detected (Fig. 5 A, lane 1). However, after thermal insult at 50°C and recovery at 24°C, c-myc-Lhs1p was
coimmunoprecipitated with anti-
-lactamase antiserum
(lanes 2-4). The total cell lysate samples (Fig. 5 A, lanes 5 and 10) show that a minor portion of Lhs1p was coprecipitated with Hsp150
-
-lactamase. Anti-
-lactamase antiserum precipitated Hsp150
-
-lactamase of ~110 kD
from all samples (Fig. 5 B, lanes 1-4). Neither Lhs1p (Fig.
5 A, lanes 6-9) nor the marker protein (Fig. 5 B, lanes 6-9)
were precipitated with preimmune serum. Thus, Lhs1p appeared to occur in association predominantly with heatdenatured marker protein molecules. Whether the association was direct or mediated by other components is not
known.
Fig. 5.
Coimmunoprecipitation of Lhs1p with Hsp150-
-lactamase. (A and B) H647 (lhs1
c-myc-LHS1+ sec18) cells were
incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2 and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes
4 and 9). The samples were lysed under mild detergent conditions
and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti-
-lactamase antiserum (
-bla; A and B, lanes 1-4) and the other
with preimmune serum (pim; A and B, lanes 6-9). The precipitates were separated by SDS-PAGE, blotted, and immunostained (IS) using anti-c-myc antibody (
-c-myc; A, lanes 1-10) or anti-
-lactamase antiserum (B, lanes 1-10). Lanes 5 and 10 in both panels contained total lysate sample. c-myc-Lhs1p (lhs1p) and Hsp150
-
-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in
lane M. (C) A cell sample incubated at 37° and 50°C, like above
in lane 2, was lysed like above and centrifuged in a 10-40% sucrose gradient as described in Fig. 2. The fractions (lanes 1-10)
and pellet material (lane P) were immunoprecipitated with anti-
-lactamase antiserum and subjected to blotting and immunostaining with anti-c-myc antibody, as in A. T, total cell lysate
sample stained with anti-c-myc antibody. Arrowhead indicates
c-myc-Lhs1p.
[View Larger Version of this Image (43K GIF file)]
-lactamase antiserum in nondenaturing conditions, c-myc-Lhs1p was detected by anti-
c-myc antibody in Western analysis mostly in fractions 7-9
and in the pellet fraction P (Fig. 5 C), i.e., in aggregates.
We showed earlier that BiP/Kar2p was found in association with heat-treated as well as native Hsp150
-
-lactamase (21). Thus, the aggregates immunoprecipitated with
anti-
-lactamase antiserum contained at least Hsp150
-
-lactamase, Lhs1p, and BiP/Kar2p.
-lactamase antiserum (Fig. 6).
Normally, CPY is synthesized as a precursor, which is N-glycosylated in the ER (pro-CPY of 67 kD), and then transported to the vacuole, where it is processed to mature
CPY of 62 kD. Similar results were obtained for pro-CPY
as for Hsp150
-
-lactamase. After incubation of H660 cells
(lhs1
c-myc-LHS1+ sec18) at 37°C, very little c-myc-
Lhs1p (Fig. 6 A, lane 1) was immunoprecipitated with proCPY (Fig. 6 B, lane 1). After treatment of the cells for 20 min at 50°C and incubation for 3 or 6 h at 24°C, much
more c-myc-Lhs1p (Fig. 6 A, lanes 2-4) was precipitated with pro-CPY (Fig. 6 B, lanes 2-4). Immunoprecipitation
with preimmune serum served as a negative control, as before (Fig. 6, A and B, lanes 5-8). Lanes 9 contain total cell
lysate. In Fig. 6 B, IgG is shown because of close migration
with pro-CPY. Thus, even in the case of pro-CPY did
Lhs1p appear to be predominantly associated with molecules that had undergone the thermal insult.
Fig. 6.
Coimmunoprecipitation of Lhs1p with pro-CPY. (A
and B) H660 (lhs1 c-myc-LHS1+ sec18) cells were incubated as
in Fig. 5 for 1 h at 37°C (lanes 1 and 5), 20 min at 50°C (lanes 2 and 6), and at 24°C with CHX for 3 h (lanes 3 and 7) or 6 h (lanes
4 and 8). After lysis of the cells under nondenaturing conditions,
the samples were divided in two. One set of samples was immunoprecipitated (IP) with anti-CPY antiserum (
-CPY, A and B,
lanes 1-4) and the other with preimmune serum (pim; A and B,
lanes 5-8). Lanes 9 contained total lysate sample. After SDSPAGE and blotting, the proteins were immunostained (IS) using
anti-c-myc antibody (
-c-myc; A, lanes 1-9) or anti-CPY antiserum (B, lanes 1-9). c-myc-Lhs1p (Lhs1p), pro-CPY (CPY), and
IgG are indicated. Molecular mass markers (kD) are indicated on
the left.
[View Larger Version of this Image (61K GIF file)]
-
-lactamase in the absence of Lhs1p in vivo, strain H621 (lhs1
sec18) was labeled with [35S]methionine/cysteine at 37°C
(Fig. 7 A, lane 1). Parallel cells were then incubated at
50°C (Fig. 7, lane 2), or thereafter at 24°C for 2-8 h (lanes
3-6). The cell lysates were immunoprecipitated with anti-
-lactamase antiserum, followed by SDS-PAGE analysis. Hsp150
-
-lactamase persisted for about 4 h at 24°C,
growing in size apparently because of some increase in
O-glycosylation. During the next 4 h, it was largely degraded. In the rescue strain H647 (lhs1
c-myc-LHS1+
sec18), the amount of the marker protein remained virtually unchanged (Fig. 7 B), as shown before for strain H393
harboring the genomic LHS1 gene (21). Degradation of
the marker in the absence of Lhs1p was not due to abrupt
cell death, since heat-treated H621 cells were capable of
protein synthesis for hours after shift to 24°C (see below).
To confirm this, we studied the stability of Hsp150
-
-lactamase in a strain lacking a functional HSP104 gene
(H534), which does not acquire thermotolerance at 50°C
in spite of preconditioning at 37°C (35) (see below). In this
strain, the thermal treatments inhibited protein synthesis
irreversibly (see below). Hsp150
-
-lactamase 35S-labeled
at 37°C remained after the 50°C treatment apparently undegraded at 24°C at least for 8 h (Fig. 7 C). These data
show that Lhs1p suppressed in vivo degradation of the
heat-denatured marker protein.
Fig. 7.
In vivo degradation of heat-denatured Hsp150-
-lactamase. (A) H621 (lhs1
sec18), (B) H647 (lhs1
c-myc-LHS1+
sec18), and (C) H534 (Hsp104
sec18) cells were preincubated
for 15 min at 37°C, labeled in SC medium lacking methionine and
cysteine with [35S]methionine/cysteine for 20 min, diluted with excess SC medium containing unlabeled methionine and cysteine,
and incubated further at 37°C for 40 min (lanes 1). Parallel cells
were incubated thereafter for 20 min at 50°C (lanes 2), or after
this at 24°C for 2 h (lanes 3), 4 h (lanes 4), 6 h (lanes 5), or 8 h
(lanes 6). The cells were lysed and immunoprecipitated with anti-
-lactamase antiserum, followed by SDS-PAGE analysis.
[View Larger Version of this Image (50K GIF file)]
-
-lactamase and Pro-CPY
-
-lactamase in more detail. Strain H602
(lhs1
) (Fig. 8 A) and its parental strain H604 (WT) (Fig. 8
B), both wild-type for secretion, were incubated at 37°C,
and the
-lactamase activities of the culture medium samples (Fig. 8, closed circles, EX) and lysed cell samples
(open circles, IN) were determined. In both strains,
-lactamase activity externalized to the culture medium increased linearly, a little faster in the parental strain than in
the lhs1
mutant. In the parental strain, little intracellular
activity accumulated (0.12 U/ml in 90 min), indicating efficient secretion of the marker enzyme. In the lhs1
mutant,
twice as much intracellular activity accumulated during the incubation, and its level in the beginning of the experiment was high. This suggests that a fraction of the newly
synthesized marker protein remained cell associated in active form and gradually built up an intracellular pool in the
absence of a functional LHS1 gene.
Fig. 8.
Secretion of -lactamase activity. Strains (A) H602
(lhs1
) and (B) H604 (WT) were incubated at 37°C. The
-lactamase activity of the culture medium (closed circles, EX) and lysed
cells (open circles, IN) was determined and plotted against incubation time.
[View Larger Version of this Image (14K GIF file)]
) cells were 35S-labeled in the
presence of DTT to allow translocation, while folding of
Hsp150
-
-lactamase was inhibited. Immunoprecipitation with anti-
-lactamase antiserum of the culture medium
sample showed that very little of the marker protein was
secreted (Fig. 9 A, lane 3). In the absence of DTT, mature
Hsp150
-
-lactamase of 145 kD was immunoprecipitated
from the culture medium (Fig. 9 A, lane 1), and some 66- and 145-kD protein from the cell lysate (lane 2). The intracellular reduced molecules were difficult to detect (Fig. 9 A,
lane 4), as noted before (43). When cells labeled under reducing conditions were washed and chased in the absence
of DTT and presence of CHX, secretion of the marker
protein was resumed, since it could be detected in the culture medium (Fig. 9 A, lane 5). Some 66-, 110-, and 145-kD
proteins remained cell associated (Fig. 9 A, lane 6). Similar
results were obtained for the parental strain H604: DTT
prevented reversibly the secretion of the marker protein
(Fig. 9 B), but less intracellular proteins were detected in
the cell lysates as compared to H602 (Fig. 9 B, lanes 2, 4,
and 6).
Fig. 9.
Conformational maturation of Hsp150-
-lactamase and pro-CPY. (A) Strains
H602 (lhs1
) and (B) H604
(WT) were preincubated for 10 min in the absence or presence
of DTT and labeled with
[35S]methionine/cysteine for 1 h
in the absence (lanes 1 and 2) or presence of the reducing agent
(lanes 3 and 4). CHX was
added and the incubations continued for 20 min. In lanes 5 and 6, the labeling was performed in the presence of DTT
as above, after which DTT was
removed and the cells incubated further in fresh medium
with CHX for 1 h. All incubations were at 37°C. The culture medium (m; lanes 1, 3, and 5)
and cell lysate samples (c; lanes 2, 4, and 6) were immunoprecipitated with anti-
-lactamase
antiserum before SDS-PAGE
analysis. Apparent molecular
masses of Hsp150
-
-lactamase-related proteins (kD) are
indicated on the right, and the
molecular mass markers (kD)
on the left. (C) Strains H602
(lhs1
) and (D) H604 (WT)
were 35S-labeled for 1 h after a
preincubation of 10 min in the
presence of DTT (lanes 1-3),
washed, and incubated with
CHX for 45 min (lanes 2) or 90 min (lanes 3). In lanes 4, the labeling was as in lanes 1, but in
the absence of DTT. All incubations were at 24°C. The cell
lysates were precipitated with
anti-CPY antiserum, followed
by SDS-PAGE analysis.
[View Larger Versions of these Images (43 + 25K GIF file)]
; Fig. 9
C) and H604 (WT; Fig. 9 D), pro-CPY was accumulated in the ER in the presence of DTT (lanes 1), and converted
similarly to CPY after DTT was replaced by CHX (lanes 2 and 3). In lanes 4, the cells were labeled in the absence of
DTT, and thus mature CPY could be detected because of
its rapid transport to the vacuole. From these data we conclude that Lhs1p had no significant role in translocation or
conformational maturation of Hsp150
-
-lactamase and
pro-CPY under our experimental conditions.
),
the parental strain H541 (WT), and strain H656 (lhs1
c-myc-LHS1+) were incubated for different times at 37°C
and thereafter for 20 min at 50°C and plated at 24°C (Fig.
10 A). More than 50% of H541 (Fig. 10 A, open circles)
and H656 cells (closed circles) formed colonies when preconditioned for 45 min at 37°C before the 50°C treatment,
whereas only 4% of H540 cells (Fig. 10 A, squares) survived. Strain H454 lacking a functional HSP104 gene (Fig.
10 A, triangles) served as a negative control: only 0.1% of
them were able to form colonies after the thermal treatments. When the assay was performed by preincubating
strains H540 (Fig. 10 B, lhs1
; squares) and H656 (lhs1
LHS1+; circles) for 45 min at 37°C followed by different
times at 50°C, lack of LHS1 again caused a 10-fold decrease in survival.
Fig. 10.
Effect of Lhs1p
on acquisition of thermotolerance. (A) Strains H541
(WT, open circles), H540
(lhs1, squares), H656 (lhs1
c-myc-LHS1+, closed circles), and H454 (hsp104
, triangles) were incubated for
different times at 37°C and
then for 20 min at 50°C, and
plated at 24°C. (B) Strains
H656 (closed circles) and
H540 (squares) were incubated for 45 min at 37°C and
then for different times at
50°C, and plated at 24°C. The colonies were counted to determine the percentage of
survival, which was plotted
against incubation times.
[View Larger Version of this Image (17K GIF file)]
cells, as compared to wild-type
cells, were able to form colonies after the thermal insult,
most of them remained biochemically active for several hours
after the 50°C treatment as shown by de novo synthesis of
invertase during the recovery period. H621 cells (lhs1
sec18; Fig. 11 A) and H393 cells (sec18; Fig. 11 B) were incubated successively for 1 h at 37°C, 20 min at 50°C, and 3 h
at 24°C. The cells were then shifted to low glucose medium
to derepress the synthesis of invertase (Fig. 11 B, arrow),
which is normally destined to the cell wall. Determination
of invertase activity in the cell wall (Fig. 11, A and B,
closed circles, EX) and inside of the cells (open circles, IN)
showed that similar amounts of the enzyme were synthesized and secreted in the presence and absence of Lhs1p.
Both strains secreted similar amounts of active invertase also at 24°C in the absence of any thermal treatments (Fig.
11, C and D). In contrast, no invertase synthesis during a
recovery period of 6 h could be detected in strain H454
(hsp104
) (not shown). We conclude that synthesis and secretion, but not repair of the heat-damaged marker protein, functioned for many hours during the recovery period
at 24°C in the absence of Lhs1p. Decreased thermotolerance
of lhs1
cells, as measured by plating efficiency, was thus
not due to abrupt cell death soon after the thermal insult,
but to later events resulting finally in failure of colony formation in >90% of the cells.
Fig. 11.
Invertase synthesis during recovery at 24°C.
(A) Strains H621 (lhs1
sec18) and (B) H393 (sec18,
indicated WT) were incubated successively for 1 h at
37°C, 20 min at 50°C, and 3 h
at 24°C in YPD medium containing 2% glucose. The cells
were shifted to YPD medium
containing 0.1% glucose (arrow in upper scheme of experimental design) and incubated further at 24°C. In C
and D, H621 and H393 cells,
respectively, were incubated
at 24°C in YPD medium containing 0.1% glucose (arrow in lower scheme). Cell samples were withdrawn for determination of intracellular
(open circles, IN) and externalized (closed circles, EX)
invertase activity. The relative activities are plotted
against incubation time.
[View Larger Version of this Image (15K GIF file)]
Discussion
-
-lactamase, aggregated and denatured by thermal insult to the
cells, failed to be solubilized and reactivated like in wildtype cells.
-
-lactamase,
whereas association with native marker protein molecules
could not be detected. Lhs1p could be immunoprecipitated even with a natural glycoprotein of S. cerevisiae, proCPY, predominantly after thermal insult. Also, pro-CPY
was damaged by the thermal insult to the cells since it became secretion incompetent (Saris, N., and M. Makarow,
to be published). Whether association of the heat-damaged marker proteins with Lhs1p was mediated by other
components is not known. Similar studies demonstrated
earlier that BiP/Kar2p also could be coimmunoprecipitated with heat-denatured Hsp150
-
-lactamase, and to a
lesser extent with the native marker protein, whereas no
association with protein disulfide isomerase could be detected (21). Heat-denatured Hsp150
-
-lactamase thus
occurred in aggregates containing at least Lhs1p and BiP/ Kar2p. The role of BiP/Kar2p in refolding of heat-damaged Hsp150
-
-lactamase could not be studied in vivo
because it is required for translocation. Under conditions
where BiP/Kar2p is nonfunctional, the marker protein is
unable to translocate into the ER, and under normal conditions the marker protein escapes from the ER immediately after translocation (not shown). An ER-located hsp70like stress protein grp170 has been found in association
with BiP and immunoglobulin in CHO cells (5, 23). Sequence comparisons suggest that grp170 could be the
mammalian homologue of yeast Lhs1p, raising the possibility that this novel molecular chaperone is ubiquitous
amongst eukaryotes.
-
-lactamase
was solubilized and part of its enzymatic activity resumed,
it remained in the ER at least for 6 h of recovery at 24°C
(21). We have found that different secretory proteins,
damaged by the thermal insult to the cells while residing in
the ER, resume secretion competence with widely varying
kinetics and efficiency (Saris, N., and M. Makarow, manuscript in preparation). Apparently, the structural features of
Hsp150
-
-lactamase were not compatible with secretion
competence at the time points examined.
-
-lactamase remained stable during the recovery period at 24°C, whereas it
was slowly degraded in vivo in the absence of Lhs1p. Lately,
degradation of mutant pro-CPY was shown to be retrotranslocated from the ER back to the cytosol for degradation
by the proteasome complex (16). Since retrotranslocation
of protein aggregates is unlikely, degradation of the heatdenatured marker protein probably occurred by some other
mechanism, perhaps in the ER lumen or by autophagy (22, 46). Recently, a misfolded soluble protein was shown to be
targeted to the vacuole for degradation (18). It has been
suggested that heat shock proteins dissolve protein aggregates and facilitate protein degradation (13, 17). Newly
synthesized misfolded proteins are degraded in the mitochondrial matrix by the PIM1 protease, if they are maintained in a soluble state by the chaperones mt-Hsp70 and
Mdj1p (49). Lhs1p did not promote degradation of heatdamaged Hsp150
-
-lactamase but suppressed its degradation. Exposed internal domains of pro-insulin were
shown to display signals for degradation in the ER of
mammalian cells. BiP has been suggested to mask these
domains, suppressing the degradation of unfolded proinsulin molecules (39).
-
-lactamase (Holkeri, H., E. Paunola, E. Jämsä, and M. Makarow, manuscript submitted for publication). In the latter studies, the marker proteins were translocated into the ER in reduced form in the presence
of DTT and did not adopt a transport-competent conformation after reestablishment of oxidizing conditions unless
BiP/Kar2p was functional. To compare the functions of
BiP/Kar2p and Lhs1p, we studied the role of Lhs1p in
folding of newly synthesized protein. Lack of Lhs1p did
not inhibit translocation, conformational maturation, or
intracellular transport of Hsp150
-
-lactamase, pro-CPY,
and invertase. Thus, Lhs1p differed from BiP/Kar2p in
that it had no significant role in folding or secretion competence of our newly synthesized marker protein, but instead was required for its conformational repair and stability after heat denaturation. This suggests that the repair
machinery in the ER may have distinct features as compared to the chaperone apparatus involved in folding of
newly synthesized polypeptides. Newly synthesized unfolded polypeptides are fed to the chaperone machinery
during translocation domain by domain, whereas the repair
machinery deals mostly with full-length protein molecules
that have been folded before stress-induced unfolding and
aggregation. Frydman and Hartl (8) found that molecular chaperones interact differently with newly translated polypeptides and their chemically denatured counterparts in
the eukaryotic cytosol. The in vivo kinetics of folding and
conformational repair of Hsp150
-
-lactamase differed
strikingly: de novo folding occurs apparently in seconds,
whereas conformational repair took several hours. Although recovery of yeast cells from thermal insult is efficient, it is remarkably slow. We found bulk protein synthesis to be inhibited for 2-3 h and fully recovered only after 6 h
(21). Inhibition of protein synthesis for hours after severe
heat stress has been noted also in mammalian cells (26).
Evidently, in the absence of protein synthesis, survival requires refolding of heat-denatured proteins that perform
vital functions.
repressor protein and luciferase were dependent on the
chaperones DnaK (Hsp70), DnaJ (Hsp40), and GrpE (9,
40). In vitro, the DnaK/DnaJ/GrpE chaperone system and
the chaperonins GroEL/GroES (Hsp60) are able to reactivate heat-damaged or chemically denatured protein (11,
45, 51). The human cytosolic chaperone Hsp90 was shown
to convert chemically denatured
-galactosidase to a folding-competent state, whereafter the substrate protein was
refolded by Hsp70 and hdj-1. It was suggested that Hsp90
maintains nonnative proteins in a soluble state in heatstressed cells, whereafter Hsp70 and hdj-1 refold them at
normal temperature (7). As BiP/Kar2p and other Hsp70 chaperones require DnaJ/Hsp40 chaperones for activity
(4, 37, 41), Lhs1p may need accessory proteins as well.
mutant cells since they were capable of protein synthesis and secretion for many hours
thereafter, and thus cell death must have occurred because
of later events. In contrast, in the hsp104
mutant protein
synthesis was completely and irreversibly inactivated by
the 50°C treatment. The upstream sequence of LHS1 contains an unfolded protein response element. Indeed, LHS1
was upregulated under conditions where unfolded proteins accumulated in the ER, and moreover, the absence
of Lhs1p caused induction of the unfolded protein response (2, 6). However, LHS1 is not upregulated at 37°C,
whereas HSP104 is strongly induced by elevated temperature (35). A mitochondrial member of the Clp/Hsp100
family of S. cerevisiae, Hsp78, was recently found to be
needed for maintenance of respiratory competence and
resumption of mitochondrial protein synthesis after thermal insult at 50°C; however, it did not affect acquisition of
cellular thermotolerance (38). Chaperones that repair vital
heat-damaged proteins evidently occur in all compartments of the cell. Lhs1p located in the ER may be part of
this fundamental survival system, which is needed for cells
to recover from heat stress causing protein denaturation.
Received for publication 6 November 1996 and in revised form 28 February 1997.
1. Abbreviations used in this paper: CHX, cycloheximide; CPY, carboxypeptidase Y; SC, synthetic complete.We thank Dr. Neil Bulleid (Manchester University, UK) for drawing our attention to YKL 073. Drs. Randy Schekman (University of California, Berkeley, CA), Susan Lindquist (Howard Hughes Medical Institute, Chicago, IL), and Sören W. Rasmussen (Carlsberg Laboratory, Denmark) generously gave us yeast strains and plasmids. Drs. Leevi Kääriäinen and Eija Jämsä from our institute had valuable suggestions on the manuscript, and Ms. Anna Liisa Nyfors provided excellent technical assistance.