From the Department of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada T2N 4N1
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ABSTRACT |
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The reovirus cell attachment protein, 1, is a
lollipop-shaped homotrimer with an N-terminal fibrous tail and a
C-terminal globular head. Biogenesis of this protein involves two
trimerization events: N-terminal trimerization, which occurs
cotranslationally and is Hsp70/ATP-independent, and C-terminal
trimerization, which occurs posttranslationally and is
Hsp70/ATP-dependent. To determine if Hsp90 also plays a
role in
1 biogenesis, we analyzed
1 synthesized in rabbit
reticulocyte lysate. Coprecipitation experiments using anti-Hsp90
antibodies revealed that Hsp90 was associated with immature
1
trimers (hydra-like intermediates with assembled N termini and
unassembled C termini) but not with mature trimers. The use of
truncated
1 further demonstrated that only the C-terminal half of
1 associated with Hsp90. In the presence of the Hsp90 binding drug
geldanamycin, N-terminal trimerization proceeded normally, but
C-terminal trimerization was blocked. Geldanamycin did not inhibit the
association of Hsp90 with
1 but prevented the subsequent release of
Hsp90 from the immature
1 complex. We also examined the status of
p23, an Hsp90-associated cochaperone. Like Hsp90, p23 only associated
with immature
1 trimers, and this association was mapped to the
C-terminal half of
1. However, unlike Hsp90, p23 was released from
the
1 complex upon the addition of geldanamycin. These results
highlight an all-or-none concept of chaperone involvement in different
oligomerization domains within a single protein and suggest a possible
common usage of chaperones in the regulation of general protein folding
and of steroid receptor activation.
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INTRODUCTION |
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It is now known that the folding of nascent proteins in the cytosol is mediated by a group of proteins known as chaperones. These chaperones are believed to be present as large macromolecular complexes whose major roles appear to be the prevention of protein aggregation and the promotion of correct folding and assembly of newly synthesized proteins. Two candidate members of the chaperone family are the heat shock proteins Hsp70 and Hsp90, of which Hsp70 has been well characterized. Through its ATPase activity and associated bind and release cycles, Hsp70 assists in the folding of a wide spectrum of denatured and nascent proteins (see Refs. 1 and 2 for reviews). In contrast, Hsp90 does not exhibit any enzymatic activity and has not been extensively probed in terms of its possible role in the folding of nascent proteins. Indeed, its activity in vitro only approximates that of a true chaperone. Specifically, Hsp90 is able to maintain some denatured proteins in a state competent for refolding by Hsp70 and the cochaperone, Hip (p48), but Hsp90 alone cannot on its own produce refolded proteins (3).
Studies on Hsp90 have focused mainly on the role of this protein in the activation of several families of protein kinases and of steroid hormone receptors. Protein kinases associated with Hsp90 have included receptor tyrosine kinases such as erbB2 (4), nonreceptor tyrosine kinases such as Wee 1 (5) and v-src (6, 7), ser/thr kinases such as Raf-1, Mek, and Cdk4 (8-10) as well as the heme-regulated eukaryotic initiation factor kinase (HRI)1 (11, 12). However, the exact role of Hsp90 in the activation of these kinases remains unclear at present. In contrast, the nature of involvement of Hsp90 in the conformational maturation of hormone receptors is much better understood.
Accumulated evidence suggests that Hsp90 is part of a multiprotein chaperone complex that interacts with steroid receptors, keeping them in a state that is competent for binding substrate yet functionally inactive (for reviews, see Ref. 13-15). In response to steroid binding, the chaperone complex is released, and the receptor becomes activated. In the case of the relatively well studied glucocorticoid receptor, maturation involves a series of complex but ordered interactions between a number of chaperones (16-20). Reconstitution experiments have shown that an Hsp90·p60·Hsp70 complex first interacts with the receptor to convert the latter to a steroid binding conformation (19). However, this intermediate complex is highly unstable and requires the additional presence of another cochaperone, p23, for stabilization (20). p23 is also capable of stabilizing a receptor·Hsp90 heterocomplex from cytosol (21). Subsequently Hsp70 and p60 leave the complex and are replaced by any of the several immunophilins such as FKBP52 and CyP-40 (22). This complex then binds hormone, and the receptor is then released as an active transcription factor (18).
An invaluable tool in the study of Hsp90 has been the benzoquinone ansamycin, geldanamycin (GA). Originally characterized as an agent responsible for inactivation of select tyrosine kinases, GA has recently been shown to bind with high affinity to a specific binding pocket within Hsp90 (23). Treatment with GA abrogates formation of Hsp90/v-src complexes (24), inhibits the function of steroid hormone receptors (16, 25, 26), disrupts interaction of Hsp90 with the HRI (11), and targets denatured luciferase and glucocorticoid receptors for proteolytic degradation (25-27). Thus GA is considered as a very specific inhibitor of Hsp90 function, although the exact mechanism is unclear. Very recently, the GA binding site was found to co-localize with an ATP binding site on Hsp90 (at the N terminus) (23, 28), and GA blocks p23 binding to Hsp90, presumably by inducing a conformational change in the p23 binding site (28). This has led to the suggestion that the ATP and GA binding site acts as a conformational switch to regulate the assembly of Hsp90-containing multichaperone complexes (28).
Although Hsp90 is generally accepted as a chaperone, its role in the
folding of newly synthesized polypeptides has not been extensively
probed until recently. Hartson et al. (29) studied the folding of the
lymphoid cell kinase p56lck translated in vitro
and demonstrated the association of Hsp90 with newly synthesized
p56lck molecules. GA was found to disrupt this association
and hence the proper folding of this kinase. A more recent study using
the heme-regulated eIF-2 kinase translated in vitro shows
that Hsp90 plays an obligatory role in this kinase acquiring and
maintaining a conformation that is competent for transformation into an
aggregation-resistant activable kinase (11). The in vitro
translation system has also been used extensively in our laboratory to
reveal the mechanisms of folding and oligomerization of the reovirus
cell attachment protein
1, a trimeric protein positioned at the 12 vertices of the icosahedral virion (30-36). The
1 trimer is highly
asymmetric, with an N-terminal fibrous tail that is anchored to the
virion, and a C-terminal globular head that interacts with the cell
receptor (37-43). These two structurally distinct domains are
separated by a protease-sensitive hinge region (42, 44) and are
generated by independent trimerization events (34), with N-terminal
trimerization preceding C-terminal trimerization. The core of the
N-terminal trimerization domain is the N-terminal one-third of the
protein, which is highly
-helical and contains an extended heptad
repeat of hydrophobic residues (45, 46), endowing this region with the
intrinsic propensity to form a triple coiled coil. During
1
biogenesis, assembly of three neighboring nascent chains occurs cotranslationally at the N terminus (35, 36). This process does not
involve Hsp70 or ATP and results in the generation of a loose triple
coiled coil (36). This occurs at around the midpoint of the polysome
where Hsp70 begins to interact with emerging residues, thereby
sterically hindering tightening of the coiled coil. As the triplex
moves down the polysome, more Hsp70 becomes associated with the
elongating C termini, preventing their misfolding and aggregation. The
immature trimer then leaves the polysome as a complex comprised of
three
1 subunits, Hsp70, and possibly other chaperones. Subsequent
ATP-dependent release of Hsp70 presumably provides the
opportunity for the loose coiled coil to quickly snap together
(tightening the coiled coil), whereas the remaining portions of the
three C termini are available for continued interaction with Hsp70 and
other chaperones. This structure, with a stably assembled N terminus
and an unassembled C terminus, is called "hydra-like intermediate,"
and it migrates as a retarded trimer in SDS-PAGE under nondissociating
conditions (34, 35). Further ATP-dependent release and
rebinding of Hsp-70 and other proposed chaperones leads to global
assembly and folding of the C terminus, generating mature
1 with the
characteristic "lollipop"-shaped structure, which migrates as an
unretarded trimer in SDS-PAGE under nondissociating conditions (34,
35). We contend that the involvement of two mechanistically distinct
oligomerization events for the same molecule, one cotranslational and
one posttranslational, may represent a common approach to the
generation of oligomeric proteins in the cytosol (35).
In the present study, we examined the possible participation of Hsp90
in 1 biogenesis in vitro. We demonstrate that Hsp90 associates with immature but not mature
1. This association occurs at the C-terminal half, but not the N-terminal half of
1. The cochaperone p23 also associates with the C terminus and immature
1.
Geldanamycin treatment, which releases p23 but not Hsp90 from
1, has
no effect on
1 N-terminal trimerization but effectively blocks
C-terminal trimerization. These observations suggest that Hsp90
actively participates in the biogenesis of the trimeric
1 protein,
and together with our previous data on Hsp70 involvement (35), are
compatible with an all-or-none concept of chaperone involvement in
different oligomerization domains within a single protein. They also
suggest a possible common usage of chaperones in the regulation of
general protein folding and assembly and of steroid receptor
activation.
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Experimental Procedures |
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In Vitro Transcription--
The plasmids encoding the
full-length and various truncated 1 products have been described
previously (34-36, 44). All transcripts were generated in
vitro using the MEGAscriptTM transcription kit (Ambion) for Sp6
RNA polymerase promoters. A typical transcription reaction involved
incubation of 1 µg of linearized plasmid with the prescribed contents
of the Ambion transcription kit (total final volume of 20 µl) for
5 h at 37 °C. The mRNA product was isolated by LiCl
precipitation followed by cleanup with the BIO 101, Inc., RNaid Kit.
The purified mRNA was then resuspended in 0.1% DEPC-treated water
to a final concentration of approximately 0.5 µg/µl and stored at
70 °C for future use. DEPC is diethyl pyrocarbonate.
In Vitro Translation and Chase-- Transcripts were translated in vitro in rabbit reticulocyte lysates (Promega) according to the manufacturer's specifications. Typically, 0.5-1.0 µg of mRNA was incubated at 37 °C with 7 µCi of [35S]methionine (Amersham Pharmacia Biotech), 1 µl of 1.0 mM methionine minus amino acids (Promega), and 18 µl of rabbit reticulocyte lysate (Promega) for the duration indicated in figure legends. The labeled product was then analyzed by SDS-PAGE.
To follow the fate of the proteins synthesized, reaction mixtures were centrifuged at 35,000 rpm for 1.0 h at 4 °C (Beckman TLA 100.1 rotor, TL-100 tabletop ultracentrifuge) to pellet ribosomes. The supernatants, which had no translation activity, were then incubated at 37 °C for various durations and subsequently analyzed by SDS-PAGE as outlined in the figure legends. Geldanamycin was prepared as a stock solution of 175 µM in 20% Me2SO. It was added (to a final concentration of 7 µM) to the reaction mixture at the onset of translation or to the postribosomal supernatant before the chase, as indicated in the figure legends. For control samples, an equal volume of 20% Me2SO was used.SDS-PAGE--
Discontinuous SDS-PAGE was performed using the
protocol of Laemmli (47). Depending upon the size of 1 products
analyzed, 10 or 12.5% polyacrylamide gels were used. Samples were
incubated in protein sample buffer [final concentration: 50 mM Tris (pH 6.8), 1% SDS, 2%
-mercaptoethanol, 10%
glycerol, and 0.01% bromphenol blue] for 30 min at either 37 or
4 °C or, alternatively, boiled for 5 min before SDS-PAGE. The
different SDS-PAGE conditions were used to differentiate between N- and
C-terminal trimerization. Under dissociating conditions, where samples
were boiled for 5 min before SDS-PAGE (carried out at room
temperature), both N- and C-terminal trimers dissociated and migrated
as monomers. Under nondissociating conditions, samples were either
incubated for 30 min at 37 °C, at which temperature only the
trimeric N-terminal domain is stable, or incubated at 4 °C, at which
temperature both the N- and C-terminal trimeric domains are stable
(SDS-PAGE was carried out at 4 °C in both cases).
Immunoprecipitation-- Initially we obtained the 3G3 monoclonal anti-Hsp90 antibody (IgM) from Dr. G. Perdew (48) and the JJ3 anti-p23 antibody (IgG1) from Dr. D. Toft (49). We subsequently purchased these antibodies from Affinity Bioreagents, Inc.
Immunoprecipitations were carried out on in vitro translations that were performed as outlined above. Typically, a 20-µl translation reaction was stopped by the addition of four volumes of TEM buffer (20-mM Tris (pH 7.4), 5 mM EDTA, 10 mM ammonium molybdate, 50 mM NaCl). To this was added primary antibody at a dilution of between 1:35 and 1:150, as empirically determined for each antibody. For primary antibodies of IgG class, samples were incubated on ice for 1 h, and 50 µl of IgGsorb (The Enzyme Center) was then added. If primary antibody was of the IgM class, then IgGsorb with preadsorbed IgG anti-IgM was used. After incubation for an additional 30 min with periodic shaking, the samples were microcentrifuged, and the pellets were washed three times with TEM-buffer containing 0.1% Triton-X. The pellets were then resuspended in protein sample buffer, then boiled for 5 min, and analyzed by SDS-PAGE and autoradiography. For detection of trimericL Cell Binding Assay-- The L cell binding assay was essentially the same as described previously (30, 34). Monolayers of mouse L cells were grown to 90% confluency on 35-mm Corning tissue culture plates in Joklik's minimum essential medium containing 5% fetal calf serum. Media was aspirated from the plates, and the cells were washed once with ice-cold phosphate-buffered saline (PBS) (pH7.4) followed by incubation at 4 °C for 20 min with an overlay of cold PBS. The PBS was then replaced with [35S]methionine-labeled translation reactions diluted 10-fold in PBS. After incubation at 4 °C for 1 h with intermittent rocking, the monolayers were washed five times with cold PBS. The cells were then lysed with 200 µl of lysis buffer (PBS containing 0.5% sodium deoxycholate, 1% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride), and the nuclei were pelleted in a microcentrifuge for 2 min at 5000 × g. The supernatant was mixed with protein sample buffer and analyzed by SDS-PAGE.
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RESULTS |
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Association of Hsp90 with Immature but Not Mature 1--
We
previously reported that Hsp70 is associated with
1 intermediates
and is actively involved in
1 biogenesis (35). To determine if Hsp90
was also part of the chaperone complex during
1 assembly,
full-length S1 transcripts encoding
1 were translated in rabbit
reticulocyte lysates for 12 min then immunoprecipitated with an
anti-Hsp90 antibody. Subsequent SDS-PAGE analysis under dissociating
conditions (Fig. 1A) revealed
that
1 coprecipitated with the anti-Hsp90 antibody but not with a
control antibody (anti-CD8), suggesting that at least some
1 was
associated with Hsp90. To identify the
1 intermediates associated
with Hsp90, similar immunoprecipitates were subjected to SDS-PAGE under
nondissociating conditions. The results (Fig. 1B) show that
both the apparent monomer and higher order forms of
1 (including the
stable hydra form) were associated with Hsp90. Previously we
demonstrated that the apparent monomer observed under these weakly
denaturing conditions actually represents an early posttranslational
form of the
1 trimer (unstable trimer form) that is SDS-sensitive
(36). It is a direct presursor of the stable hydra form, which in turn
is a direct precursor of mature
1 (compact form) (34, 35). The
association of Hsp90 with the two active intermediate forms of
1,
but not with mature
1 (compact form), suggests that Hsp90 may be
playing a dynamic role in
1 trimerization.
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Mapping of Domains on 1 That Associate with Hsp90--
We have
previously shown that the N-terminal coiled-coil domain of
1 can
trimerize independently of ATP and that the triple coiled coil, after
denaturation using guanidine hydrochloride, can spontaneously
reassemble upon subsequent dialysis (36). This suggests that N-terminal
trimerization is independent of chaperone involvement. In contrast, the
C-terminal domain of
1 does not fold in the absence of ATP, nor does
it spontaneously refold upon denaturation/renaturation (reviewed in
Ref. 50). These data are consistent with the observation that Hsp70
only interacts with regions downstream of the
-helical coiled coil (35). It was therefore of interest to determine if Hsp90 has the same
bias in terms of
1 domain association. To this end, various
C-terminal-truncated
1 mutants were probed for their association
with Hsp90 using the same coimmunoprecipitation approach as above. All
the mutants examined have been previously characterized and found to
form N-terminal stable trimers during in vitro translation (35, 36). However, none of these trimers, with the exception of
full-length
1, is functional for host cell binding because of the
lack of C-terminal assembly (42). The results of the coprecipitation
experiment are shown in Fig. 2. Only the
full-length protein and d90 (lacking the C-terminal 90 amino acids)
were efficiently coprecipitated with Hsp90. Some d204 was
coprecipitated, but considerably less than was seen for d90; little or
no coprecipitation was detected with further deletions. These findings
demonstrate that as is the case with Hsp70, Hsp90 association is
through the C-terminal half of
1. The lack of Hsp90 association at
the N-terminal one-third of
1 is again congruent with the notion
that formation of the triple coiled coil is a chaperone-independent
process.
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GA Interferes with C-terminal Trimerization but Not N-terminal
Trimerization of 1--
Our demonstration that Hsp90 was associated
with
1 intermediates suggests that it probably plays a role in the
1 assembly pathway. That this association was confined to the
C-terminal half of
1 further implicates its potential involvement in
the formation of the globular head of
1. To determine if this was in
fact the case, the effect of the Hsp90-specific inhibitor GA on
1
assembly was examined. Accordingly, protein
1 was translated in vitro in the presence of 7 µM GA for 25 min, and the products were then analyzed by SDS-PAGE under
nondissociating conditions. The results (Fig.
3A) show that
1 synthesis
proceeded normally at 7 µM GA (higher concentrations
caused some inhibition of translation); however, the formation of the
mature compact
1 form was clearly blocked (compare lane 1 with lane 2). Results from cell binding assays agreed with
this conclusion (compare lane 3 with lane 4). Importantly, inhibition of mature
1 formation by GA corresponded to
an accumulation of the stable hydra form, a direct precursor of mature
1 (compare lane 1 with lane 2). This suggests
that Hsp90 is not involved in the formation of the N-terminal coiled coil and is required only in the final step of
1 biogenesis, namely, formation of the globular head.
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GA Does Not Affect Hsp90 Association with 1 but Prevents Its
Subsequent Release from
1--
The interference of C-terminal
assembly by GA could be because of the inability of GA-bound Hsp90 to
interact with the C-terminal domain on
1. Alternatively, GA-bound
Hsp90 could still associate with
1 but was unable to be subsequently
released such that assembly of the globular head could ensue. To see if
GA interfered with Hsp90-
1 association,
1 synthesized in
vitro in the presence or absence of GA was subjected to
coprecipitation analysis using the anti-Hsp90 antibody. The results
(Fig. 4) show that essentially the same
amount of
1 coprecipitated with Hsp90 in the two reactions, suggesting that GA did not affect Hsp90 association with
1 and that
it likely interfered with a subsequent
1 maturation step such as
dissociation of Hsp90 from the
1 complex.
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Association of the p23 with 1 Intermediates--
p23 is an
Hsp90-associated protein that is part of the chaperone system involved
in steroid receptor maturation (20, 21, 51). Whether p23 represents a
universal chaperone involved in the folding and assembly of newly
synthesized cytosolic proteins in general remains to be seen. We
therefore wished to determine if p23 was also part of the chaperone
complex during
1 biogenesis. To this end, in vitro
translated
1 was subjected to coprecipitation analysis using an
anti-p23 antibody. The results (Fig.
6A) revealed a clear
association of p23 with
1; however, the amount of
1 coprecipitated was consistently found to be somewhat less than that
using the anti-Hsp90 antibody (compare lanes 2 and
3). Although the reason for this is unclear at present, it
is compatible with the binding of p23 to an existing Hsp90·
1
complex at a late stage of
1 maturation. To see if p23, like Hsp90,
only associated with intermediate
1 forms, immunoprecipitates were
analyzed by SDS-PAGE under nondissociating conditions. Fig.
6B shows that indeed, like Hsp90, p23 associated with both
the apparent monomer (unstable hydra) and the stable hydra form of
1
but not with mature
1.
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GA Releases p23 from the 1 Complex--
Recent studies have
shown that the Hsp90-p23 association is disrupted in the presence of GA
(28). This observation, coupled with our current demonstration that GA
blocks the release of Hsp90 from the
1 complex (Fig. 5B),
led to the interesting question as to the effect of GA on p23-
1
association. Accordingly,
1 translated in the presence or absence of
GA was subjected to coprecipitation analysis using the anti-p23
antibody. Interestingly, GA, which had no effect on Hsp90 association
with
1, inhibited the association of p23 with the
1 complex (Fig.
8A). In view of the reported disruptive effect of GA on Hsp90-p23 association, our data suggest that
the association of p23 with
1 is an indirect one, most likely via Hsp90.
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DISCUSSION |
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Protein 1 is a fiber-with-knob structure located at the 12 vertices of the reovirus icosahedron. The two morphologically distinct
domains serve different functions: the C-terminal globular head
contains a conformation-dependent receptor binding domain, whereas the N-terminal fibrous tail anchors
1 to the virion and serves as a stable extension, which presumably facilitates access of
the globular head to the host cell receptor. Recent evidence indicates
that binding of virion
1 to the cellular receptor (sialic acid)
induces a conformational change in the globular head that progresses to
the fibrous region of the protein and subsequently spreads to other
capsid proteins (52). Thus, despite their pronounced structural and
functional differences, there is communication between the two termini
during the early stages of reovirus infection.
The discovery that 1 is a trimer (31), coupled with the
demonstration that functional trimeric
1 can be generated in an in vitro translation system (42, 53) has made
1 an
interesting model system for the study of protein oligomerization and
folding mechanisms. Generation of
1 involves two independent
trimerization events (34). The first event, which leads to the
formation of a triple coiled coiled at the N terminus, involves
neighboring nascent chains that interact cotranslationally after the
ribosomes associated with these chains have traversed past the
mid-point of the S1 transcript (35). This event has previously been
shown to be ATP- and Hsp70-independent and is believed to occur
spontaneously (36). Indeed, truncated trimers representing the
N-terminal one-third of
1, which had been dissociated using
guanidine hydrochloride, renatured very efficiently upon subsequent
dialysis (36). Furthermore, sucrose gradient analyses of in
vitro translation products have revealed that, whereas full-length
1 intermediate forms are associated with a large molecular weight
complex, no such association is demonstrable with the translation
product representing the N-terminal one-third of
1.2 The present study
further demonstrates the lack of involvement of other chaperones such
as Hsp90 or p23 in
1 N-terminal trimerization. It therefore seems
safe to conclude that N-terminal trimerization of
1 is a spontaneous
event that involves no other participants.
Formation of the C-terminal globular head follows a totally different
strategy. First of all, it is global in nature and therefore necessarily occurs posttranslationally. The global nature of this process is suggested by the following observations. First, deletion of
as few as four amino acids from the C terminus totally abrogates the
cell binding function of 1 (42) as does the single substitution of
certain conserved amino acids at the C-terminal half of
1 (53). In
both cases, the N-terminal half of the protein remains intact (trimeric
and protease-resistant), whereas the C-terminal half is grossly
misfolded (unassembled and protease-sensitive). Second,
1
heterotrimers comprised of two wild-type subunits and a mutant subunit
with deletions or substitutions at the C terminus are invariably
nonfunctional and manifest C-terminal misfolding (34). These
observations have led to the prediction that trimerization of the C
terminus can proceed only when the C terminus of all three subunits are
intact and is accordingly a posttranslational and global event. That
this is in fact the case was subsequently demonstrated by following the
fate of
1 intermediates in the postribosomal fractions (35). Overall
C-terminal trimerization contrasts sharply with N-terminal
trimerization in terms of temporality, stringency, and Hsp70 and ATP
requirements.
In the present study, we provide evidence for the involvement of Hsp90
in 1 C-terminal but not N-terminal assembly. (i) Hsp90 is associated
with
1 intermediates including the stable hydra form with a stably
assembled N terminus and an unassembled C terminus, (ii) Hsp90
association sites are found on the C-terminal half but not the
N-terminal half of
1, and (iii) the Hsp90 inhibitor geldanamycin
blocks C-terminal trimerization but not N-terminal trimerization. The
involvement of Hsp90 in
1 folding and assembly is therefore
reminiscent of that of Hsp70 previously observed (35). Because Hsp70
and Hsp90 interaction sites on
1 overlap and because both chaperones
are involved with a late stage of
1 maturation, it seems reasonable
to suggest that they function cooperatively to generate mature
1.
Although we do not yet have direct evidence that Hsp70 and Hsp90
interact during
1 biogenesis, the fact that they are often found as
a complex in the cytosol suggests that physical association between
these two proteins is likely and is part of their chaperoning function.
In the case of steroid receptors, recent evidence from reconstitution
experiments suggests that a foldosome (comprised of Hsp90, Hsp70, and
an additional component called p60) is first formed that then
associates with (and hence activates) the steroid receptor (19, 20).
Whether a similar foldosome is involved in
1 C-terminal assembly
remains to be seen. In this regard, it would clearly be of interest to first determine if p60 (or other chaperone-associated proteins such as
Hip and the immunophilins) is part of the
1 complex during
1
maturation, and if so, whether it also maps to the same region on
1
as Hsp70 and Hsp90. The involvement of a foldosome in
1 folding and
assembly would have significant implications, because it would unify
concepts pertaining to steroid receptor activation and those pertaining
to chaperone-assisted folding of cytosolic proteins in general.
Another analogy to the steroid receptor activation mechanism is the
association of p23 with 1 intermediates. Recent evidence suggests
that p23 associates with Hsp90 in the glucocorticoid receptor-Hsp90-p60-Hsp70 assembly intermediate, thereby stabilizing a
conformation of Hsp90 that mediates the steroid binding activity of the
glucocorticoid receptor (20). Our present observation that p23 and
Hsp90 association sites map to the same regions on
1 is compatible
with the concept of p23 associating with
1 via Hsp90, although
direct binding of
1 by p23 has not been ruled out. Whether p23 also
plays a stabilizing role in the
1 assembly process remains to be
seen. However, the fact that it is part of the
1 complex and that it
associates only with the C terminus of
1 makes it likely that it is
important for
1 maturation. The additional observation that GA
causes release of p23 from the
1 complex while blocking C-terminal
assembly is compatible with this view. At present we do not yet have
any concrete information on the relative temporal aspects of Hsp90 and
p23 involvement in
1 assembly. However, the observation that GA
blocks the association of p23 but not the association of Hsp90 to
1
intermediates is compatible with the sequential binding of Hsp90 to the
1 complex followed by that of p23 (possibly to Hsp90). Preliminary
data (not shown) from time course studies appear to concur with this notion. These results, if confirmed, would again be reminiscent of the
steroid receptor system, further contributing to the concept of shared
chaperoning mechanisms between biogenesis of cytosolic proteins and
steroid receptor activation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gary Perdew for the 3G3 anti-Hsp90 antibody and Dr. David Toft for the JJ3 anti-p23 antibody.
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FOOTNOTES |
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* This work was supported by the Medical Research Council of Canada (to P. W. K. L.).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.
Recipients of the Alberta Heritage Foundation for Medical Research
Studentship.
§ To whom correspondence should be addressed: Dept. of Microbiology and Infectious Diseases, University of Calgary Health Sciences Centre, Calgary, Alberta, Canada T2N 4N1. Tel: 403-220-7548; Fax: 403-270-8520; E-mail: plee{at}acs.ucalgary.ca.
1
The abbreviations used are: HRI, heme-regulated
inhibitor, also called the heme-regulated eukaryotic initiation
factor-2 (eIF2
) kinase; GA, geldanamycin; PAGE, polyacrylamide
gel electrophoresis; PBS, phosphate-buffered saline.
2 G. Leone and P. W. K. Lee, unpublished data.
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REFERENCES |
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