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(Received for publication, February 1, 1996)
From the Department of Pharmacology, The University of Michigan
Medical School, Ann Arbor, Michigan 48109
ABSTRACT Rabbit reticulocyte lysate contains a
multiprotein system that assembles steroid receptors into a
heterocomplex with hsp90. In the case of the glucocorticoid receptor
(GR), the receptor must be bound to hsp90 to bind steroid, and assembly
of the GR·hsp90 complex restores the hormone binding domain of the
receptor to the steroid binding conformation. Using both direct assay
of heterocomplex assembly by Western blotting and indirect assay of
assembly by steroid binding, it has previously been determined that the
assembly system is both ATP/Mg2+-dependent and
K+-dependent and that hsp70 and an acidic
23-kDa protein (p23) are required to form a functional GR·hsp90
complex. It is also thought that a 60-kDa protein (p60) may be required
for progesterone receptor·hsp90 heterocomplex assembly, but a
complete heterocomplex assembly system has never been reconstituted
from individual components. In this work, we separate the proteins of
rabbit reticulocyte lysate into three fractions by DEAE chromatography
and then reconstitute the GR·hsp90 heterocomplex assembly system in a
manner that requires the presence of each fraction. Fraction A contains
most of the hsp70 and all of the p60 in lysate, and elimination of p60
by immunoadsorption inactivates this fraction, with bioactivity being
restored by the addition of bacterially expressed human p60. The
activity of fraction A is replaced by a combination of highly purified
rabbit hsp70 and lysate from p60-expressing bacteria. Fraction B
contains hsp90, and its activity is replaced by purified rabbit hsp90.
Fraction C contains p23, and its activity is replaced in the recombined
system by highly purified bacterially expressed human p23. A minimal
GR·hsp90 heterocomplex assembly system was reconstituted with
purified rabbit hsp70 and hsp90 and bacterially expressed human p23 and
p60. This reports the first reconstitution of this apparently
ubiquitous protein folding/heterocomplex assembly system.
INTRODUCTION In cytosols prepared from hormone-free mammalian cells, steroid
receptors exist in multiprotein complexes that contain
hsp90,1 an immunophilin (either
FKBP52/hsp56 or CyP-40), a 23-kDa acidic protein, p23, and, often,
substoichiometric amounts of hsp70 (for review see Refs. 1 and 2). This
multiprotein receptor heterocomplex can be assembled under cell-free
conditions by incubating immunoadsorbed, hormone-free receptors
(preincubated with salt to strip them free of associated proteins) with
rabbit reticulocyte lysate (3, 4). This heterocomplex assembly system
is versatile in that reticulocyte lysate has been used to assemble
heterocomplexes containing the oncogenic protein kinases Src or Raf
with hsp90 and p50 (5, 6). The heterocomplex assembly system appears to
be ubiquitous, in that concentrated lysates from other mammalian,
insect, and even plant cells are able to assemble the glucocorticoid
receptor (GR) into a complex with hsp90 (7). The GR must be associated
with hsp90 for it to bind steroid (8), and conversion of the GR to a
steroid binding form constitutes a rapid assay for heterocomplex
assembly (4, 9).
Over the past five years, several conditions required for steroid
receptor·hsp90 heterocomplex assembly by reticulocyte lysate have
been defined. Assembly is an ATP/Mg2+-dependent
process that requires the presence of a monovalent cation, such as
K+ or NH+4 (9, 10). Removal
of hsp70 (11) or its inactivation with antibody (10) inactivates the
assembly process, and the activity is restored by purified hsp70 (11),
proving that hsp70 is required to form complexes with hsp90. p23, a
conserved, widely distributed protein that is neither a heat shock
protein nor an immunophilin (12), binds directly to hsp90 in an
ATP-dependent manner (13) and is required for assembly of a
functional GR·hsp90 heterocomplex (14, 15). It is thought that a
60-kDa protein of reticulocyte lysate is also required for
heterocomplex assembly. This p60 was originally observed in
reconstituted progesterone receptor complexes when ATP was limiting
(10) or at early stages of assembly (16). p60 is a protein that was
identified by Honoré et al. (17) to be up-regulated by
viral transformation, and it is a homolog of the nonessential yeast
heat shock protein, Sti1 (18). It is established that p60, hsp90, and
hsp70 interact with each other (19), and they are thought to act in a
cooperative manner in receptor heterocomplex assembly.
The immune adsorption of hsp90 from reticulocyte lysate and other cell
extracts copurifies several proteins, including hsp70, p60, p23, and
the immunophilins FKBP52/hsp56 (also called p59) and CyP-40 (19, 20, 21).
The immunoadsorbed hsp90 with its coimmunoadsorbed proteins contains
all the components of reticulocyte lysate required to reassociate the
GR with hsp90 and to restore the receptor to its steroid binding state
(22). The immunophilin components of this foldosome
machinery are not required for GR·hsp90 heterocomplex assembly (23),
but a loosely associated component that is easily washed away is
required to form a functional (i.e. steroid binding)
GR·hsp90 complex (22). This weakly bound component has been
identified as p23 (15).
It is thought that the core complex for receptor heterocomplex assembly
may be the hsp90·p60·hsp70 unit and that p60 dissociates after
hsp70 and hsp90 bind to the receptor, with p23 becoming tightly bound
to the receptor heterocomplex at that time. To define the mechanism of
heterocomplex assembly in greater detail, the system must first be
reconstituted from purified components. In this work, we have separated
rabbit reticulocyte lysate into three fractions that contain the four
identified components of the heterocomplex assembly system. When the
fraction containing hsp70 and p60 is reconstituted with two other
fractions containing hsp90 and p23, respectively, the heterocomplex
assembly activity of reticulocyte lysate is reconstituted. Substitution
of the reticulocyte lysate fractions with purified rabbit hsp90 and
hsp70 and bacterially expressed human p23 and p60 yields a system that
assembles a GR·hsp90 complex with steroid binding activity. Although
it is likely that other proteins (e.g. rDnaJ, p48) are
required for optimal heterocomplex assembly in intact cells, this work
identifies a minimal system for receptor heterocomplex assembly under
cell-free conditions.
EXPERIMENTAL PROCEDURES Materials
[6,7-3H]Triamcinolone acetonide (42.8 Ci/mmol) and
125I-conjugated goat anti-mouse and anti-rabbit IgGs were
obtained from DuPont NEN. Untreated rabbit reticulocyte lysate was from
Green Hectares (Oregon, WI). Protein A-Sepharose, Sepharose CL-6B, and
goat anti-mouse and anti-rabbit IgG horseradish peroxidase conjugates
were from Sigma. Phenyl-Sepharose was purchased from Pharmacia Biotech
Inc. The rabbit antiserum against hsp70 and hsp90 (24) was a generous
gift from Dr. Ettore Appella (National Cancer Institute). The BuGR2
monoclonal IgG antibody against the GR and the anti-cyclophilin 40
(C-Terminal Peptide) rabbit antiserum were from Affinity Bioreagents
(Neshanic Station, NJ). The JJ3 monoclonal IgG against p23 (12) and
purified, bacterially expressed human p23 were gifts from Dr. David
Toft (The Mayo Clinic). The DS14F5 monoclonal IgG against p60 (19) was
kindly provided by Dr. David Smith (University of Nebraska Medical
School). The IgM monoclonal antibody against p50 (25) and the UPJ56
rabbit antiserum against FKBP52/hsp56 (26) were generous gifts from
Drs. Gary Perdew (Pennsylvania State University) and Karen Leach (The
Upjohn Co., Kalamazoo, Michigan), respectively. Rabbit antiserum
(anti-YDJ1) against the yeast DnaJ protein (27) also recognizes rabbit
DnaJ (rDnaJ) and was kindly provided by Dr. Avrom Caplan (Mount Sinai
Medical Center).
Methods
Prior to immunoadsorption, the
BuGR antibody was prebound to protein A-Sepharose pellets by incubating
40 µl of a 20% slurry of protein A-Sepharose for 1 h at 4 °C with
40 µl of antibody at a concentration of 100 µg/ml and 150 µl of
TEG buffer (10 mM TES, 50 mM NaCl, 4
mM EDTA, 10% glycerol, pH 7.6), followed by centrifugation
and washing with TEG. Glucocorticoid receptors were immunoadsorbed from
250-300-µl aliquots of L cell cytosol (11) by rotation for 2 h at
4 °C with 8 µl of protein A-Sepharose prebound with BuGR antibody.
Prior to incubation with reticulocyte lysate, immunoadsorbed receptors
were stripped of associated hsp90 by incubating the immunopellet an
additional 2 h at 4 °C with 0.5 M NaCl followed by one
wash with 1 ml TEG and a second wash with 1 ml of HEPES buffer (10
mM HEPES, pH 7.4).
BuGR
immune pellets (8 µl of protein A-Sepharose) containing GR stripped
of hsp90 were incubated with 50 µl of rabbit reticulocyte lysate,
with combinations of purified proteins, or with fractions A-C of
reticulocyte lysate (each at 10 µl) and adjusted to 50 µl with HKD
buffer (10 mM HEPES, 100 mM KCl, 5
mM dithiothreitol, pH 7.35). Dithiothreitol (1 µl) was
added to each incubation to a final concentration of 5 mM,
and 5 µl of an ATP-regenerating system (50 mM ATP, 250
mM creatine phosphate, 20 mM MgOAc and 100
units/ml creatine phosphokinase) were added to all assays to yield a
final assay volume of 56 µl. The assay mixtures were incubated for 20
min at 30 °C with resuspension of the pellets by shaking the tubes
every 5 min. At the end of the incubation, one-fourth of the suspension
was removed for assay of steroid binding as described previously (9),
and the remainder was used for Western blotting of receptor and
associated proteins. The portion of the immunopellet used for steroid
binding assay was washed one time with 1 ml of iced TEGM (TEG buffer
plus 20 mM sodium molybdate), whereas the portion used for
Western blotting was washed four times with 1 ml of TEGM. As noted
previously (9), 100 µl of L cell cytosol contains ~60,000 cpm of
[3H]triamcinolone acetonide binding capacity, and with 1
µg of BuGR/100 µl of L cell cytosol (the binding conditions
employed here), we immunoadsorb about 50% of the glucocorticoid
receptor. Thus, ~30,000 cpm represents 100% of receptors reactivated
to the steroid binding form.
For
assay of GR and associated proteins, immune pellets were boiled in SDS
sample buffer with 10% Rabbit
reticulocyte lysate (25 ml) was adsorbed to a 2.5 × 20-cm column of
DE52 equilibrated with HE buffer (10 mM HEPES, 1
mM EDTA, pH 7.35), the column was washed with 150 ml of HE
buffer with 20 mM sodium molybdate followed by 150 ml of HE
buffer alone, and the proteins were eluted with a 400-ml gradient of
0-0.5 M KCl. hsp90, hsp70, p60, FKBP52/hsp56, CyP-40, and
p23 were detected by resolving an aliquot of each fraction by SDS-PAGE
and Western blotting with appropriate antibodies. Fractions were
combined in three pools designated A-C (see Fig. 1). Pooled fractions
were dialyzed against HKD buffer, concentrated to 1 ml (1/25 the
original volume of lysate), and flash-frozen in small aliquots.
ATP-agarose-treated lysate, which does not contain hsp70, was made as
described previously (11).
The bacterial expression of human p23
and its purification have been described (14). Briefly, p23 is soluble
in bacterial lysates, and its abundance and high affinity for
DEAE-cellulose allowed purification to 90% purity by chromatography on
DEAE-cellulose. The protein was concentrated by precipitation with
ammonium sulfate at 80% of saturation. It was dissolved and dialyzed
into 10 mM Tris, 100 mM KCl, and 10% glycerol,
pH 7.4 and stored at hsp70 was purified from the DE52 fraction pool A of rabbit reticulocyte
lysate by chromatography on ATP-agarose and elution with ATP followed
by ammonium sulfate precipitation exactly as described previously (11).
hsp90 was purified from the DE52 fraction pool B of either rabbit
reticulocyte lysate or rabbit brain cytosol by chromatography on
hydroxylapatite followed by chromatography over ATP-agarose exactly as
described in Hutchison et al. (11). After removal of hsp70
by ATP-agarose, the hsp90-containing drop-through fractions were
pooled, contracted to 1.0 ml, dialyzed against HKD buffer, flash
frozen, and stored at For bacterial lysates containing p60,
cDNA for the 60-kDa human protein (IEF SSP 3521) cloned by
Honoré et al. (17), which is the homolog of the rabbit
p60 (19), was subcloned into a pET 23C vector (Novagen) using the
EcoRI and NotI
sites.2 This construct was used to
transform (Escherichia coli strain BL21 (DE3), which harbors
an integrated T7 polymerase gene. Control E. coli and
bacteria expressing p60 (kindly provided by Dr. David Smith) were grown
to an A600 of 0.6, induced with
isopropyl-1-thio- RESULTS The elution of hsp90 and
its associated proteins from a DE52 column is shown in Fig.
1. Fractions were combined into three pools designated
A, B, and C, as indicated by the brackets shown under the
Western blots of the individual proteins. The distribution of proteins
in DE52 fraction pools A-C are shown in Fig.
2A, and the ability of each pool and
combination of pools to assemble GR·hsp90 heterocomplexes is shown in
Fig. 2B. When the stripped GR (lane 1) was
incubated with DE52 pool A, B, or C (lanes 3, 4,
and 5, respectively), there was no heterocomplex assembly,
and very little assembly was seen with combinations of two fractions
(lanes 6-8). However, when the stripped GR immune pellet
was incubated with the combination of DE52 pools A, B, and C
(lane 9), there was substantial GR·hsp90 heterocomplex
assembly, comparable with the unfractionated lysate control in
lane 2. It should be mentioned that immunoblots of larger
volumes of the DE52 pools than that analyzed in Fig. 2A show
that pool B always contains some hsp70 and trace amounts of p23,
although as shown in Fig. 2A, the great majority of each of
these proteins segregates to pools A and C, respectively. Also, DE52
pool C contains small amounts of hsp90.
To determine the components of DE52
pool A required for reconstitution of GR·hsp90 heterocomplex
assembly, we first asked whether the activity of pool A could be
replaced by hsp70. As shown in Fig. 3A,
removal of hsp70 by passing reticulocyte lysate through ATP-agarose
inactivates heterocomplex assembly activity (cf. lane
3 with lane 2). The addition of the
ATP-agarose-retained material (lane 5) or purified rabbit
hsp70 (lane 4) restores the heterocomplex assembly activity.
We have demonstrated previously that the hsp70 is purified to near
homogeneity and that neither the purified hsp70 nor the
ATP-agarose-retained fraction of lysate have any heterocomplex assembly
activity when present alone (11). In the experiment of Fig.
3B, the addition of purified hsp70 to the the combination of
DE52 pools B and C (lane 9) produced a small increment in
activity over that seen with the combination of B and C alone
(lane 8), whereas addition of DE52 pool A to the combination
of B and C (lane 6) yielded nearly the same heterocomplex
assembly activity as unfractionated lysate (lane 2). Because
the addition of larger amounts of purified hsp70 to the combination of
B and C does not yield any greater activation than that seen in
lane 9 (data not shown), it seems likely that another
component (or components) of DE52 pool A is required for heterocomplex
assembly.
A likely candidate for this additional factor is p60, and we wanted to
determine the effect of p60 depletion on the activity of pool A. To
facilitate depletion, selected DE52 fractions were combined such that
fractions from the half of pool A containing the majority of p60 were
in one subfraction designated A1, and selected fractions
from the other half of pool A containing a minority of p60 were in a
subfraction designated A2. Half of A1 and
A2 was extracted with nonimmune antibody, and the other
half of A2 was extracted with protein A-Sepharose prebound
with F5 antibody to extract the remaining p60. The distributions of
selected proteins in the subfractions of pool A are shown in Fig.
4A, where it can be seen that the amount of
p60 in A2 is low relative to A1 and p60 is
eliminated from A2 by immunoextraction with F5 antibody. As
shown in Fig. 4B, the combination of subfraction
A1 with B and C (lane 9) yields the same GR
heterocomplex assembly as unfractionated reticulocyte lysate
(lane 2), whereas subfraction A2 is less active
(lane 10) and F5-extracted A2 (lane
11) has only a low activity comparable with that of hsp70 alone
added to B and C (Fig. 3, lane 9).
The experiment of Fig. 5 was performed to determine if
bacterially expressed human p60 would restore heterocomplex assembly
activity to the p60-depleted DE52 A2 subfraction of
reticulocyte lysate. Fig. 5A shows the presence of p60 by
Coomassie Blue staining (lane 2) and Western blotting
(lane 4) in lysate of E. coli expressing p60
versus control bacterial lysate (lanes 1 and 3).
In Fig. 5B, stripped receptors (lane 1) were
incubated with various fractions of reticulocyte lysate and bacterial
lysates. Neither the A2 subfraction alone (lane
3) nor the combined DE52 pools B and C (lane 4) had
heterocomplex assembly activity, but the combination of A2,
B, and C (lane 5) resulted in substantial activity. The
combination of B and C with F5-extracted (p60-depleted) A2
yielded only a small amount of heterocomplex assembly (lane
6), and the addition of control bacterial lysate to the mixture
was without effect (lane 7). However, addition of extract
from p60-expressing bacteria to the mixture yielded a
concentration-dependent increase in heterocomplex assembly
(lanes 8-10).
Fig. 6 shows that the receptor activating activity of
the DE52 pool A of reticulocyte lysate can be replaced by the
combination of highly purified rabbit hsp70 and lysate from
p60-expressing E. coli. It can be seen that purified hsp70
alone (lane 6), bacterial lysate containing p60 alone
(lane 7), and the combination of hsp70 and p60 (lane
8) were inactive at heterocomplex assembly. Also, the addition of
either p60 alone (lane 9) or hsp70 alone (lane
10) to the combination of DE52 pools B and C was without effect.
However, the addition of both purified rabbit hsp70 and bacterial
lysate containing human p60 to the combined DE52 pools B and C
(lane 11) yielded steroid binding activity that was close to
that achieved with the combination of DE52 pools A, B, and C
(lane 5).
In the
experiment shown in Fig. 7, DE52 pool B was substituted
with purified rabbit hsp90. It can be seen in Fig. 7B that
purified hsp90 does not bind to the GR or activate steroid binding
activity when incubated alone with stripped receptors (lane
6). However, in the presence of DE52 pools A and C, which together
yield only a small amount of heterocomplex reconstitution (lane
4), the addition of purified hsp90 (lane 7) yields
GR·hsp90 complex formation and steroid binding activity comparable
with that achieved with the combination of DE52 pools A, B, and C
(lane 5). The addition of purified hsp90 to pool A alone
does not yield steroid binding activity (lane 9).
Fig.
8 shows that the activity of DE52 pool C can be replaced
with purified p23. In preliminary experiments (data not shown) we found
that rabbit p23 purified from the DE52 pool C of reticulocyte lysate
could substitute for the activity of pool C in the recombined system.
As shown in Fig. 8A, purified human p23 (shown in Fig.
8B, lane 1), which does not itself activate
steroid binding activity (lane 6) produces a
concentration-dependent activation when added to the
combination of rabbit reticulocyte lysate DE52 pools A and B
(lane 7-10). Fig. 8C shows that the incubation
of GR with purified p23 and lysate pools A and B (lane 7)
yields more GR·hsp90 heterocomplex (see Western blot) than
pools A and B without p23 (lane 4), with both steroid
binding activity and heterocomplex formation being equivalent to that
achieved with the combination of DE52 pools A, B, and C (lane
5).
To
optimize the concentrations of the various components of the assembly
system, concentration curves like that shown for p23 in Fig.
8A were performed for p60 (Fig.
9A), hsp70 (Fig. 9B), and hsp90
(Fig. 9C). The heterocomplex assembly system was then
reconstituted in the experiment of Fig. 10, utilizing
the optimal concentrations of components determined in Figs.
8A and 9. As shown in lanes 3-5 of Fig. 10, GR
incubated with p60 and p70 (lane 3) or with purified hsp90
(lane 4) or with p23 (lane 5) are not converted
to the steroid binding conformation. Incubation of receptors with p60,
hsp70, and hsp90 (lane 7) or with p23 and hsp90 (lane
9) results in little GR·hsp90 heterocomplex formation and
steroid binding activity. But the combination of all four components
(lane 9) results in nearly the same level of heterocomplex
formation and steroid binding activity as was obtained with
unfractionated reticulocyte lysate (lane 2).
DISCUSSION The components of the rabbit reticulocyte receptor·hsp90
heterocomplex assembly system were identified by analyzing the proteins
that were bound to the chicken progesterone receptor (PR) at early and
late stages of assembly of the PR·hsp90 heterocomplex (10, 12, 14,
19). Although there is evidence that hsp70, p60, and p23 are each
required for PR·hsp90 heterocomplex assembly (10, 14, 19), the
proteins have not previously been reconstituted into a receptor·hsp90
heterocomplex assembly system.
Herein, we have divided rabbit reticulocyte lysate into three DE52
fraction pools (Fig. 1), each of which must be present in the
incubation mixture for substantial assembly of GR·hsp90
heterocomplexes and reactivation of steroid binding activity (Fig. 2).
The ability of DE52 fraction pool A to activate the steroid binding
activity of the mouse GR was replaced with purified rabbit hsp70 and
bacterial lysate containing human p60 (Figs. 3, 4, 5, 6), and pools B and C
were replaced with purified rabbit hsp90 and purified human p23,
respectively (Figs. 7 and 8). When these four preparations are
combined, the mixture assembles GR·hsp90 heterocomplexes and restores
the receptors to the steroid binding conformation (Fig. 10).
Reconstitution of the system with these p60, hsp70, hsp90, and p23
preparations does not establish that these are the only proteins
required for receptor·hsp90 heterocomplex assembly. The Smith
laboratory has shown that a 48-kDa protein of reticulocyte lysate is
recovered with PR·hsp90 complexes at early times of assembly (16) or
when assembly is blocked at an intermediate stage by geldanamycin (28),
an hsp90-binding benzoquinone ansamycin (29). This 48-kDa protein
appears to be the same as Hip, the hsc70-interacting protein identified
by Hohfeld et al. (30), and it could be present as a
contaminant in the purified rabbit hsp70 and/or hsp90. Whether Hip is
required for GR·hsp90 heterocomplex assembly or whether it
facilitates assembly has not yet been determined.
It is known that mammalian cells contain homologs of the bacterial DnaJ
(31). The DnaJ homolog Ydj1 has been found in GR·hsp90
heterocomplexes from yeast, and genetic experiments show that Ydj1 is
required for the GR to be hormone-responsive in yeast (32). DnaJ
homologs could also be present as contaminants in our purified rabbit
hsp70 and/or hsp90 preparations and might facilitate assembly.
Two FK506-binding immunophilins, FKBP52 and p54, as well as a
cyclosporin A-binding immunophilin, CyP-40, are present in
receptor·hsp90 heterocomplexes reconstituted with reticulocyte lysate
(33, 34, 35). We established by immunoblotting (data not shown) that the
purified preparations of hsp70, hsp90, and p23 used in our
reconstitution of the GR·hsp90 heterocomplex assembly system do not
contain any of these mammalian immunophilins. These immunophilins all
have peptidylprolyl isomerase activity (36), but that activity does not
seem to be required for GR·hsp90 heterocomplex assembly. Reticulocyte
lysate also assembles the oncogenic protein kinases Src and Raf into
heterocomplexes that contain hsp90 and a 50-kDa protein, p50 (5, 6). We
have also demonstrated by immunoblotting (data not shown) that p50 is
not present in the purified proteins we have used for GR·hsp90
heterocomplex reconstitution. Thus, like the immunophilins, it is not
required for heterocomplex assembly.
This work presents the first demonstration of p60 activity in a
biochemical assay and the first reconstitution of the GR·hsp90
heterocomplex assembly system. The availability of a reconstituted
system should greatly facilitate study of the mechanism of the assembly
process.
FOOTNOTES Acknowledgments We thank Ettore Appella, Avrom Caplan, Karen
Leach, Gary Perdew, David Smith, and David Toft for providing
antibodies.
REFERENCES
Volume 271, Number 22,
Issue of May 31, 1996
pp. 12833-12839
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
§,,
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES
-mercaptoethanol, and proteins were resolved
on 7% SDS-polyacrylamide gels (12% for resolving p23) as described
previously (22). Proteins were then transferred to Immobilon-P
membranes and probed with 2 µg/ml BuGR monoclonal antibody for the
glucocorticoid receptor, 0.05% rabbit antiserum for hsp70 and hsp90,
0.1% F5 anti-p60 mouse ascites for p60, 0.1% UPJ56 rabbit antiserum
for FKBP52/hsp56, 0.1% anti-CyP-40 antiserum for CyP-40, 0.1%
anti-YDJ1 antiserum for rDnaJ, and 0.1% JJ3 mouse ascites for p23. The
immunoblots were then incubated a second time with the appropriate
125I-labeled or horseradish peroxidase-conjugated
counterantibody to visualize the immunoreactive bands.
Fig. 1.
Preparation of reticulocyte lysate fractions
used for reconstitution of heterocomplex assembly. Rabbit
reticulocyte lysate (25 ml) was chromatographed on a column of DE52 as
described under ``Methods'' (solid line, absorbance at 280
nm; dotted line, KCl gradient). Aliquots (20 µl) of every
other fraction were resolved by SDS-PAGE and immunoblotted for the
proteins indicated below the graph. Fractions were pooled
(A-C) as indicated by the brackets under the immunoblots.
Fraction pools A, B, and C were dialyzed against HKD buffer,
concentrated to 1 ml, and stored at 
70 °C.
70°. Rabbit p23 was purified by adding solid
ammonium sulfate to fraction pool C (see Fig. 1) from DE52
chromotography of rabbit reticulocyte lysate to achieve a final
concentration of 1.5 M. After centrifugation, the
p23-containing supernatant was applied to a small column containing 5
ml of phenyl-Sepharose, which was washed with 50 ml of 1.5
M ammonium sulfate in 10 mM phosphate buffer,
pH 7.4. The column was then eluted with 150 ml of a descending gradient
of 1.5-0 M ammonium sulfate in phosphate buffer. Fractions
containing p23 were identified by SDS-PAGE and Western blotting with
JJ3 antibody. p23-containing fractions were pooled, contracted to 1 ml,
dialyzed against HKD buffer, flash frozen, and stored at
70 °C.
70 °C.
-D-galactopyranoside for 3 h at
25 °C, and harvested. Bacterial lysates were prepared by sonication
in phosphate-buffered saline, and aliquots were flash frozen and stored
at 70 °C.
Fig. 2.
DE52-resolved fraction pools A, B, and C are
all required for reconstitution of GR·hsp90 complexes and generation
of steroid binding activity. GR was immunoadsorbed to protein
A-Sepharose from replicate aliquots of L cell cytosol, and the
immunopellets were stripped of hsp90 with salt. Immune pellets were
then incubated with 50 µl of normal unfractionated reticulocyte
lysate or with 10 µl of DE52 fraction pools (A-C) of reticulocyte
lysate in the presence of 100 mM KCl and an ATP-generating
system. Receptor and hsp90 were assayed in each sample by SDS-PAGE and
Western blotting, and a portion of the immunopellet was incubated with
50 nM [3H]triamcinolone acetonide to
determine steroid binding activity (bar graph).
A, Western blot of selected proteins in 2-µl aliquots of
DE52 fraction pools A, B, and C. B, GR, hsp90, and steroid
binding activity for samples of immunoadsorbed, stripped receptor
incubated with the same DE52 fraction pools shown in A.
Conditions are: lane 1, immune pellet with stripped GR;
lane 2, stripped GR incubated with unfractionated lysate;
lane 3, stripped GR incubated with DE52 fraction pool A;
lane 4, GR incubated with pool B; lane 5, GR
incubated with pool C; lane 6, GR plus A and B; lane
7, GR plus A and C; lane 8, GR plus B and C; lane
9, GR plus A, B, and C.
Fig. 3.
Purified hsp70 is required for heterocomplex
assembly, but it alone does not replace DE52 fraction pool A of
reticulocyte lysate for reconstitution of receptor steroid-binding
activity. A, purified hsp70 promotes reconstitution of
steroid binding activity in ATP-agarose-extracted (i.e.
hsp70-depleted) lysate. Reticulocyte lysate was depleted of hsp70 by
extraction of hsp70 onto a matrix of ATP-agarose exactly as described
by Hutchison et al. (11). Immunoadsorbed, stripped receptors
were incubated with mock-extracted lysate, ATP-agarose extracted
lysate, or ATP-agarose-extracted lysate plus either highly purified
hsp70 or the ATP-agarose-retained material that was eluted from the
column with ATP. Samples were then assayed for steroid binding
activity. Conditions are: lane 1, stripped receptors;
lane 2, stripped receptors plus mock-depleted lysate;
lane 3, stripped receptors plus ATP-agarose-extracted
lysate; lane 4, stripped receptors plus
ATP-agarose-extracted lysate and 10 µg of purified hsp70; lane
5, stripped receptors plus ATP-agarose-extracted lysate and the
ATP-agarose-retained fraction of lysate. B, purified hsp70
alone does not substitute for DE52 fraction pool A. Immunoadsorbed,
stripped receptors were incubated with whole (i.e.
unfractionated) lysate or DE52 fraction pools of lysate with or without
10 µg of purified hsp70. Samples were then assayed for steroid
binding activity. Lane 1, stripped receptors; lane
2, stripped receptors plus unfractionated lysate; lane
3, stripped receptors plus DE52 fraction pool A; lane
4, stripped receptors plus B; lane 5, stripped
receptors plus C; lane 6, stripped receptors plus A, B, and
C; lane 7, stripped receptors plus purified hsp70 alone;
lane 8, stripped receptors plus DE52 fraction pools B and C;
lane 9, stripped receptors plus purified hsp70 and DE52
fraction pools B and C.
Fig. 4.
Immunoadsorption of p60 from DE52 pool A of
reticulocyte lysate inactivates reconstitution of receptor steroid
binding activity. Stripped GR immunopellets were incubated in the
presence of 100 mM KCl and an ATP-generating system with
normal unfractionated reticulocyte lysate or with various combinations
of reticulocyte lysate DE52 pools B and C and subfractions of the DE52
pool A that had been extracted twice with either nonimmune ascites
(A1 and A2) or with an F5 (i.e.
anti-p60-bound) immunopellet (A2-extr) to extract p60.
Samples were then assayed for steroid binding activity. A,
Western blot of selected proteins in DE52 subfractions A1,
A2, and p60-depleted A2 (A2-extr).
B, ability of DE52 fraction pools to assemble GR
heterocomplexes. lane 1, stripped GR; lane 2, GR
incubated with unfractionated lysate; lane 3, GR plus
unfractionated pool A; lane 4, GR plus B and C; lane
5, GR plus A, B and C; lane 6, GR plus subfraction
A1; lane 7, GR plus A2; lane
8, GR plus A2 after extraction with F5 antibody to
deplete p60; lane 9, GR plus A1, B and C;
lane 10, GR plus A2 together with B and C;
lane 11, GR plus p60-depleted A2 together with B
and C.
Fig. 5.
Bacterially expressed human p60 replaces
rabbit p60 for reconstitution of receptor steroid binding
activity. Stripped GR immunopellets were incubated in the presence
of 100 mM KC1 and an ATP-generating system with normal
unfractionated reticulocyte lysate or various combinations of DE52
fraction pool A2, pools B and C together, p60-depleted
A2, and increasing amounts of lysate from bacteria
expressing p60, or control bacterial lysate. Samples were then assayed
for steroid binding activity. A, expression of p60 in
E. coli. Lanes 1 and 2, Coomassie
Blue-stained SDS-PAGE of lysate (2 µl) from control bacteria
(lane 1) and bacteria expressing p60 (lane 2);
lanes 3 and 4, Western blot of control bacterial
lysate (lane 3) and lysate from bacteria expressing p60
(lane 4). B, GR heterocomplex reconstitution
assay of bacterially expressed p60. Lane 1, stripped GR;
lane 2, GR incubated with unfractionated lysate; lane
3, GR plus A2; lane 4, GR plus B and C; lane
5, GR plus A2, B and C; lane 6, GR plus B,
C and p60-depleted A2; lane 7, GR plus B, C, and
p60-depleted A2 plus 1 µl of control bacterial lysate;
lanes 8-10, GR plus B, C, and p60-depleted A2
plus 0.01 µl (lane 8), 0.1 µl (lane 9) or 1.0
µl (lane 10) bacterial lysate containing p60.
Fig. 6.
DE52 pool A of lysate can be replaced by
highly purified rabbit hsp70 and lysate from bacteria expressing human
p60. Stripped GR immunopellets were incubated with the indicated
additions and steroid binding was assayed. Conditions are: lane
1, stripped GR; lane 2, GR incubated with
unfractionated reticulocyte lysate; lane 3, GR plus A;
lane 4, GR plus B and C; lane 5, GR plus A, B,
and C together; lane 6, GR plus 10 µg purified hsp70;
lane 7, GR plus 1 µl of lysate from bacteria expressing
p60; lane 8, GR plus hsp70 and bacterial lysate containing
p60; lane 9, GR plus B and C and bacterial lysate containing
p60; lane 10, GR plus B and C and purified hsp70; lane
11, GR plus B and C, purified hsp70, and bacterial lysate
containing p60.
Fig. 7.
Purified rabbit hsp90 replaces DE52 pool B in
the reconstituted heterocomplex assembly system. A, purified
rabbit brain hsp90. Lanes 1 and 2, Coomasie Blue-stained
SDS-PAGE of DE52 fraction B and purified rabbit hsp90, respectively;
lanes 3 and 4, the same samples immunobloted with antiserum
against hsp90 and hsp70. B, GR·hsp90 heterocomplex
assembly with purified rabbit hsp90 added to pools A and C of lysate.
Stripped GR immunopellets were incubated with the indicated additions
and GR, hsp90, and steroid binding activity were assayed. Conditions
are: lane 1, stripped GR; lane 2, GR incubated
with unfractionated lysate; lane 3, GR plus B; lane
4, GR plus A and C; lane 5, GR plus A, B, and C
together; lane 6, GR plus 12 µg of purified hsp90;
lane 7, GR plus A, C, and 12 µg of purified hsp90;
lane 8, GR plus A; lane 9, GR plus A and 12 µg
of purified hsp90.
Fig. 8.
Purified human p23 replaces DE52 pool C in
the reconstituted heterocomplex assembly system. A,
activation of steroid binding activity with purified, bacterially
expressed human p23 added to DE52 pools A and B of lysate. Stripped GR
immunopellets were incubated with the indicated additions, and steroid
binding was assayed. Conditions are: lane 1, stripped GR;
lane 2, GR incubated with unfractionated reticulocyte
lysate; lane 3, GR plus A and B; lane 4, GR plus
C; lane 5, GR plus A, B and C together; lane 6,
GR plus 6 µg of purified p23; lanes 7-10; GR plus A and B
and 0.2 µg (lane 7), 0.6 µg (lane 8), 2 µg
(lane 9), or 6 µg (lane 10) of purified human
p23. B, purified human p23. Lanes 1 and
2, Coomassie blue-stained SDS-PAGE of purified human p23 and
the DE52 pool C of rabbit reticulocyte lysate, respectively.
Lanes 3 and 4, immunoblots of the same fractions
with the JJ3 antibody against p23. C, GR·hsp90
heterocomplex assembly with purified human p23 added to DE52 pools A
and B of lysate. Stripped GR immunopellets were incubated with the
indicated additions and GR, hsp90, and steroid binding activity were
assayed. Conditions are: lane 1, stripped GR; lane
2, GR incubated with unfractionated reticulocyte lysate;
lane 3, GR plus C; lane 4, GR plus A and B;
lane 5, GR plus A, B, and C together; lane 6, GR
plus 6 µg of purified human p23; lane 7, GR plus A, B, and
6 µg of purified human p23.
Fig. 9.
Concentration dependance of p60, hps70, and
hsp90 in the reconstituted heterocomplex assembly system. A,
activation of steroid binding activity with bacterially expressed p60
added to purified hsp70 and DE52 fractions B and C. Stripped GR
immunopellets were incubated with the indicated additions, and steroid
binding was assayed. Conditions: lane 1, stripped GR;
lane 2, GR plus unfractionated reticulocyte lysate;
lane 3, GR plus A; lane 4, GR plus B and C;
lane 5, GR plus A, B, and C; lane 6, GR plus 1.5
µl of bacterial lysate containing p60 and 20 µg of hsp70;
lanes 7-10; GR plus B, C, 20 µg of hsp70 and 1.5 µl
(lane 7), 3.0 µl (lane 8), 6.0 µl (lane
9), or 20 µl (lane 10) of bacterial lysate containing
p60. B, activation of steroid binding activity with purified
rabbit hsp70 added to p60 and DE52 fractions B and C. Conditions:
lane 1, stripped GR; lane 2, GR plus
unfractionated reticulocyte lysate; lane 3, GR plus A;
lane 4, GR plus B and C: lane 5, GR plus A, B,
and C; lane 6, GR plus 3 µl of bacterial lysate with p60
and 20 µg of hsp70; lanes 7-10; GR plus B, C, 3 µl of
bacterial lysate with p60, and 10 µg (lane 7), 20 µg
(lane 8), 30 µg (lane 9), or 40 µg
(lane 10) of purified hsp70. C, activation of
steroid binding activity with purified hsp90 added to DE52 fractions A
and C. Conditions: lane 1, stripped GR; lane 2,
GR plus unfractionated lysate; lane 3, GR plus B; lane
4, GR plus A and C; lane 5, GR plus A, B, and C;
lane 6, GR plus 12 µg of hsp90; lanes 7-10; GR
plus A, C, and 1.2 µg (lane 7), 6 µg (lane
8), 12 µg (lane 9), or 24 µg (lane 10)
of purified hsp90.
Fig. 10.
Reconstitution of the GR heterocomplex with
bacterially expressed p60, purified human p23, and purified rabbit
hsp70 and hsp90. Stripped GR immunopellets were incubated with the
indicated additions, and GR, hsp90, and steroid binding activity were
assayed. Conditions are: lane 1, stripped GR; lane
2, GR incubated with unfractionated lysate; lane 3, GR
plus 3 µl of bacterial lysate with p60 and 20 µg of purified hsp70;
lane 4, GR plus 12 µg of purified hsp90; lane
5, GR plus 6 µg of purified human p23; lanes 6 and
7, nonimmune (lane 6) and immune (lane
7) GR plus p60, hsp70, and hsp90; lanes 8 and
9; nonimmune (lane 8) and immune (lane
9) GR plus p23 and hsp90; lanes 10 and 11,
nonimmune (lane 10) and immune (lane 11), GR plus
p60, hsp70, p23, and hsp90.
These authors contributed equally to this work and are considered
equivalent as first author.
§
Trainee under Pharmacological Science Training Program Training
Grant GM07767 from the National Institutes of Health.
¶
Trainee of the Cancer Biology Training Program Grant
T32CA09676 from the National Cancer Institute.
To whom correspondence should be addressed: Dept. of
Pharmacology, 1301 Medical Science Research Bldg. III, University of
Michigan Medical School, Ann Arbor, MI 48109-0632. Tel.: 313-764-5414;
Fax: 313-763-4450.
1
The abbreviations used are: hsp, heat shock
protein; GR, glucocorticoid receptor; PR, progesterone receptor; FKBP,
FK506 binding protein; CyP-40, the 40 kDa cyclosporin
A-binding protein; TES,
2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethane
sulfonic acid; PAGE, polyacrylamide gel electrophoresis.
2
W. P. Sullivan and D. O. Toft, unpublished
observations.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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B. K. Meyer, M. G. Pray-Grant, J. P. Vanden Heuvel, and G. H. Perdew Hepatitis B Virus X-Associated Protein 2 Is a Subunit of the Unliganded Aryl Hydrocarbon Receptor Core Complex and Exhibits Transcriptional Enhancer Activity Mol. Cell. Biol., February 1, 1998; 18(2): 978 - 988. [Abstract] [Full Text]
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K. D. Dittmar, D. R. Demady, L. F. Stancato, P. Krishna, and W. B. Pratt Folding of the Glucocorticoid Receptor by the Heat Shock Protein (hsp) 90-based Chaperone Machinery. THE ROLE OF p23 IS TO STABILIZE RECEPTOR·hsp90 HETEROCOMPLEXES FORMED BY hsp90·p60·hsp70 J. Biol. Chem., August 22, 1997; 272(34): 21213 - 21220. [Abstract] [Full Text] [PDF]
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N. Xiao and D. B. DeFranco Overexpression of Unliganded Steroid Receptors Activates Endogenous Heat Shock Factor Mol. Endocrinol., August 1, 1997; 11(9): 1365 - 1374. [Abstract] [Full Text]
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B. Segnitz and U. Gehring The Function of Steroid Hormone Receptors Is Inhibited by the hsp90-specific Compound Geldanamycin J. Biol. Chem., July 25, 1997; 272(30): 18694 - 18701. [Abstract] [Full Text] [PDF]
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A. M. Silverstein, M. D. Galigniana, M.-S. Chen, J. K. Owens-Grillo, M. Chinkers, and W. B. Pratt Protein Phosphatase 5 Is a Major Component of Glucocorticoid Receptor·hsp90 Complexes with Properties of an FK506-binding Immunophilin J. Biol. Chem., June 27, 1997; 272(26): 16224 - 16230. [Abstract] [Full Text] [PDF]
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 W. B. Pratt and D. O. Toft Steroid Receptor Interactions with Heat Shock Protein and Immunophilin Chaperones Endocr. Rev., June 1, 1997; 18(3): 306 - 360. [Abstract] [Full Text]
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K. D. Dittmar and W. B. Pratt Folding of the Glucocorticoid Receptor by the Reconstituted hsp90-based Chaperone Machinery. THE INITIAL hsp90·p60·hsp70-DEPENDENT STEP IS SUFFICIENT FOR CREATING THE STEROID BINDING CONFORMATION J. Biol. Chem., May 16, 1997; 272(20): 13047 - 13054. [Abstract] [Full Text] [PDF]
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L. A. Carver and C. A. Bradfield Ligand-dependent Interaction of the Aryl Hydrocarbon Receptor with a Novel Immunophilin Homolog in Vivo J. Biol. Chem., April 25, 1997; 272(17): 11452 - 11456. [Abstract] [Full Text] [PDF]
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L. F. Stancato, A. M. Silverstein, J. K. Owens-Grillo, Y.-H. Chow, R. Jove, and W. B. Pratt The hsp90-binding Antibiotic Geldanamycin Decreases Raf Levels and Epidermal Growth Factor Signaling without Disrupting Formation of Signaling Complexes or Reducing the Specific Enzymatic Activity of Raf Kinase J. Biol. Chem., February 14, 1997; 272(7): 4013 - 4020. [Abstract] [Full Text] [PDF]
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M.-S. Chen, A. M. Silverstein, W. B. Pratt, and M. Chinkers The Tetratricopeptide Repeat Domain of Protein Phosphatase 5Mediates Binding to Glucocorticoid Receptor Heterocomplexes and Acts as a Dominant Negative Mutant J. Biol. Chem., December 13, 1996; 271(50): 32315 - 32320. [Abstract] [Full Text] [PDF]
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Y. Morishima, P. J. M. Murphy, D.-P. Li, E. R. Sanchez, and W. B. Pratt Stepwise Assembly of a Glucocorticoid Receptor{middle dot}hsp90 Heterocomplex Resolves Two Sequential ATP-dependent Events Involving First hsp70 and Then hsp90 in Opening of the Steroid Binding Pocket J. Biol. Chem., June 9, 2000; 275(24): 18054 - 18060. [Abstract] [Full Text] [PDF]
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T. Rajapandi, L. E. Greene, and E. Eisenberg The Molecular Chaperones Hsp90 and Hsc70 Are Both Necessary and Sufficient to Activate Hormone Binding by Glucocorticoid Receptor J. Biol. Chem., July 14, 2000; 275(29): 22597 - 22604. [Abstract] [Full Text] [PDF]
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P. J. M. Murphy, K. C. Kanelakis, M. D. Galigniana, Y. Morishima, and W. B. Pratt Stoichiometry, Abundance, and Functional Significance of the hsp90/hsp70-based Multiprotein Chaperone Machinery in Reticulocyte Lysate J. Biol. Chem., August 3, 2001; 276(32): 30092 - 30098. [Abstract] [Full Text] [PDF]
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