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J Biol Chem, Vol. 273, Issue 45, 29727-29737, November 6, 1998
From the The 70-kDa heat shock proteins, hsp70, are highly
conserved among both prokaryotes and eukaryotes, and function as
chaperones in diverse cellular processes. To elucidate the function of
the yeast cytosolic hsp70 Ssa1p in vivo, we characterized a
Saccharomyces cerevisiae ssa1 temperature-sensitive mutant
(ssa1-134). After shifting to the restrictive temperature
(37 °C), ssa1-134 mutant cells showed abnormal
distribution of nuclei and accumulated as large-budded cells with a 2 N DNA content. We observed more prominent mutant phenotypes
using nocodazole-synchronized cells: when cells were incubated at the
restrictive temperature following nocodazole treatment, viability was
rapidly lost and abnormal arrays of bent microtubules were formed.
Chemical cross-linking and immunoprecipitation analyses revealed that
the interaction of mutant Ssa1p with Ydj1p (cytosolic DnaJ
homologue in yeast) was much weaker compared with wild-type Ssa1p.
These results suggest that Ssa1p and Ydj1p chaperone activities play
important roles in the regulation of microtubule formation in M phase.
In support of this idea, a ydj1 null mutant at the
restrictive temperature was found to exhibit more prominent phenotypes
than ssa1-134. Furthermore, both ssa1-134 and
ydj1 null mutant cells exhibited greater sensitivity to
anti-microtubule drugs. Finally, the observation that SSA1
and YDJ1 interact genetically with a The hsp701 family of
molecular chaperones is highly conserved from bacteria to mammals.
Although initially identified as the proteins which are rapidly induced
under stress conditions, they are now known to play essential roles in
diverse cellular functions under normal conditions as well, including:
1) assisting the folding of newly synthesized proteins; 2) facilitating
protein translocation across organelle membranes at the both sides of
membranes; 3) promoting assembly or disassembly of oligomeric proteins;
and 4) facilitating protein degradation of malfolded or denatured proteins. Hsp70 is known to function by transiently binding the hydrophobic peptide of unfolded or newly synthesized proteins and
prevent misfolding by masking their hydrophobic regions. These interactions are assisted by the action of Escherichia coli
DnaJ or its eukaryotic DnaJ homologue hsp40 (1).
In Saccharomyces cerevisiae, 12 homologues of the hsp70
family have been isolated and characterized so far (2-6). They are classified into at least five subfamilies based on their structural and
functional similarities and cellular localization. The SSA subfamily exhibits the highest homology to mammalian hsp70, and is an
essential group of proteins predominantly localized in the cytosol.
This subfamily consists of four functionally redundant members
(SSA1 to SSA4), each of which can substitute for
the others (7). The effect of various combinations of SSA
subfamily null alleles on cell growth have been extensively studied by
Dr. Craig's group (7). Disruption of the SSA1 and
SSA2 genes rendered cells temperature-sensitive for growth
at 35 °C or higher, while the additional disruption of the
SSA4 gene produced cells which were not viable at any
temperature (7). This result shows that expression of SSA3
alone under the control of its own promoter is insufficient to support
viability. Experiments in which Ssap expression could be controlled by
the GAL1 promoter revealed that Ssa proteins are involved in
the translocation of a few proteins into the endoplasmic reticulum and
mitochondria in vivo (8). Further analysis using a
temperature-sensitive ssa1 mutant produced similar results
(9). However, as previously pointed out (10, 11), Ssa1p may be involved
in one or more essential functions in addition to protein translocation
for the following reasons. First, the translocation defect of the
ssa1 mutant has been observed to affect only three out of
nine proteins examined, In this paper, we found that some Ts Yeast Strains, Plasmids, Materials, and Microbial
Techniques--
The S. cerevisiae strains and plasmids used
in these experiments are indicated in Table
I. Strain MO211 used in the synthetic lethality test was obtained as follows. A tub4-1 strain
(Strain ESM208) carrying pRS314(TRP1) was crossed to
HFSA/316P-SSA and sporulated. A tetrad was isolated with the genotype
ssa1::HIS3 ssa2::LEU2 ssa4::LYS2
tub4-1 and carrying the SSA1 gene on the single-copy
plasmid (316P-SSA1(URA3)) (using auxotroph, red color, temperature sensitivity of tub4-1 allele, and 5-FOA
sensitivity as markers). Strains MO203 (tub2-401), MO204
(tub2-402), and MO205 (tub2-403) were crossed to
strain HFSA/316P-SSA and sporulated to obtain strains MO207, MO208, and
MO209, respectively. A polymerase chain reaction (PCR) fragment
carrying the YDJ1 gene was cloned into the
BamHI-XhoI site of pRS316 to create plasmid
316-YDJ1. The BglII fragment of pASZ11 (39) carrying the
ADE2 gene was cloned into the BamHI site of
pRS315 to create plasmid 315-ADE2. A PCR fragment carrying the
TUB4 gene was cloned into the
XhoI-PstI site of 315-ADE2 to generate plasmid
315-ADE2-TUB4. Yeast cell culture and genetic manipulations were
performed essentially as described (40). Benomyl was added to medium
from a 20 mg/ml stock in dimethyl sulfoxide. Thiabendazole was added
from a 40 mg/ml stock in dimethyl sulfoxide. For cell cycle arrest in S phase, 0.1 M hydroxyurea (stock solution, 2 M
in H2O) was added to log-phase cells in liquid medium at
23 °C for 4 h. For disassembly of microtubules and cell cycle
arrest in M phase, nocodazole (Nacalai Tesque, Inc. Kyoto, Japan)
(stock solution, 3.3 mg/ml in dimethyl sulfoxide) was added to a final
concentration 20 µg/ml in liquid medium and cells were incubated at
23 °C for 3 h. Usually, more than 80% of the cells appeared as
large-budded cells, except strain DYJ1 and DM45 (50 and 60%,
respectively).
Isolation of Temperature-sensitive Mutant--
A 2.0-kilobase
BamHI-SphI DNA fragment containing
SSA1 was cloned into pUC 118. Then the SphI site
was converted to a XhoI site by insertion of a
XhoI linker. The resulting BamHI-XhoI
DNA fragment was randomly mutagenized by PCR according to a previously described procedure (41). The PCR reaction consisted of 20 fmol of
template DNA, 1 µM primers (sequences:
5'-CAGGAAACAGCTATGACCATG-3' and 5'- GTTTTCCCAGTCACGACGTTG-3'), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 0.01%
gelatin, 7.0 mM MgCl2, 0.5 mM
MnCl2, 0.2 mM dATP, 0.2 mM dGTP,
1.0 mM dCTP, 1.0 mM dTTP, and 5 units of
Taq polymerase and was performed with an automatic Thermal
Cycler for 30 cycles (94 °C for 1 min, 45 °C for 1 min, 72 °C
for 2 min). The PCR product fragments were cut at BamHI and
XhoI restriction sites and inserted into the
BamHI-SalI site of the pGAP-SSA1 plasmid (42) to
generate a plasmid library containing pGAP-SSA1M(TRP1),
which carries a temperature-sensitive allele of the ssa1
gene. This construct placed the PCR-mutagenized SSA1 genes
under the control of the glyceraldehyde-3-phosphate dehydrogenase
promoter. This library was introduced into the yeast strain HFSA-11
which carries disruptions of the chromosomal SSA1, SSA2, and
SSA4 genes and is complemented by a single copy plasmid,
pGAL-SSA1(URA3) expressing wild-type SSA1 from
the GAL1 promoter. Trp+ transformants were
obtained on medium. Trp+ Ura+ transformants
were selected on media containing galactose and transferred to media
containing glucose, which represses expression of
pGAL-SSA1(URA3) and rendered cells dependent on mutagenized pGAP-SSA1M(TRP1) for growth. Transformants which grew on
glucose at the permissive temperature (23 °C), but not at the
nonpermissive temperature (37 °C) were chosen for further analysis.
Plasmid DNA was recovered and reintroduced into strain HFSA-11.
Transformants were subsequently replica plated onto 5-fluoroorotic acid
(5-FOA) plates to obtain Trp+ Ura Protein Labeling, Chemical Cross-linking and
Immunoprecipitation--
Radiolabeling of yeast cells and
immunoprecipitaion of radiolabeled cell extracts were carried out
essentially as described (43). Yeast cells were incubated for 3 h
at 23 or 37 °C and then radiolabeled with EXPRESS
[35S]Protein labeling mixture (DuPont) (400 µCi/OD600 cells) for 20 min. Extracts were prepared by
resuspending cells in 200 µl of lysis buffer (100 mM
potassium phosphate, pH 7.2, 1 mM EDTA, 1 mM
EGTA, 1 mM phenylmethylsulfonyl fluoride) followed by
addition of glass beads, and vortexing four times, 30 s each.
After addition of DSP, a cross-linking agent, lysates were incubated
for 30 min in the presence or absence of 10 mM ATP, and
diluted with 1 volume of bovine serum albumin (10 mg/ml) and 4 volume
of immunoprecipitation dilution buffer (60 mM Tris-Cl, pH
7.5, 1.25% Triton X-100, 190 mM NaCl, 6 mM
EDTA). Background binding was reduced by preincubation with preimmune
rabbit antisera and Protein A-Sepharose for 1 h at 4 °C
followed by centrifugation in a microcentrifuge for 5 min. The
supernatant fraction was transferred to fresh siliconized tubes and
incubated with 2 µl of rabbit anti-Ssa1p polyclonal antibodies
overnight at 4 °C. After incubating the sample with Protein
A-Sepharose beads at 4 °C for 1 h, beads were collected by
brief centrifugation and washed three times with IP buffer (50 mM Tris-Cl, pH 7.5, 1% Triton X-100, 0.2% SDS, 150 mM NaCl, 5 mM EDTA). Finally, the
immunoprecipitates were dissolved in SDS-PAGE sample buffer at 95 °C
for 5 min and analyzed by 10% SDS-PAGE.
Purification of His-tagged Ssa1p Complex and
Immunoblotting--
Yeast cells were cultured to log-phase in YPD
medium. Yeast cells were washed once with sterile H2O and
resuspended in lysis buffer (100 mM potassium phosphate, pH
7.2, 1 mM EDTA, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride), followed by glass beads lysis (44).
Cross-linking was performed with 0.5 mM DSP for 30 min at
30 °C. Yeast lysates were diluted with equal volume of binding buffer (20 mM Tris-Cl, pH 7.9, 250 mM NaCl, 60 mM imidazole) and incubated with pre-equilibrated
nickel-charged resin for 1 h at 4 °C followed by centrifugation
in a microcentrifuge for 10 s. The supernatant fractions were used
as the "unbound fraction." After washing nickel-charged resin three
times with binding buffer, resin-bound protein complexes were eluted
with elution buffer (20 mM Tris-Cl, pH 7.9, 250 mM NaCl, 300 mM imidazole), concentrated with
Centriprep-10 (Amicon, Inc), and used as the "bound fraction." Both
fractions were dissolved in SDS-PAGE sample buffer at 95 °C for 5 min, analyzed by 10% SDS-PAGE, and subjected to immunblotting analysis
(44). Rabbit anti-Ssa1p polyclonal antibodies (1:1000 dilution) and
rabbit anti-Ydj1p polyclonal antibodies (1:1000 dilution) were used as
primary antibodies in the respective experiments.
Immunofluorescence Microscopy--
For microtubule staining,
cells were fixed by addition of formaldehyde directly to the culture to
a final concentration of 3.7% and incubation at room temperature for
2 h. Fixed cells were washed with potassium phosphate buffer (0.1 M potassium phosphate, pH 6.5) and converted into
spheroplasts by incubation with 50 µg/ml zymolyase-100T (Seikagaku
Corp. Tokyo, Japan) in 1.2 M sorbitol, 0.1 M
potassium phosphate, pH 6.5, and 0.2% DNA Sequence Analysis of the ssa1 Alleles--
DNA sequences of
the ssa1 alleles cloned into pGAP-SSA1M were determined by
the dideoxy nucleotide triphosphate termination method using an
automated procedure involving differential fluorescent labeling. All
sequencing reactions and electrophoresis of the sequencing gels were
performed according to the ABI manual for automated sequencing on a
373A DNA sequencer (Perkin-Elmer Japan Co., Ltd., Osaka, Japan).
Isolation and Characterization of Temperature-sensitive ssa1 Mutant
Cells--
To study the role of S. cerevisiae Ssa1p, we
generated a temperature-sensitive allele of ssa1. We used a
strain HFSA-11 as a parental strain, which is disrupted for the
chromosomal SSA1, SSA2, and SSA4 genes and is
rescued by the expression of pGAL-SSA1, a single copy vector which
utilizes the galactose-inducible and glucose-repressible
GAL1 promoter for conditional expression of wild-type
SSA1 gene. To isolate temperature-sensitive ssa1
alleles, this strain was transformed with another single copy plasmid
carrying a PCR-mutagenized SSA1 gene under the
transcriptional control of the constitutive glyceraldehyde-3-phosphate
dehydrogenase promoter. Transformants were screened for growth on
glucose-based media at 23 and 37 °C, conditions under which the
wild-type SSA1 gene was not expressed. After counter
selecting for pGAL-SSA1(URA) on 5-FOA plates and several precise
experiments as described under "Materials and Methods," we obtained
some temperature-sensitive ssa1 strains designated as
HFSA/AM. Each strain of HFSA/AM carries a single copy plasmid,
pGAP-SSA1M, which expresses a temperature-sensitive ssa1
gene, whereas the control strain designated as HFSA/AW carries pGAP-SSA1 which expresses an unmutagenized SSA1 gene.
Microscopic and flow cytometric analyses showed that some
ssa1 mutants accumulate as large-budded cells containing 2 N DNA content at the nonpermissive temperature of 37 °C
(data not shown). We chose ssa1-134 cells (strain HFSA/A34)
for further analysis because it exhibited the most prominent phenotype
compared with the wild-type SSA1 control cells (strain
HFSA/AW), which did not show any significant changes in bud
morphologies following the temperature shift (Fig.
1, A and B). To
visualize nuclei we stained cells with DAPI (Fig. 1C). In
the large-budded wild-type SSA1 cells, nuclei were located
close to the bud neck and relocated accordingly, one each to the mother
and daughter cells, soon after nuclear division. However,
ssa1-134 mutant cells exhibited a different cell morphology
after shift to 37 °C: both nuclei migrated to the mother cell, but
none to the daughter cell (Fig. 1C, lane e). This led us to
conclude that ssa1-134 cells may be defective in nuclear
migration. Furthermore, in a large portion of ssa1-134 cells
a single nucleus was observed to be localized randomly within the
mother cell (Fig. 1C, lane d). In these cells, the nuclei did not migrate to the bud-neck. Fewer SSA1 cells were
observed with this morphology than ssa1-134 cells. The
percentage of cells with both nuclei correctly distributed and a single
nucleus properly located at the bud-neck was dramatically lower in
ssa1-134 cells compared with SSA1 cells (Fig.
1C, lanes b and c). These results imply that
Ssa1p plays an important role in the orientation and migration of
nuclei during mitosis.
Aberrant Microtubular Structure Observed in ssa1 Mutant Cells after
Nocodazole Treatment--
Abnormal nuclear migration is a phenotype
frequently observed in mutants of tubulin or its functionally related
proteins, such as motor proteins or chaperonins (20, 46-51). We
speculate that the chaperone activity of Ssa1p affects tubulin
molecules such that it results in the abnormal assembly of
microtubules. Therefore, we examined the in vivo microtubule
re-assembling ability of ssa1 cells following disassembly of
microtubules by nocodazole. Nocodazole treatment is known to cause
disassembly of microtubules resulting in the accumulation of
large-budded cells with single nucleus and duplicated spindle pole
bodies (SPB) (Fig. 2, A and B) (52). Cells were treated for 3 h with nocodazole at
23 °C, after which nocodazole was completely removed and cells were
rapidly transferred to YPD medium prewarmed to 23 or 37 °C.
Microtubules started to reassemble as soon as 5 min after the removal
of nocodazole (data not shown). 2 h later, both normally dividing
cells as well as large-budded cells were observed. We focused our
analysis on large-budded cells, comparing the morphology of the mitotic
spindles. In SSA1 cells we observed the reassembly of
straight mitotic spindles elongated along the axis between two divided
nuclei and normal distribution of nuclei between the mother and
daughter cells after 2 h of incubation at either 23 or 37 °C
(Fig. 2, C and D). In contrast, in the
large-budded cells of ssa1-134 cells, abnormal arrays of
spindle microtubules were observed to have re-assembled after 2 h
of incubation at 37 °C (Fig. 2, G, H, I, and
J). This abnormal phenotype could not be observed at any
stage of the normal cell cycle (45). Concomitant with the appearance of
these abnormal arrays of spindle microtubules, we observed that
microtubules in ssa1-134 cells deformed into bent
microtubules located at the periphery of cells (Fig. 2, I
and J). These bent spindle microtubules elongated only
within the mother cells of ssa1-134 cells and did not
elongate through the bud-neck toward the daughter cell as in the
SSA1 cells. As a result, their nuclei did not correctly segregate into the mother and daughter cells. At the permissive temperature (23 °C), such abnormal assemblies of microtubules were
not observed (Fig. 2, E and F).
To examine whether the aberrant microtubule structures observed in
ssa1-134 cells after nocodazole treatment are a phenotype common to genetic defects in chaperone activity, we examined in vivo microtubule reassembly activity in Cct chaperonin mutant (tcp1- Mutant Ssa1p Showed Reduced Association with 45-kDa
Protein--
We examined molecules which associate with Ssa1p by
cross-linking with the chemical cross-linker DSP followed by
immunoprecipitation. We identified two molecules (85 and 45 kDa) which
associated with Ssa1p in the presence of 0.5 or 2.5 mM DSP
(Fig. 4). In the absence of DSP, these
molecules still associated very weakly with Ssa1p. The 85-kDa molecules
dissociated from Ssa1p in the presence of 10 mM ATP. In
contrast, association of the 45-kDa molecule with Ssa1p was as good if
not better in the presence of 10 mM ATP. In
ssa1-134 cell extracts we observed an
ATP-dependent association between Ssa1p and the 85-kDa
molecule similar to that seen in wild-type SSA1 cells,
whereas we observed a reduced association between Ssa1p and the 45-kDa
molecule at either both 23 and 37 °C (Fig.
5).
45-kDa Molecules Identified as Ydj1p--
The hsp70 chaperone
family is highly conserved and its binding/release cycle is modulated
by several associated proteins. DnaJ/hsp40 interacts with hsp70 through
a highly conserved J-domain and stimulates ATP hydrolysis. Based on its
similar molecular mass, we considered the possibility that the
Ssa1p-associated 45-kDa protein we detected may be the yeast homologue
of DnaJ/hsp40, Ydj1p. To examine this possibility we used His-tagged
Ssa1p molecules, which contain six histidine residues at their carboxyl
terminus (53). Cell lysates prepared from strain HFSA/AWH (expressing His-tagged wild-type Ssa1p) and HFSA/A34H (expressing His-tagged mutant
Ssa1p) were divided into bound and unbound fractions, depending on
their binding to nickel beads, and immunoblotted with
anti-Ydj1p and anti-Ssa1p antibodies. These results showed that
the molecule associated with wild-type Ssa1p is
indeed Ydj1p and the interaction is further enhanced in a
DSP-dependent manner (Fig.
6A). The interaction between
wild-type Ssa1p and Ydj1p was observed in the absence of DSP and could
be further enhanced by the addition of DSP. In contrast, no interaction
between mutant Ssa1p and Ydj1p was observed in the absence of DSP, and
only a small amount could be observed in the presence of DSP. This
lower association was not due to decreased expression of Ydj1p, since
expression levels in the unbound fraction (Fig. 6B) and
whole extract (data not shown) were similar to those seen with the
wild-type extract. These results show that mutant Ssa1p is somehow
defective in its interaction with Ydj1p. We hypothesize that the
failure of Ssa1p to interact with Ydj1p is related to the abnormal
microtubular structure and function observed in ssa1-134
mutant cells (Fig. 2, F and H).
Aberrant Microtubular Structure Observed in ydj1 Mutant Cells after
Nocodazole Treatment--
YDJ1 is known to genetically interact with
SSA1 in vivo; and a deletion mutant allele of ydj1 shows
synthetic lethality in combination with a ssa1 mutant allele
(9). Examination of in vivo microtubule re-assembly in
ydj1 null cells following release from nocodazole treatment
revealed a more prominent mutant phenotype than observed in
ssa1 mutants. At 37 °C ydj1 cells had
elongated mitotic spindles and spindle arrays oriented in an abnormal
direction (Fig. 7, E-H): as
much as 60% of the large-budded cells had double nuclei located in the
mother cell (Table II). These
observations lead us to hypothesize that the Ssa1p·Ydj1p complex
might be involved in the regulation of microtubular function. In
addition, a significant fraction of ydj1 null mutant cells
after synchronization in S phase by hydroxyurea showed phenotypes
similar to that seen in cells synchronized in M phase by nocodazole,
i.e. aberrant microtubule formation and accumulation of
large budded cells containing double-nuclei in the mother cell,
although this fraction was smaller than that seen in
nocodazole-synchronized cells (Table II). These observations suggest
that one of the important roles of Ydj1p is the regulation of spindle
microtubule assembly, although Ydj1p has other pleiotropic effects on
cellular functions.
Hypersensitivity of ydj1 Disrupted Cells to Microtubule
Depolymerizing Drug--
Mutations that cause defects in the function
of microtubules are often found to cause increased or decreased
sensitivity to microtubule-depolymerizing drugs. Thus we studied the
sensitivity of the HFSA/AW (wild-type SSA1 control),
HFSA/A34 (mutant ssa1-134), YPH499 (Wild-type
YDJ1 control), and DYJ1 (ydj1 null mutant)
strains to the microtubule-depolymerizing drugs thiabendazole and
benomyl. Equal numbers of each strain were spotted on YPD plates
containing different concentrations of drugs (Fig.
8). ssa1-134 cells were found
to be slightly sensitive to 120 µg/ml thiabendazole and were more
sensitive to 30 µg/ml benomyl compared with wild-type SSA1
cells. ydj1 null mutant cells showed a marked
hypersensitivity to both thiabendazole and benomyl. Whereas wild-type
cells (strain YPH499) were able to grow on plates containing 80 µg/ml
thiabendazole or 20 µg/ml benomyl, ydj1 null mutants
failed to grow. The hypersensitivity of ssa1
versus ydj1 mutant cells to microtubule
depolymerizing drugs correlated with their in vivo
phenotypes, as their role in vivo, because ydj1
disrupted cells exhibited a more pronounced aberrant microtubular
structure phenotype compared with ssa1 mutant cells.
Synthetic Lethality of ssa1 Mutant and ydj1 Null Mutant Cells with
tub4-1--
To understand how Ssa1p is involved in the functional
regulation of microtubules, we examined genetic interactions between ssa1 mutant alleles and some conditional lethal tubulin
mutant alleles ( In this study, we observed that a temperature-sensitive mutant of
ssa1-134, a yeast cytosolic homologue of hsp70, underwent cell cycle-specific arrest leading to the accumulation of large-budded cells following incubation at the restrictive temperature of 37 °C
(Fig. 1B). Hsp70 has been shown to be an important factor in multiple cellular processes including protein translocation across the
membrane of endoplasmic reticulum and mitochondria, protein folding,
and regulation of heat shock responses (54). However, in our
preliminary experiment in which we obtained and tested several
temperature-sensitive mutants of ssa1, we did not find a
close relationship between temperature-sensitive cell growth and
inhibitory effects on protein translocation. For instance, F1 The use of nocodazole has enabled us to investigate the function of
Ssa1p in more detail. Nocodazole treatment completely disassembles
microtubules and causes cell-cycle arrest at the G2/M
phase, and cells are forced to reassemble mitotic spindles and
cytoplasmic microtubules once nocodazole is removed. Thus, use of
nocodazole enabled us to observe more pronounced phenotypes in mutant
cells which could not be clearly detected in asynchronous yeast cells
due to the heterogeneity of their microtubule staining patterns. The
appearance of aberrant microtubules in ssa1-134 mutant cells
following nocodazole treatment suggests that Ssa1p might affect
assembly/disassembly of microtubules (Fig. 2).
We found that wild-type Ssa1p was associated with Ydj1p in
vivo in chemical cross-linker experiments (Figs. 4 and 6). This interaction was very weak in the case of mutant Ssa1p (Figs. 5 and 6).
As Ydj1p is known to play an important role in the regulation of Ssa1p
chaperone activities by stimulating the ATPase activity or by
modulating the affinity for ATP (55-57), we speculate that its
interaction with Ssa1p contributes to the ability of Ssa1p to regulate
microtubular function. This idea was supported by the following
evidence in which ydj1 disrupted cells exhibited a more
pronounced aberrant microtubule phenotype, i.e. aberrant microtubule formation after nocodazole treatment, hypersensitivity to
benomyl and thiabendazole (Figs. 7 and 8). In ssa1-134
cells, a very weak interaction between Ydj1p and Ssa1p was still
observed in the presence of chemical cross-linker (Fig. 6), but in the case of ydj1 null mutant cells there was no interaction.
Thus, there is a correlation between normal microtubular function and the association between Ssa1p and Ydj1p. These results suggest that the
chaperone activities of the Ssa1p·Ydj1p complex may be necessary for
the proper function of microtubules. Another possible role of Ydj1p is
to target Ssa1p to a specific site(s) within the cell. Ydj1p is known
to be farnesylated and targeted to the membrane (58), and this membrane
localization is required for its function at elevated temperature
(37 °C).
Although numerous observations suggest an interaction between hsp70 and
microtubules (27-35), the physiological role of this interaction has
not been elucidated. Our results suggest that Ssa1p and Ydj1p function
is important for the proper assembly of microtubules. Gupta et
al. (59) found proteins which are specifically altered in mutant
mammalian cells resistant to microtubule inhibitors, and identified one
of these as a homologue of hsp70 (60). Similarly, we found that some
mutant ssa1 cells show resistance to microtubules
depolymerizing drugs.3 These
observations suggest that there is an intimate functional relationship
between hsp70 and tubulin molecules.
How might the ssa1 mutant affect the functions of
microtubules? The first possibility is that a mutation in
ssa1 affects the nucleation of microtubules at the
microtubules organizing center (MTOC), a body which provides a
microtubule nucleation site initiating self-assembly of tubulin (61).
One of the components of the MTOC is A second possibility is that a mutation in hsp70 affects the
organization of the MTOC. Hsp70 has been shown to localize to the
centrosome (36, 37, 38) or associate with the centrosomal compartment
(68). Brown et al. (38) reported that hsp70 has an essential
role in the repair of centrosomes following heat shock treatment, which
leads to the fragmentation of centrosomes (69, 70). In our experiments,
cells were heat shocked at the restrictive temperature following
nocodazole treatment, thus the possible effect of heat-shock on the
MTOC could not be eliminated. However, we note that SPB is embedded in
the nuclear envelope and may be more structurally rigid than the
centrosome. There is also a possibility that hsp70 participates in the
duplication of SPB, similar to hsp90 (71). Indeed, we observed abnormal monopolar spindle formation in some ssa1 mutant cells (data
not shown), which suggest that SPB duplication did not occur
properly. However, we emphasize that the data presented here indicates
a role of hsp70 in the assembly and/or nucleation of microtubules after
arrest of the cell cycle by nocodazole, a point at which the SPB has
already duplicated (52).
A third possible role for Ssa1p is as a molecular chaperone in the
microtubule assembly/disassembly process. Microtubules exist in a
dynamic state in which their deployment is dramatically affected by
changes in polymerization and depolymerization rate (72, 73). We
speculate that mutations in Ssa1p/Ydj1p may affect the kinetics of
microtubule polymerization/depolymerization. For example, association
between Ssa1p-Ydj1p and tubulin might prevent the rapid polymerization
of tubulin, such that loss of Ssa1p-Ydj1p function results in increased
and rapid polymerization of tubulin leading to the formation of
abnormally extended microtubules. This possibility is supported by the
observation that hsp70 binds to the carboxyl terminus of tubulin (34),
which is also the site where microtubule-associated proteins bind. Thus
Ssa1/hsp70 may antagonize microtubule-associated proteins function by
competitive binding (12). We could not exclude the possibility that
Ssa1p-Ydj1p (hsp70-hsp40) acts indirectly by affecting the folding or
stability of some factors which interact with microtubules.
Finally, the above possibilities suggest that molecular chaperones may
contribute to the proper assembly of macromolecular complexes, such as
SPB or microtubules. It is of great interest to understand how
macromolecular complexed assemble into precise structural arrangements.
It is tempting to speculate that molecular chaperones participate in
the proper assembly of such complexes.
We are grateful to Drs. A. Horwich (Yale
University), D. Botstein (Stanford University), E. Shiebel (Max-Planck
Institut für Biochemie), and Y. Kimura (Tokyo Metropolitan
Institute of Medical Science) for the gifts of the yeast strains. We
also thank I. Yahara (Tokyo Metropolitan Institute of Medical Science)
and E. Shiebel for helpful discussions; R. Ando for technical
assistance; and M. Lamphier for manuscript preparation.
*
This work was supported by a Grants-in-Aid from the Ministry
of Education, Science, Sports, and Culture of Japan.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
Research fellow of Japan Science and Technology Corporation.
**
Present address: Institute for Protein Research, Osaka University,
3-2 Yamada-oka, Suita, Osaka 565, Japan.
§§
To whom correspondence should be addressed: Research and
Education Center for Genetic Information, Nara Institute of Science and
Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.:
81-743-72-5640; Fax: 81-743-72-5649; E-mail:
kkouno{at}bs.aist-nara.ac.jp.
The abbreviations used are:
Cct, chaperonin
containing t complex; 5-FOA, 5-fluoroorotic acid; DAPI, 4,6-diamino-2-phenylindole; DSP, dithiobis(succinimidylpropionate); PCR, polymerase chain reaction; MTOC, microtubules organizing center; SPB, spindle pole body.
2
M. Nakai and T. Endo, unpublished data.
3
M. Oka and K. Kohno, unpublished results.
Loss of Hsp70-Hsp40 Chaperone Activity Causes Abnormal
Nuclear Distribution and Aberrant Microtubule Formation in M-phase
of Saccharomyces cerevisiae*
§¶,
**,,
,§, and§§§
Research and Education Center for Genetic
Information, Nara Institute of Science and Technology, 8916-5 Takayama,
Ikoma, Nara 630-0101, Japan, the Faculty of Science, Nagoya
University, Furo-cho, Chikusa-ku, Nagoya, Aichi 464-8602, Japan, and
§ CREST, Japan Science and Technology
Corporation, Tokyo, Japan
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-tubulin,
TUB4, supports the idea that they play a role in the
regulation of microtubule formation.
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
-factor, proteinase A, and mitochondrial 
-subunit of F1-ATPase (F1), and this
translocation defect was partial (9). Second, an extragenic suppressor
of the temperature-sensitive ssa1 ssa2 strain,
exa2-1, suppressed temperature sensitivity but did not
suppress the defect in protein translocation (10). Therefore, the
essential role of Ssa proteins (Ssap) may involve more than its
translocation function. Recently there is a growing body of evidence
suggesting an intimate relationship between molecular chaperones and
the cytoskeleton (12). Chaperonin containing t complex (Cct), which is
a cytosolic mammalian homologue to the hsp60 chaperonin (13), acts as a
chaperone for a wide range of cytoskeletal components including -,
-, -tubulin, and actin (14-18). Furthermore, a portion of Cct
has been shown to localize to the centrosome of HeLa cells and is
essential for the growth of microtubules after nocodazole treatment
(19). The yeast S. cerevisiae CCT genes consist of
eight independent genes (CCT1-8), one of each of which is
contained in the heteroligomeric yeast Cct complex (20-25). Mutant
cct yeasts are abnormal in their nuclear distribution and
have aberrant microtubule structures (20, 23, 26). CCT has
also been shown to genetically interact with -, -tubulin
(TUB1, 2) and actin (ACT1) (26). On the other
hand, the roles of hsp70s in the function of cytoskeletal components have not been elucidated despite numerous reports indicating their association with microtubules (27-35). In addition, hsp70s have been
shown to be localized to the centrosome in various species, but their
function remains obscure (36-38).
ssa1
mutant cells are defective in microtubular function and form aberrant
microtubular structures at the restrictive temperature. Furthermore,
mutant Ssa1p was shown to associate poorly with co-chaperone Ydj1p. We discuss a possible role of this putative Ssa1p·Ydj1p complex in the
assembly/disassembly of microtubules.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
strains,
and the temperature-sensitive phenotype conferred by the plasmid was
re-tested and confirmed. Plasmid DNA from one transformant, designated
HFSA/A34, was recovered and the sequence of the ssa1 allele,
ssa1-134, was determined. The following amino acid
substitutions were found: Q254P, R339G, A633V, stop codon 643K + PIGAAIDNNENVF. A wild-type control strain, designated strain HFSA/AW,
was derived by transforming the starting strain, HFSA-11 with the
non-mutagenized pGAP-SSA1 plasmid.
-mercaptoethanol at 30 °C
for between 15 and 40 min. Immunofluorescence was performed essentially
as described (45). The rat monoclonal anti-yeast -tubulin antibody,
YOL1/34 (Accurate Chemical and Scientific Corporation, NY), was used as
the primary antibody and rhodamine-conjugated goat anti-rat IgG
antiserum (Cappel Research Products, ICN, Inc., Tokyo, Japan) was used
as the secondary antibody. DNA was stained with 1 µg/ml DAPI for 3 min at room temperature before mounting. Preparations were viewed in a
Zeiss Axiophoto fluorescence microscope.
RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (17K):
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Fig. 1.
ssa1 mutant cells show abnormal nuclear
migration and accumulate as large-budded cells at nonpermissive
temperature. Growth curve, cell morphology, and nuclear
distribution in ssa1-134 mutant cells (strain HFSA/A34) and
wild-type SSA1 control cells (strain HFSA/AW) grown in
liquid medium. Cells were grown in YPD at 23 °C to log-phase and
shifted to 37 °C at time 0. At the indicated times, aliquots of
cells were harvested, fixed, and observed by microscope. Cells were
also stained with DAPI to visualize nuclei. A, cell growth
of the wild-type control cells (open circles) and
ssa1-134 mutant cells (closed circles).
OD600 was measured at the indicated times. B,
cell morphology as determined by bud size and C, nuclear
distribution in large-budded cells. More than 200 cells were counted
for each culture. Data is presented as percentage of total cell
number.
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Fig. 2.
Aberrant microtubular structure observed in
ssa1-134 mutant cells after nocodazole treatment.
SSA1 and ssa1-134 cells were grown at 23 °C in
YPD to log-phase and then nocodazole was added to the culture (20 µg/ml). After 3 h, cells were washed with sterile water and
transferred to fresh medium preincubated at 23 or 37 °C. Just prior
to (A and B) or 2 h after removal of
nocodazole (C-J) cells were harvested, fixed, and stained
with anti-tubulin antibodies to show microtubule formation (B, D,
F, H, J), and DAPI to show DNA (A, C, E, G, and I).
A and B, SSA1 cells, at time 0;
C and D, SSA1 cells, 37 °C, 2 h; E and F, ssa1-134 cells, 23 °C,
2 h; G-J, ssa1-134 cells, 37 °C, 2 h.
) cells. The Cct chaperonin complex acts as a
chaperone facilitating tubulin molecule folding in vitro and
the assembly of microtubules in vivo. At the restrictive
temperature tcp1- mutant cells were arrested during the
late G2 to M phase of the cell cycle and exhibited abnormal
tubulin staining patterns, suggesting that mitotic spindle formation
and function are impaired in this mutant strain (23). When
tcp1- mutant cells were released from nocodazole
treatment and incubated at the restrictive temperature (37 °C), only
very short or virtually no visible spindles nucleating from the SPB
could be observed in 80% of the cells (Fig.
3, C and D). In
contrast, at the permissive temperature (23 °C) only 35% of cells
exhibited this phenotype (Fig. 3, A and B). These results suggest that Cct has an essential role in nucleation and/or assembly of microtubules in yeast, but that its role is distinct from
that of Ssa1p.
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Fig. 3.
tcp1- mutant cells are defective in
microtubule reassembly following nocodazole treatment.
tcp1- mutant cells were grown at 23 °C in YPD to
log-phase and then nocodazole was added to the culture (20 µg/ml).
After 3 h, cells were washed with sterile water and transferred to
fresh medium preincubated at 23 °C (A and B)
or 37 °C (C and D). 2 h after the removal
of nocodazole cells were harvested, fixed, and stained with
anti-tubulin antibodies to show microtubule formation (B and
D) and DAPI to show DNA (A and
C).
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Fig. 4.
Association of wild-type Ssa1p with 45- and
85-kDa molecules revealed by chemical cross-linking.
Immunoprecipitation of cross-linked complexes was performed using
anti-Ssa1p antibodies. SSA1 cells were radiolabeled with
[35S] at 30 °C for 20 min. Cell extracts were prepared
by disrupting cells with glass beads. Cross-linking was performed using
0.5 mM DSP (lanes 2 and 5), 2.5 mM DSP (lanes 3 and 6), or without
DSP (lanes 1 and 4), in the absence (lanes
1-3) or presence of 10 mM ATP (lanes 4-6)
for 30 min at 30 °C and immunoprecipitated with anti-Ssa1p
antibodies. Immunoprecipitates were resolved by electrophoresis on 10%
SDS-PAGE.
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Fig. 5.
Mutant Ssa1p showed reduced association with
the 45-kDa molecule. Immunoprecipitation of cross-linked complex
was performed using ssa1-134 mutant and wild-type
SSA1 control cells. Cells were radiolabeled with
[35S] at 23 °C for 20 min. Cell extracts from
SSA1 cells (lanes 1-4) and ssa1-134
(lanes 5-8) were cross-linked with 0.5 mM DSP
in the absence (lanes 1, 2, 5, and 6) or presence
of 10 mM ATP (lanes 3, 4, 7, and 8)
for 30 min at 23 °C (lanes 1, 3, 5, and 7) or
at 37 °C (lanes 2, 4, 6, and 8). Cross-linked
complexes were immunoprecipitated with anti-Ssa1p antibodies, dissolved
in electrophoresis sampling buffer, and resolved by electrophoresis on
10% SDS-PAGE.
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Fig. 6.
Identification of the 45-kDa molecule as
Ydj1p. Strain HFSA/AWH and HFSA/A34H cells were grown to
log-phase, and cell extracts were prepared by glass beads disruption.
Cross-linking was performed as in Fig. 4 and histidine-tagged Ssa1p was
purified with nickel-charged resin. Resin bound (A) and
unbound (B) fractions were resolved by electrophoresis on
10% SDS-PAGE and immunoblotted with anti-Ssa1p antibodies and
anti-Ydj1p antibodies.
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Fig. 7.
Aberrant microtubular structure observed in
ydj1 null mutant cells after nocodazole treatment.
YDJ1 (YPH499 strain) and ydj1 (DYJ1 strain) cells
were grown in YPD at 23 °C to log-phase. Nocodazole was added to the
culture (20 µg/ml) and after 3 h cells were washed with sterile
water and transferred to fresh medium preincubated at 23 or 37 °C.
2 h after the removal of nocodazole, cells were harvested, fixed,
and stained with anti-tubulin antibodies to reveal microtubule
formation (B, D, F, and H) and DAPI to show DNA
(A, C, E, and G). A and B,
wild-type cells, 37 °C, 2 h; C and D,
ydj1 null mutant cells, 23 °C, 2 h; E-H,
ydj1 null mutant cells, 37 °C, 2 h.
Schematic orientation of microtubules and nuclei after nocodazole
treatment
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[in a new window]
Fig. 8.
Hypersensitivity of ssa1-134 and
ydj1 null mutant cells to thiabendazole and benomyl. A
5-fold serial dilution (starting from 5000 cells/spot) of HFSA/AW
(wild-type SSA1 control), HFSA/A34 (ssa1-134),
YPH499 (wild-type YDJ1 control), and DYJ1 (ydj1)
cells were spotted on YPD plates (plate A) or YPD containing
either thiabendazole (80 µg/ml, plate C; 120 µg/ml,
plate D) or benomyl (20 µg/ml, plate E; 30 µg/ml, plate F) and incubated at 30 °C for 3 days. For
thermosensitivity tests, equal numbers of cells were spotted on YPD
plates and incubated at 37 °C for 2 days (plate B).
-tubulin (tub4) and -tubulin
(tub2))(see "Yeast Strains" under "Materials and
Methods"). To minimize the effects of variable genetic background, we
made isogenic variants of the strain MO211, which contains three
deleted SSA genes (ssa1, ssa2, and
ssa4) and a tub4-1 mutation, and expresses the
wild-type SSA1 gene from the ADH1; alcohol
dehydrogenase 1 promoter on a single copy plasmid (316P-SSA1(URA3)) to rescue lethality. Isogenic
ssa1 variants were generated by cotransformation with six
combinations of plasmids: i.e. wild-type SSA1
(pGAP-SSA1(TRP1)) and wild-type TUB4
(315-ADE2-TUB4), wild-type SSA1 (pGAP-SSA1(TRP1))
and the empty vector pRS315-ADE2, mutant ssa1-134
(pGAP-ssa1-134(TRP1)) and wild-type TUB4
(315-ADE2-TUB4), mutant ssa1-134
(pGAP-ssa1-134(TRP1)) and the empty vector pRS315-ADE2, the
empty vector pRS314(TRP1), and wild-type TUB4
(315-ADE2-TUB4), and the empty vector pRS314(TRP1) and the
empty vector pRS315-ADE2. All 6 strains were subsequently tested for
growth on plates with or without 5-FOA. 5-FOA is toxic to cells
expressing the URA3 gene product and thus selects for the
loss of URA3-based SSA1 plasmid
(316P-SSA1(URA3)). All strains grew and formed colonies when
incubated on SD medium without 5-FOA (Fig.
9A, left). As expected, cells carrying wild-type SSA1 on the TRP1 vector
(pGAP-SSA1(TRP1)) could grow in the presence of 5-FOA,
regardless of whether it was TUB4 or tub4-1 (Fig.
9A, right, second lane). However, the Ura
strain carrying ssa1-134 could grow only in the presence of
wild-type TUB4 but not with the mutant tub4-1
allele (Fig. 9A, right, third lane). This
indicates that ssa1-134 is synthetically lethal in combination with tub4-1 within the range of temperatures
from 16 to 30 °C (Fig. 9A). This genetic interaction is
allele-specific, as synthetic lethality was not observed with some
other Ts ssa1 alleles (data not shown). In
contrast, we found no synthetic lethality between ssa1 and
any of the -tubulin mutant alleles tested (tub2-401, 40, 403) (see "Materials and Methods"), although their temperature
sensitivity was slightly enhanced (data not shown). To examine
synthetic lethality between a ydj1 null allele and
tub4-1, the tub4-1 strain ESM208 was transformed
with the plasmid 316-YDJ1(URA3), crossed to the
ydj1 strain DYJ1, and sporulated. The ydj1 tub4-1
double mutants, which carried the wild-type YDJ1 gene on the
plasmid, were tested and found to be unable to grow on 5-FOA plates at
either 16 and 30 °C, indicating that ydj1 is synthetically lethal with tub4-1 (Fig. 9B). This
genetic interaction was further confirmed by the observation that the
viability of these cells depended on the presence of the plasmid-borne
wild-type TUB4 gene.
View larger version (60K):
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Fig. 9.
Both ssa1-134 and ydj1
null mutant showed synthetic lethality with yeast -tubulin
mutant, tub4-1. A, synthetic interaction
between tub4 and ssa1. The double mutants
ssa1-134 and tub4-1 exhibited a synthetic lethal
phenotype at 30 °C (top) and 16 °C (bottom)
in the absence of the plasmid-borne wild-type TUB4 gene.
Strain MO211 containing wild-type SSA1, ssa1-134,
or control plasmid were further transformed with plasmid 315-ADE2
(control) or 315-ADE2-TUB4 (wild-type TUB4), and
grown in liquid YPD medium repeatedly and equal numbers of cells were
spotted onto SD or 5-FOA plates. B, synthetic interaction
between tub4 and ydj1. Double mutants
ydj1 and tub4-1 show a synthetic phenotype at
30 °C (top) and 16 °C (bottom) in the
absence of wild-type TUB4 gene. ydj1 and
tub4-1 double mutants carrying wild-type YDJ1
gene on plasmid (316-YDJ1) were further transformed with plasmid
315-ADE2 or 315-ADE2-TUB4, and grown in liquid YPD medium repeatedly
and equal numbers of cells were spotted onto SD or 5-FOA plates.
DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
protein import into mitochondria was slightly inhibited but translocation of prepro--factor to the endoplasmic reticulum was not
affected in ssa1-134 cells at the nonpermissive
temperature.2 We believe that
the defects observed in ssa1-134 mutant cells are not simply
due to a general breakdown in these activities, since this mutant
arrested at a specific stage in the cell cycle rather than at various
stages. Furthermore, ssa1-134 mutant cells exhibited
abnormal nuclear distribution and/or became binucleated when
incubated at the restrictive temperature (Fig. 1C). The
observation that nuclear division results in the uneven segregation of
nuclei to mother and daughter cells suggested that at the restrictive temperature microtubules in this mutant are defective. Benomyl and
thiabendazole sensitivity subsequently supported this idea (Fig.
8).
-tubulin (62, 63), which is a
member of the tubulin superfamily along with - and -tubulin.
Tub4p, a -tubulin-like molecule found in S. cerevisiae,
associates with the inner and outer plaques of the yeast MTOC, the SPB,
and may act as a direct link between microtubule ends and the SPB
(64-66). We found that mutant tub4-1 exhibits synthetic
lethality in combination with some mutant alleles of SSA1,
viz. ssa1-134 (Fig. 9A). Furthermore, the ydj1 null mutant allele also exhibits synthetic
lethality with tub4-1 (Fig. 9B). Attachment of
nuclear microtubules to the SPB is apparently weak in tub4-1
mutants, as the SPB has no or few detectable microtubules and the
remaining attached microtubules are severely disorganized (66). Thus,
we speculate that Ssa1p and Ydj1p play essential roles in the proper
nucleation of microtubules mediated by -tubulin. It is possible that
loss of proper -tubulin function leads to a decrease in SPB
nucleation sites, resulting in the development of aberrant and
unusually long spindle arrays due to the excess formation of tubulin
heterodimers per nucleation site. Indeed, similar elongated
microtubules were observed when the interaction between Tub4p and
Spc98, one component of SPB, was weakened (67).
ACKNOWLEDGEMENTS
FOOTNOTES
Fellow from the Japan Society for the Promotion Science for
Japanese Junior Scientists.
REFERENCES
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
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