 |
INTRODUCTION |
The 90-kDa heat shock protein
(Hsp90)1 is a highly
conserved, abundant, and constitutively expressed homodimeric molecular chaperone of the eukaryotic cytosol. It is specifically involved in the
folding and conformational regulation of a limited subset of proteins.
Almost all natural substrates of Hsp90 are medically relevant signal
transduction molecules, including the nuclear receptors for steroid
hormones, several protooncogenic kinases, and disparate proteins such
as nitric-oxide synthase (1-4). In order to fulfill its cellular role,
Hsp90 cooperates with a cohort of cofactors such as Sti1/Hop/p60, the
immunophilins FKBP51/54 and FKBP52, cyclophilin 40, cdc37/p50,
Sba1/p23, and CHIP (5-8) and acts as a part of a multichaperone
machine together with Hsp70 (3, 9).
Several Hsp90-associated cofactors contain tetratricopeptide repeat
motifs, a degenerate 34-aa sequence, that mediate binding to the
C-terminal EEVD motif of the molecular chaperone (10). Other cofactors
of Hsp90, like Sba1/p23 and cdc37/p50, lack tetratricopeptide repeats
and are believed to use unique sequences to associate with the
molecular chaperone.
The basic mechanism of Hsp90 function is still not fully understood.
Recent crystal structures of the N-terminal domain of Hsp90 (11, 12)
have identified a conserved binding site for ATP. Based on this
structural evidence for nucleotide binding, it was demonstrated that
Hsp90 can hydrolyze ATP and that this activity of the molecular
chaperone is essential in vivo (13, 14). Thus, Hsp90 was
unequivocally identified as an ATP-dependent molecular chaperone.
Here we demonstrate binding of the novel cofactors Aha1 and Hch1 to the
middle domain of Hsp90, currently a poorly investigated region in the
molecular chaperone. The intrinsic ATPase activity of Hsp90 can be
stimulated 5-fold by the cofactor Aha1. Deletion of the genes encoding
Aha1 and Hch1 in Saccharomyces cerevisiae impaired
activation of the heterologous Hsp90-dependent substrate protein v-Src and interfered with cell viability under non-optimal conditions.
 |
EXPERIMENTAL PROCEDURES |
Plasmid Construction and Yeast Two-hybrid Experiments--
Yeast
Hsp90 (yHsp90) and fragments thereof were amplified from pTGPD/P82 (a
gift from S. Lindquist) and yeast Aha1 (yAha1); Hch1 and Sti1 were
amplified from wild type yeast DNA. PCR products were inserted into
pProExHTa expression vector to generate an N-terminal His6
sequence followed by a tobacco etch virus cleavage site. The
point mutation E381K was generated using the QuickChange site-directed
mutagenesis kit (Stratagene). Desired exchanges were confirmed by DNA sequencing.
Human Aha1 (hAha1) was amplified from a human brain cDNA library
and cloned into pAS2-1 vector (Clontech). Human
Hsp90FL (hHsp90) and hHsp90 fragments were amplified from human
Hsp90
cDNA (StressGen, accession number X15183) (15) and cloned
into pACT2 vector (Clontech). Yeast two-hybrid
screening was performed as described previously (13, 16) using a human
brain library based on the pACT2 vector (Clontech).
Positive candidate cDNA inserts were further analyzed by analytical
PCR and by nucleotide sequencing. For assaying protein-protein
interactions, cotransformants were grown on SD/
Trp,
Leu plates. The
interaction of the bait construct with the target construct was judged
by the ability of cotransformants to grow on SD/
Trp,
Leu,
His
plus 25 mM 3-amino-1,2,4-triazole selection plates.
Protein Purification--
Expression constructs in pProExHTa
were transformed into Escherichia coli BL21(DE3)pLysS cells.
Bacteria were grown at 18 °C in LB medium supplemented with 100 mg/liter ampicillin and 34 mg/liter chloramphenicol to
A600 = 1, and protein expression was
induced for 5 h with 0.25 mM
isopropyl-1-thio-
-D-galactopyranoside. After harvesting,
proteins were enriched from cell pellets by Ni-NTA chromatography at pH
8.0 essentially as described (13). Proteins were further purified by
ion-exchange chromatography on ResourceQ and by gel filtration on
Superose 12 (Amersham Biosciences).
Protein Interaction Assays--
For gel filtration analysis,
500-µl samples containing the indicated combinations of yeast
proteins yHsp90 (5 µM), yHsp90NM (5 µM),
yHsp90N (10 µM), yAha1 (5 µM), Hch1 (5 µM), or Sti1 (5 µM) were incubated for 10 min at room temperature in 40 mM HEPES-KOH, pH 7.4, 100 mM KCl, 2 mM MgCl2 and then
separated on a Superose 12 HR10/30 or on a Superdex 75 HR10/30 column
equilibrated in the same buffer using an ÄKTA chromatography
system (Amersham Biosciences). 500-µl fractions were collected for
analysis beginning at 6 ml of elution volume.
For pull-down assays, yHsp90 protein fragments (10 µM)
were bound to Ni-NTA resin in buffer PB (25 mM HEPES-KOH,
pH 7.4, 5% glycerol, 5 mM MgCl2, 0.2% Tween
20, 100 mM KCl, 10 mM imidazole) supplemented
with 2% (w/v) bovine serum albumin and 2% fish skin gelatin to
minimize unspecific protein interactions. yAha1 or Hch1 proteins (10 µM), which had been cleaved off from their
His6 tags by tobacco etch virus protease treatment,
were added, and the mixture was agitated at 4 °C for 30 min. After
two washes with buffer PB, specifically bound protein was eluted from
yHsp90 fragments with 500 mM KCl in the same buffer and
analyzed by SDS-PAGE.
Assay of Hsp90 ATPase Activity--
Recombinant yHsp90 protein
(5 µM) or the mutant yHsp90 E381K (5 µM)
were incubated at 30 °C or at temperatures indicated with or without
addition of yAha1 or Hch1 in 40 mM HEPES-KOH, pH 7.4, 2 mM MgCl2 supplemented with 2 mM
[
-32P]ATP (4 µCi/mM, Amersham
Biosciences) for 15 min in a final volume of 20 µl. Aliquots of each
reaction were stopped at time points by the addition of 5 mM EDTA and freezing in liquid nitrogen (13). Separation of
ADP from ATP was achieved by thin layer chromatography on
polyethyleneimine-cellulose sheets (Merck) in 0.5 M LiCl
and 0.5 M formic acid. ATPase activity was monitored by
quantitation of [
-32P]ADP in a liquid scintillation
counter. Steady state ATPase rates were calculated from the linear
range of the reactions, and ATP hydrolysis activity of 5 µM yHsp90 at 30 °C was set to 100%. For specific
inhibition of the ATPase activity, geldanamycin was added to the
reaction at a final concentration of 180 µM in 1%
Me2SO. Control reactions received Me2SO alone.
Yeast Methods and Manipulations--
Yeast strains W303 and
ILEP1 (2, 17) were used throughout this study, and standard methods for
growth, transformation, and manipulation were employed. Cells were
grown on YPD or on SD, SRaf, and SGal selective minimal medium (0.67%
yeast nitrogen base supplemented with 2% glucose, raffinose, or
galactose, respectively, and with nucleotides and amino acids depending
on auxotrophy).
The HCH1 gene was amplified by PCR from yeast genomic DNA
and cloned into pBluescript vector via SacI/XhoI.
A disruption allele was generated by cutting a substantial part of the
ORF and inserting the TRP1 gene via
StyI/HincII. The disruption fragment was released from the plasmid by digestion with SacI/XhoI and
transformed into haploid ILEP1 (a gift from Susan Lindquist) or haploid
W303 cells. Transformants were selected on SD/
Trp plates. The
AHA1::KanMX4 disruption cassette was
generated by PCR amplification from genomic DNA isolated from the
AHA1 knockout strain Y03573 (EUROSCARF), and transformants
were selected on YPD agar plates containing 200 mg/l G418. After
isolation of genomic DNA from transformants and from wild type cells,
disruption of wild type alleles was analyzed by PCR using specific
flanking primers. The correct replacement of the respective target gene
by its deletion allele in transformed cells was verified by the
appearance of PCR products of the expected size and absence of PCR
products corresponding to the wild type allele as described (18).
Expression and Detection of v-Src--
Yeast cells were grown
overnight in SRaf/
Ura medium, diluted to A600 = 0.3 into the same medium, and supplemented with 2% galactose for
6 h at 25 °C to induce v-Src expression. Cell viability was
assessed by spotting serial dilutions onto SD/
Ura plates at 25 °C.
Cultures were collected by centrifugation, resuspended for 10 min in
150 mM NaCl, 50 mM HEPES, pH 8.0, 1% Triton
X-100, 1% sodium deoxycholate, 0.1% SDS, 10 mM EDTA, 1 mM Na3VO4, 5 units/µl lyticase
and lysed by ultrasonification for 10 s on ice. Proteins were
separated by SDS-PAGE on 7.5% gels (19). Immunoblotting was performed
following the protocol of Towbin et al. (20). Antibodies
4G10 (Upstate Biotechnology, Inc.) and EC10 (Upstate Biotechnology,
Inc.) were used to detect phosphotyrosine residues or v-Src protein,
respectively. The ECL reagent kit (Amersham Biosciences) was used to
visualize immunocomplexes.
Miscellaneous Procedures--
Protein concentrations were
determined by the Bio-Rad protein assay kit.
 |
RESULTS |
Identification of Aha1 and Hch1 as Binding Partners of
Hsp90--
HCH1 (ORF YNL281w) had been identified as a
high copy Hsp90 suppressor of the
temperature-sensitive Hsp90 mutant E381K in S. cerevisiae
(21). We used the 153-aa sequence of the Hch1 protein (Swiss-Prot
accession number P53834) as a bait to screen the EMBL and NCBI protein
data bases with the Blast and Fasta3 server. As a result, we found that
Hch1 shares sequence homology to a short 151-aa protein present in
Candida albicans (Swiss-Prot accession number O93993) and to
the N-terminal half of the 350-aa S. cerevisiae protein Aha1
(activator of Hsp90 ATPase,
Swiss-Prot accession number Q12449, ORF YDR214w), which is
conserved from yeast to mammals (Swiss-Prot accession numbers Q9P782
for Schizosaccharomyces pombe, 336 aa; Q93168 for
Caenorhabditis elegans, 343 aa; Q9LHL7 for Arabidopsis
thaliana, 360 aa; Q9V9Q4 for Drosophila melanogaster,
361 aa; Q8R3E6 for Mus musculus, 338 aa; and O95433 for
Homo sapiens, 338 aa). Aha1 proteins therefore consist of a
putative N-terminal domain, which is homologous to Hch1 and an
additional putative C-terminal domain. Human Aha1 (hAha1) shares 23%
sequence identity and 38% sequence homology to S. cerevisiae Aha1 (yAha1) (Fig. 1).
Interestingly, S. cerevisiae is unique in a way that it
harbors Hch1 and Aha1, whereas all other organisms investigated, with
the exception of C. albicans, contain only homologues of
Aha1.

View larger version (52K):
[in this window]
[in a new window]
|
Fig. 1.
Sequence comparison aligning S. cerevisiae Hch1 (yHch1) to S. cerevisiae Aha1 (yAha1) and to human Aha1
(hAha1). Amino acids identical in all three
sequences are marked by asterisks; identical residues found
in two sequences are indicated by colons; gaps in
the sequence are denoted by periods.
|
|
In their original study, Lindquist and co-workers (21) suspected that
Hch1 might act as a cochaperone or modulator of the Hsp90 system. This
idea implies that Hch1 and possibly Aha1 due to its sequence similarity
could interact with Hsp90. To test this hypothesis, we cloned the
cDNA for human Aha1 (hAha1) into the pAS2-1 vector and used it as
the bait in a yeast two-hybrid screen against a human brain cDNA
library. Screening of ~1.5 million cotransformants yielded 129 positive clones, all of them harboring hHsp90 sequences as demonstrated
by analytical PCR using Hsp90-specific primers. Strikingly, besides
hHsp90, no other interacting proteins were identified. Similarly, when
recombinant yAha1 or hAha1 was added to S. cerevisiae or
mammalian cell extracts, yHsp90 or Hsp90 could be coprecipitated (data
not shown).
The Middle Domain of Hsp90 Is the Specific Binding Site for Aha1
and Hch1--
Sequencing of 16 of the 129 positive hHsp90 cDNA
clones revealed 10 sequences starting within a region (aa 220-291)
that links the N-terminal and the middle domain of hHsp90 (Fig.
2A). This result proposed that
regions in the middle domain or in the C-terminal domain of hHsp90 were
involved in binding to hAha1. The remaining cDNAs were full length
or started very close to the N terminus.

View larger version (22K):
[in this window]
[in a new window]
|
Fig. 2.
Interaction of Aha1 and of Hch1 with
Hsp90 and Hsp90 fragments established by the yeast two-hybrid
technology. A, schematic representation of the domain
organization of human Hsp90 deduced from proteolytic data (12) and of
the Hsp90 fragments used in this study; numbers refer to the
sequence of human Hsp90 . B, hHsp90FL and fragments of
hHsp90 in the pACT2 vector or empty pACT2 vector serving as a control
(vertical) were cotransformed into S. cerevisiae (strain Y190) with the cDNAs encoding hAha1 and
Hch1 in the pAS2-1 vector or with empty vector as a control
(horizontal). To monitor protein-protein interactions,
cotransformants from SD/ Trp, Leu plates were restreaked on
SD/ Trp, Leu, His plates containing 25 mM
3-amino-1,2,4-triazole for 8 days at 30 °C. Growth was observed only
for combinations of hAha1 and Hch1 with hHsp90FL and with
hHsp90M.
|
|
To delineate the binding site further, we tested hHsp90FL and the
fragments hHsp90N, hHsp90M, and hHsp90C by yeast two-hybrid analysis
for binding to hAha1 and to Hch1 (Fig. 2B). Although hHsp90FL and hHsp90M exhibited binding to hAha1, hHsp90N and hHsp90C did not. A similar result was obtained for Hch1 (Fig. 2B).
These data indicate that the interaction with hAha1 and Hch1 is
mediated by the middle domain of hHsp90 and therefore distinct from the binding of TPR-containing cofactors like Sti1/p60/Hop to the C-terminal domain of Hsp90 (10, 16) or of Sba1/p23 where regions in the N-terminal
domain and in the C terminus of Hsp90 are involved (22).
Biochemical binding assays using purified yAha1 protein were performed
to support and extend the outcome of our two-hybrid experiments. First,
we tested yHsp90FL and the protein fragment yHsp90NM for their ability
to interact with the yAha1 protein by gel filtration chromatography.
yAha1, clearly migrating as a monomer, yHsp90FL, and yHsp90NM showed
different elution profiles on a Superose 12 column (Fig.
3, A, B, and
D). We combined yHsp90FL and yHsp90NM with yAha1 and
analyzed the mixtures for protein interactions (Fig. 3, C
and E). When a mixture of yHsp90FL and yAha1 was run on a
Superose 12 gel filtration column, a significant amount of yAha1
coeluted with yHsp90FL (Fig. 3C, fractions
7-12), consistent with the formation of a complex between the two
proteins. This binding was independent of ATP and could be disrupted by increasing the salt concentration to 500 mM KCl (not
shown).

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 3.
The middle domain of Hsp90 is the specific
binding site for Aha1. Purified yHsp90 and the fragments yHsp90NM
and yHsp90N were incubated with yAha1 and fractionated by gel
filtration chromatography on a Superose 12 column (A-E) or
on a Superdex 75 column (F-H). Fractions were analyzed by
SDS-PAGE. Marker proteins are shown on top (thyroglobulin
667 kDa, bovine serum albumin 67 kDa, ribonuclease A 14 kDa).
A, yAha1. B, yHsp90FL. C, yAha1
incubated with yHsp90FL. D, yHsp90NM. E, yAha1
incubated with yHsp90NM. F, yAha1. G, yHsp90N.
H, yAha1 incubated with yHsp90N.
|
|
When yHsp90NM and yAha1 were incubated together and run on a Superose
12 column, again a certain amount of yAha1 comigrated with this yHsp90
fragment (Fig. 3E, fractions 10-12) indicating that the C-terminal domain of the molecular chaperone is dispensable for the interaction. Binding of yAha1 to yHsp90 M is not shown in Fig.
3, and because complexes could not be resolved from either protein
alone, we instead used a pull-down experiment to demonstrate this
interaction (see below). However, when using a Superdex 75 column
instead of a Superose 12 column, yAha1 could be separated from yHsp90N
(Fig. 3, F and G). A mixture of the two proteins was analyzed by gel filtration chromatography, but no shift in the
migration of yHsp90N was observed (Fig. 3H), consistent with the view that the N-terminal domain of yHsp90 is not involved in
binding to yAha1.
As an independent verification of these domain mapping studies, yAha1
was assayed for interacting with purified N-terminal, middle, and
C-terminal domains of yHsp90 immobilized on Ni-NTA agarose in a
pull-down experiment as described under "Experimental Procedures."
Mixtures of yAha1 and yHsp90 fragments were incubated to allow binding
and were finally recovered by centrifugation. Protein that had
specifically bound to yHsp90 fragments was eluted by 500 mM
KCl and analyzed by SDS-PAGE. yAha1 bound to yHsp90M and could be
specifically eluted from this protein fragment but was not retained on
yHsp90N or on yHsp90C (Fig. 4).

View larger version (31K):
[in this window]
[in a new window]
|
Fig. 4.
Aha1 binds to the middle domain of Hsp90 but
not to Hsp90N or to Hsp90C. yHsp90 fragments were bound to Ni-NTA
agarose via their His6 tags and incubated with non-tagged
yAha1 protein as described under "Experimental Procedures."
Specifically bound protein was eluted with 500 mM KCl and
analyzed by SDS-PAGE. The position of marker proteins with sizes
indicated in kDa is shown.
|
|
Similar results were obtained when Hch1 or the corresponding N-terminal
fragment yAha1-(1-156) was used in this pull-down experiment. In
contrast, the C-terminal yAha1-(157-350) fragment did not bind to
yHsp90 (data not shown). Thus, the biochemical data are in full
agreement with the observation of our yeast two-hybrid assays and
demonstrate that the middle domain of Hsp90 is necessary and sufficient
to bind Aha1 and Hch1.
Sti1 Competes with yAha1 for Binding to yHsp90--
Sti1/p60/Hop
is bound in "intermediate" Hsp90 chaperone complexes to the
C-terminal domain of the molecular chaperone by one of its TPR clamp
domains and also contacts the N-terminal domain of Hsp90 thereby
blocking access to the ATP-binding site (23). This observation prompted
us to ask whether Sti1 interferes with binding of yAha1 to yHsp90, and
we therefore analyzed a mixture of Sti1, yHsp90, and yAha1 for complex
formation by gel filtration chromatography. Sti1 and the yHsp90-yAha1
complex showed different elution profiles on a Superose 12 column (Fig.
5, A and B). Upon addition of Sti1 to yHsp90FL and yAha1 at equimolar amounts, the yHsp90FL-yAha1 complex was apparently almost completely disrupted (Fig.
5C). This situation resembles the competition of cdc37/p50 by Sti1/p60/Hop (24, 25) and suggests a steric hindrance caused by a
three-dimensional overlap of the cofactors or their binding sites on
Hsp90.

View larger version (21K):
[in this window]
[in a new window]
|
Fig. 5.
Sti1 competes with yAha1 for binding to
yHsp90FL. Purified yHsp90FL was incubated with Sti1 and yAha1 and
fractionated by gel filtration chromatography on a Superose 12 column.
A, Sti1. B, yHsp90FL incubated with yAha1
(identical to B in Fig. 3). C, yHsp90FL incubated
with yAha1 and Sti1. Marker proteins are shown on top
(thyroglobulin 667 kDa, bovine serum albumin 67 kDa).
|
|
yAha1 Stimulates the Intrinsic ATPase Activity of yHsp90--
ATP
hydrolysis has been shown to be essential to the in vivo
function of the molecular chaperone Hsp90 (13, 14). Therefore, we were
curious as to whether this capacity of yHsp90 could be affected by
yAha1. We titrated increasing amounts of yAha1 into 5 µM
yHsp90 (corresponding to 2.5 µM dimer) and measured
ATPase activity as described under "Experimental Procedures." The
ATP hydrolysis rate for various combinations of yHsp90 and yAha1 is plotted in Fig. 6A. Obviously,
yAha1 was able to stimulate the intrinsic ATPase activity of yHsp90 up
to 5-fold. Next we asked whether Hch1, which is homologous to the
putative N-terminal domain of yAha1, would also be able to influence
yHsp90 ATPase activity. Interestingly, although Hch1 bound to Hsp90
(Fig. 2B), it was not able to stimulate yHsp90 ATPase
activity significantly even at 50 µM, a 10:1 molar ratio
relative to yAha1 (Fig. 6B). Moreover, when yAha1 was
dissected into two fragments, one comprising the putative N-terminal
domain (aa 1-156) and the other one encompassing the putative
C-terminal domain (aa 157-350), the intrinsic ATP hydrolysis rate of
yHsp90 could not be stimulated by any of these truncated proteins (Fig.
6B). This result indicates that the entire yAha1 polypeptide
is required for stimulating the ATPase activity of yHsp90.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 6.
yAha1 stimulates the intrinsic ATPase
activity of yHsp90. ATP hydrolysis rate was measured as described
under "Experimental Procedures," and the ATPase activity of 5 µM yHsp90 at 30 °C without additional
proteins was set to 100%. All data are corrected for unspecific
background activity which could not be inhibited by addition of
geldanamycin. Activities are averages of at least three independent
experiments, and error bars are indicated. A,
yHsp90 ATPase activity stimulated by increasing amounts of yAha1 at
30 °C. Note that yAha1 could stimulate yHsp90 up to 5-fold.
B, yAha1 but neither of its fragments (aa 1-156 and aa
157-350) nor Hch1 could stimulate ATP hydrolysis of yHsp90. yHsp90 was
incubated alone or with indicated amounts of yAha1, yAha1-(1-156),
yAha1-(157-350), or Hch1 and assayed for ATPase activity at 30 °C.
C, ATPase activity of wild type yHsp90 and of the E381K
mutant at 25 and 37 °C. The ATPase activity of the E381K mutant
reached nearly wild type levels.
|
|
Since Hch1 was not able to stimulate the ATP hydrolysis rate of yHsp90
but suppressed the temperature-sensitive yHsp90 mutant E381K (21), we
assumed that the defect of this mutant at the non-permissive
temperature is not due to a major reduction of ATPase activity. To test
this assumption rigorously, we determined the rate of ATP hydrolysis
for the E381K mutant at 25 and 37 °C, the non-permissive
temperature, and compared it to wild type yHsp90. The shift to 37 °C
did not harm the ATPase activity of the E381K mutant. Instead, the
ATPase activity of the E381K mutant was raised with temperature and
reached a value comparable with wild type yHsp90 (Fig. 6C).
Thus, the loss of function due to the E381K mutation at the
non-permissive temperature presumably results from defects in other
aspects of Hsp90 biochemistry, which can be restored by the assistance
of Hch1.
Aha1 and Hch1 Are Non-essential but Confer Thermotolerance to Yeast
Cells When Intracellular Levels of Hsp90 Are Limiting--
Due to the
relationship of both proteins (Fig. 1), Hch1 and Aha1 could serve
overlapping functions and be redundant in S. cerevisiae.
Therefore, we created knockouts
HCH1,
AHA1,
and the
HCH1/
AHA1 double knockout in the
wild type yeast strain W303 as described under "Experimental
Procedures." The relative growth rate of wild type cells and of these
three knockout strains was assessed using a plate assay. There was no
obvious difference in the growth rate at 25, 35.5, or at 37 °C on
YPD agar plates (Fig. 7). Thus a complete
loss of Hch1 and Aha1 protein activity did not affect viability of W303
cells at normal and at elevated temperatures. A comparable mild effect
at 37 °C has been described previously for the deletion of the gene
encoding Sba1/p23 (26, 27).

View larger version (63K):
[in this window]
[in a new window]
|
Fig. 7.
Aha1 and Hch1 are non-essential but confer
thermotolerance to yeast cells when intracellular levels of Hsp90 are
limiting. Viability of ILEP1 and W303 wild type (wt) or
HCH1, AHA1, and
HCH/ AHA1 deletion strains at 25, 35.5, and
37 °C. Cells were grown overnight in YPD medium, adjusted to 1 × 108 cells per ml, and 10-fold serially diluted. 2-µl
portions were spotted on YPD agar plates and incubated for 48 (at
25 °C) or 36 h (at 35.5 and 37 °C).
|
|
We next asked whether deletions of Hch1 and Aha1 would harm cells more
severely if intracellular levels of Hsp90 were significantly reduced.
To investigate such a situation we made use of the W303-derived strain
ILEP1, a kind gift from Susan Lindquist. In ILEP1 cells expression of
Hsp90 was controlled by a partially deleted promoter, which results in
a reduction of Hsp90 to ~1/20 of the normal level (17). The same
deletions as described above for W303 were generated in ILEP1, and
cells were again assessed for viability. No difference in growth
phenotype was observed at 25 and at 35.5 °C. However, when viability
was tested at 37 °C, the ILEP1
HCH1/
AHA1
double knockout cells exhibited obvious growth defects compared with the parental strain and with the single knockout strains (Fig. 7).
Deletion of Genes Encoding Aha1 and Hch1 Interferes with v-Src
Activation in Vivo--
The tyrosine kinase v-Src is active in a
manner that is dependent on the molecular chaperone Hsp90 and its
cofactors/cochaperones. In wild type cells like W303, expression of
v-Src leads to greatly increased levels of phosphotyrosine. Mutations
affecting Hsp90 itself or deletions of genes encoding cofactors of the
Hsp90 system resulted in a decrease of v-Src activity accompanied with
lower levels of tyrosine phosphorylation (28-31) that provides a
handle to estimate the activity of the Hsp90 system in vivo.
Since v-Src expression is toxic in yeast cells, v-Src was expressed
from a galactose-regulated plasmid.
We observed in a control experiment that most of the strains generated
in this study grew equally well on glucose, galactose, and raffinose
medium. As an exception,
HCH1/
AHA1 double
deletion cells displayed a clearly delayed growth phenotype on
galactose as the sole carbon source. When these
HCH1/
AHA1 cells were transformed with the
v-Src expression plasmid and induced by transfer from SRaf/
Ura to
SGal/
Ura medium, v-Src levels were strongly decreased compared with
wild type or to the single deletion strains (data not shown). In
contrast, addition of galactose to
HCH1/
AHA1 cells growing on raffinose medium
had no negative effect on their growth rate. Therefore, W303 and ILEP1
wild type and deletion strains were transformed with the v-Src
expression plasmid, grown in SRaf/
Ura medium overnight, and diluted
with the same medium to an A600 = 0.3, and v-Src
expression was induced at 25 °C for 6 h by addition of 2%
galactose to the SRaf/
Ura medium (26) as described under
"Experimental Procedures." Under these conditions v-Src protein
accumulated to similar levels in wild type,
HCH1,
AHA1, and
HCH1/
AHA1 cells (Fig.
8A). However, compared with W303 cells v-Src accumulation in ILEP1 cells was reduced, likely reflecting the lower amount of Hsp90 in this strain (Fig.
8A).

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 8.
Deletion of genes encoding Aha1 and Hch1
interfere with v-Src activation in vivo. Cell
lysates were prepared from normal or mutant ILEP1 and W303 cells
expressing v-Src (lanes 2-5 and 7-10) or from
empty vector control cells (lanes 1 and 6) as
described under "Experimental Procedures." Proteins were separated
by PAGE on 7.5% gels and transferred to nitrocellulose membranes.
Equal protein loading was confirmed by the Bio-Rad protein assay and by
Coomassie staining of a corresponding SDS gel. A, Western
blot analysis of v-Src expression with antibody EC10. B,
Western blot analysis of phosphotyrosine activity with antibody 4G10 in
the strains corresponding to A. The position of marker
proteins with sizes indicated in kilodaltons is shown. C,
quantification of phosphotyrosine levels from the Western blot in
B, phosphotyrosine activity of W303 wild type
(wt) was set to 100%.
|
|
When protein tyrosine phosphorylation was monitored, all three mutant
W303 strains showed a moderate reduction of v-Src activity to ~50%
of the W303 wild type cells (Fig. 8, B and C, lanes
6-10). Next, we examined ILEP1 strains for protein tyrosine
phosphorylation. Compared with W303, v-Src activity in ILEP1 was
reduced as expected from the lower level of v-Src accumulation.
However, deletions of the genes encoding Hch1 and Aha1 had a more
pronounced effect, which resulted in a decrease of v-Src activity in
ILEP1
HCH1/
AHA1 as low as ~10% compared
with the parental ILEP1 cells (Fig. 8, B and
C, lanes 1-5). From this result we conclude that
Hch1 and Aha1 became important for client protein activation under
non-optimal conditions at higher temperatures when Hsp90 levels are
limiting as observed in ILEP1 cells.
 |
DISCUSSION |
An increasing number of cochaperones/cofactors are now known to
regulate the Hsp90/Hsp70 chaperone machine for fulfilling different
tasks in the cell. A defined group of cofactors like Sti1/p60/Hop, the immunophilins FKBP51/54 and FKBP 52, cyclophilin 40, protein phosphatase PP5, and CHIP bind to the C-terminal EEVD motif of
the Hsp90 component of this system by virtue of their modular TPR clamp
domains (10). In the case of p23, regions in the N-terminal ATPase
domain and induced dimerization of Hsp90 are required for binding
(22).
The results presented here demonstrate that Aha1 and Hch1, in
remarkable contrast, associate with the middle domain of Hsp90, whereas
C- and N-terminal domains of the molecular chaperone are dispensable.
As a consequence of this interaction, the cofactor Aha1 stimulates the
inherent ATPase rate of Hsp90 5-fold, comparable with the 7-fold
stimulation of Hsp70, the other major chaperone of the eukaryotic
cytosol, by the J-domain protein Hsp40 (32). In association with
our results, a stimulating effect of Aha1 on the ATPase activity of
Hsp90 has also been reported by the recent work of Panaretou and
co-workers (44). When we used Hsp90 and Aha1 at a 1:1 molar ratio,
Hsp90 ATPase was almost fully stimulated, whereas Hch1 and the
N-terminal Aha1 fragment displayed no stimulating effect under the same
conditions (Fig. 6B). When the Hch1:Hsp90 ratio
was increased from 1:1 to 10:1, a slight stimulation of ATP hydrolysis
could be observed (Fig. 6B). Panaretou and co-workers (44)
raised this molecular excess further and could thereby stimulate Hsp90
ATPase by Hch1 and by the N-terminal Aha1 fragment, however, only to
~30% that achieved by full-length Aha1. These in vitro
data suggest that Aha1 is by far a more potent stimulator of Hsp90
ATPase activity than Hch1. Another point questions a function of Hch1
as an authentic stimulator of Hsp90 ATPase. Remarkably, Hch1 suppressed
the growth defect of the Hsp90 mutant T22I in S. cerevisiae
(21). Since the toxicity of this mutant was attributed to its 6-fold
increased ATPase activity (43), a pivotal role of Hch1 as a stimulator
of Hsp90 ATPase seems to be virtually unlikely.
Like Sba1/p23 (26, 27), Aha1 and Hch1 are not essential in S. cerevisiae for general chaperone-mediated protein folding under
normal conditions. However, Aha1 and Hch1 turned out to be crucial when
growth temperatures were raised to 37 °C and Hsp90 levels were
limiting (Fig. 7). Under conditions where Hsp90 becomes limiting, both
cofactors contributed to the activation of the substrate protein v-Src
by Hsp90 (Fig. 8); thus our study and the work of Panaretou and
co-workers (44) identify Aha1 and Hch1 as cofactors crucial for the
activation of a client protein in yeast. Compared with wild type and
single deletion strains,
HCH1/
AHA1 cells
accumulated slight levels of v-Src protein when expression was
induced by transfer from raffinose to galactose medium as mentioned
under "Results" and reported by Panaretou and co-workers (44). This
may be due to rapid degradation (44) or by lower synthesis of the v-Src
protein under those conditions. However, we realized that
HCH1/
AHA1 cells were sensitive to galactose
as the sole carbon source and that this stress situation might
interfere with the analysis of v-Src activation. Therefore, we induced
v-Src expression by addition of galactose to cells growing on raffinose
medium, rather then by transfer of cells from raffinose to galactose
medium. Under those conditions v-Src protein reached comparable levels
in wild type,
HCH1,
AHA1, and in
HCH1/
AHA1 strains (Fig. 8).
At higher temperatures, Hsp90 is thought to prevent unfolding of
substrates and allow recovery of sensitive client proteins which had
been partially unfolded (33, 34). Remarkably, Aha1 and to a lower
extent Hch1 is up-regulated by heat stress to the same degree as Sti1
and Hsp82, the inducible form of Hsp90 in S. cerevisiae
(data from expression profiling analysis may be obtained from the
Saccharomyces Genome Data base at genome-www4.stanford.edu). Aha1 and Hch1 cannot prevent aggregation of unfolded proteins (data not
shown) and are therefore no chaperones on their own. Instead, they add
to the efficiency of the Hsp90 system, which becomes crucial under
non-optimal conditions. Whether the stimulating effect of Aha1 on the
activation of client proteins like v-Src is a direct consequence of its
ability to stimulate Hsp90 ATPase has yet to be demonstrated. For this
purpose, rather than a wholesale disruption of Aha1, mutants of Aha1
would be helpful that bind indifferently to Hsp90 but fail to stimulate
its ATPase activity. However, such mutants are not readily available.
It is generally believed that unfolded substrate proteins associate
first with the Hsp70 system and are then targeted to Hsp90 via
Sti1/p60/Hop that binds both Hsp70 and Hsp90 (1, 3, 35-38). This
interaction leads to the formation of an "intermediate complex" in
which Sti/p60/Hop inhibits the ATPase activity of Hsp90 and locks the
molecular chaperone in a conformation allowing substrate protein to
bind (23). Further remodeling and dissociation of Sti1/p60/Hop, Hsp70
and its cochaperones then leads to the "mature complex," the state
in which Hsp90 consumes ATP (23) and actively contributes to the
activation of client proteins. Our data suggest that binding of Aha1
and Sti1 to Hsp90 is mutually exclusive (Fig. 5C), probably
due to steric hindrance. In contrast, Panaretou and co-workers (44)
proposed in their recent work that Aha1 and Sti1 can coexist in Hsp90
complexes. To resolve this potential inconsistency it is important to
point out that this group used the C-terminal fragment cSti1 (aa
237-589) in their experiments, whereas in our studies natural
full-length Sti1 was used. It is plausible that the N-terminal
truncation prevents a three-dimensional overlap of cSti1 with Aha1, and
therefore both cofactors can bind simultaneously to Hsp90, whereas
full-length Sti1 competes with Aha1 for Hsp90 binding. The affinity of
Aha1 for Hsp90 seems to be too weak to replace Sti1 directly from Hsp90 complexes at physiological salt conditions (Fig. 5C), but it
might be increased by modifications or by the presence of a client
protein, although there is no experimental evidence for such a
regulation. On the other hand, Panaretou and co-workers (44) have shown that Aha1 can coexist in Hsp90 complexes with cochaperones of the
"mature" type like full-length Cpr6. Thus, displacement of Sti1 by
Cpr6 during the transition from the intermediate to the mature
Hsp90 chaperone complex allows Aha1 to enter the Hsp90 complex at this
stage. Hence, the cell can keep Aha1 at moderate levels as Aha1 does
not have to replace Sti1 directly from Hsp90. Therefore, it is
conceivable that Aha1 binds to the middle domain of Hsp90 and
contributes to protein folding at this stage of the mature complex once
Sti1 has been displaced by the mechanism described. Interestingly, it
has been suggested recently that in addition to the N- and C-terminal
domains (39, 40), the middle domain of Hsp90 can interact with client
proteins and serves as a molecular scaffold for their activation (41).
Aha1 and Hch1 bind to the same domain that would enable them to act on
this process.
The more complex the organism from which the Hsp90 protein is derived,
the less efficient the ATP hydrolysis rate, which predicts that the
molecular chaperone would require cofactors to regulate ATPase activity
or binding of substrate especially in higher organisms (42). Compared
with more complex organisms, Hsp90 from S. cerevisiae has a
relatively high ATPase activity, and consequently further stimulation
would not be urgently required. This may explain the relative
redundancy of Aha1 and Hch1 in S. cerevisiae. On the other
hand, in higher organisms tuning of Hsp90 ATPase activity by Aha1
according to the demands of the client protein might become important
to trigger substrate activation and account for the absence of an Hch1
homologue. However, it has still to be demonstrated experimentally that
Hsp90 systems of humans and other higher organisms can indeed be
stimulated by their respective Aha1 cofactors similar to yeast Hsp90 by
yeast Aha1.
The mechanism of ATPase stimulation by Aha1 is elusive. A direct
interaction of Aha1 with the catalytic N-domain of Hsp90 could not be
demonstrated in this study (Figs. 3 and 4); however, a transient
interaction could contribute. Alternatively, Aha1 might give rise to a
conformational transition within the Hsp90 dimer and thereby induce
contacts of the N-terminal domains required for significant ATPase
activity (43).