Plant Physiol. (1998) 118: 1395-1401
High-Molecular-Weight FK506-Binding Proteins Are Components of
Heat-Shock Protein 90 Heterocomplexes in Wheat Germ Lysate1
Ramachandra K. Reddy,
Isaac Kurek,
Adam M. Silverstein,
Michael Chinkers,
Adina Breiman, and
Priti Krishna*
Department of Plant Sciences, University of Western Ontario,
London, Ontario, Canada N6A 5B7 (R.K.R., P.K.); Department of Botany,
The George S. Wise Faculty of Life Sciences, Tel Aviv University, Tel
Aviv 69978, Israel (I.K., A.B.); Department of Pharmacology, The
University of Michigan Medical School, Ann Arbor, Michigan 48109 (A.M.S.); and The Vollum Institute, Oregon Health Sciences University,
Portland, Oregon 97201-3098 (M.C.)
 |
ABSTRACT |
In animal cell lysates the
multiprotein heat-shock protein 90 (hsp90)-based chaperone complexes
consist of hsp70, hsp40, and p60. These complexes act to convert
steroid hormone receptors to their steroid-binding state by assembling
them into heterocomplexes with hsp90, p23, and one of several
immunophilins. Wheat germ lysate also contains a hsp90-based chaperone
system that can assemble the glucocorticoid receptor into a
functional heterocomplex with hsp90. However, only two
components of the heterocomplex-assembly system, hsp90 and hsp70, have
thus far been identified. Recently, purified mammalian p23 preadsorbed
with JJ3 antibody-protein A-Sepharose pellets was used to isolate a
mammalian p23-wheat hsp90 heterocomplex from wheat germ lysate (J.K.
Owens-Grillo, L.F. Stancato, K. Hoffmann, W.B. Pratt, and P. Krishna
[1996] Biochemistry 35: 15249-15255). This heterocomplex was found
to contain an immunophilin(s) of the FK506-binding class, as judged by
binding of the radiolabeled immunosuppressant drug
[3H]FK506 to the immune pellets in a specific manner. In
the present study we identified the immunophilin components of this
heterocomplex as FKBP73 and FKBP77, the two recently described
high-molecular-weight FKBPs of wheat. In addition, we present evidence
that the two FKBPs bind hsp90 via tetratricopeptide repeat domains. Our
results demonstrate that binding of immunophilins to hsp90 via
tetratricopeptide repeat domains is a conserved protein interaction in
plants. Conservation of this protein-to-protein interaction in both
plant and animal cells suggests that it is important for the biological
action of the high-molecular-weight immunophilins.
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INTRODUCTION |
Immunophilins are immunosuppressant drug-binding proteins, which,
based on their affinity for two different drugs, cyclosporin A or FK506
and rapamycin, are classified as cyclophilins or FKBPs (for reviews,
see Schreiber, 1991
; Walsh et al., 1992). All immunophilins have
peptidylprolyl isomerase activity, which suggests a role for these
proteins in protein folding (Schmid, 1993). It has been demonstrated
that immunophilins affect protein folding both in vitro (Bose et al.,
1996; Freeman et al., 1996) and in vivo (Lodish and Kong, 1991;
Steinmann et al., 1991). The high-Mr
immunophilin FKBP52 is a component of the steroid-receptor complexes in
mammalian cells (Pratt and Toft, 1997). The recent demonstration that
the chaperone activity of FKBP52 is not affected by the presence of immunosuppressant drugs that inhibit its peptidylprolyl isomerase activity suggests that the chaperone activity of FKBP52 is independent of its peptidylprolyl isomerase activity (Bose et al., 1996).
Several lines of evidence suggest that
low-Mr immunophilins such as FKBP12 and
cyclophilins A and B in complex with immunosuppressant drugs inhibit
the activity of calcineurin, a
Ca2+/calmodulin-dependent protein phosphatase,
thereby blocking the signaling pathway for T-cell activation (Liu et
al., 1991; Clipstone and Crabtree, 1992).
High-Mr immunophilins such as FKBP51,
FKBP52, and Cyp40 have been identified as components of
steroid-receptor complexes (for reviews, see Pratt, 1997; Pratt and
Toft, 1997). Although it remains to be demonstrated that these
immunophilins affect receptor action or heterocomplex assembly in any
way (Hutchison et al., 1993; Dittmar et al., 1996), there is some
evidence to suggest that FKBP52 may play a role in trafficking of the
receptor to the nucleus (Czar et al., 1995; Owens-Grillo et al.,
1996a).
The high-Mr immunophilins possess a TPR
domain and a calmodulin-binding domain in their C-terminal half
(Callebaut et al., 1992). The TPR domains consist of 34 amino acid
repeats with a degenerate consensus and are believed to be sites for
protein-protein interactions (Sikorski et al., 1990). The immunophilins
in steroid-receptor complexes bind to a common site on hsp90 via their
TPR domains (Radanyi et al., 1994; Owens-Grillo et al., 1995). It is
predicted that mammalian hsp90 has a universal TPR-domain-binding
region that permits it to bind to immunophilins and other
TPR-domain-containing proteins such as PP5 and the stress-related
protein p60 (Owens-Grillo et al., 1996a; Silverstein et al.,
1997).
Both FKBPs and cyclophilins have been identified in plants (for review,
see Boston et al., 1996). Genes encoding cyclophilin homologs have been
isolated from tomato, maize, oilseed rape, and Arabidopsis (Gasser et
al., 1990; Hayman and Miernyk, 1994). Luan et al. (1994) isolated a
high-Mr FKBP from broad bean using a FK506
affinity column. More recently, genes encoding
high-Mr FKBPs were isolated from
Arabidopsis (Vucich and Gasser, 1996) and wheat (Blecher et al., 1996;
Kurek et al., 1999). The deduced amino acid sequences of FKBPs from
both plant species show the presence of TPR motifs and a putative
calmodulin-binding domain.
In a previous study we used purified p23, a component of the mammalian
steroid-receptor heterocomplex, preadsorbed to JJ3 antibody to
isolate a mammalian p23-wheat hsp90 heterocomplex following incubation
with wheat germ lysate (Owens-Grillo et al., 1996b). The immunopurified
complex bound [3H]FK506 in a specific manner,
suggesting the presence of one or more FKBPs in the complex. Here we
show that the two recently identified
high-Mr wheat FKBPs, FKBP73 and FKBP77, are
components of this complex and that they interact with hsp90 via TPR
domains.
| |
MATERIALS AND METHODS |
Materials
Wheat germ lysate, horseradish peroxidase-conjugated anti-mouse
IgG, and horseradish peroxidase-conjugated anti-rabbit IgG were from
Promega. Protein A-Sepharose was from Pharmacia, and mouse IgG1 
(MOPC-21) clarified ascites was from Sigma. Enhanced chemiluminescence
western blotting detection reagents were from Amersham. The bacterial
strain expressing human p23 and the JJ3 monoclonal antibody against p23
(Johnson et al., 1994) were kindly provided by Dr. D.O. Toft
(Rochester, MN). Geldanamycin was kindly provided by Dr. W.B. Pratt
(Ann Arbor, MI). The source of geldanamycin was as described by
Owens-Grillo et al. (1996b). The rabbit R2 antiserum (Krishna et al., 1997) was used to detect wheat hsp90, and a
polyclonal antiserum against recombinant wheat FKBP73 (Blecher et al.,
1996) was used for detection of FKBP73. A polyclonal antibody raised
against a 24-mer peptide corresponding to amino acids 545 to 568 of
wheat FKBP77 (Kurek et al., 1999) was used to detect FKBP77. The amino
acid sequence between residues 545 to 568 is unique to FKBP77. As would
be expected, the anti-FKBP77 antibody reacted specifically with FKBP77
and showed no cross-reactivity with FKBP73 in the western assay (I. Kurek, K. Aviezer, N. Erel, E. Herman, and A. Breiman, unpublished
data).
Purification of p23
The bacterial expression of human p23 and its purification were
described previously (Johnson and Toft, 1994). p23 is soluble in
bacterial lysates, and its abundance and high affinity for DEAE-cellulose allowed purification to 90% by chromatography. The
protein was concentrated by precipitation with ammonium sulfate at 80%
saturation. It was dissolved and dialyzed into 10 mM
Tris-HCl, pH 7.4, 100 mM KCl, and 10% glycerol and stored
at 
70°C.
Preparation of Insect Cell Lysates
Sf9 cells were maintained in Grace's medium supplemented with
lactalbumin hydrolysate, Yeastolate (Life Technologies), gentamicin, and 10% fetal calf serum. Cells were infected with a recombinant baculovirus expressing the FLAG epitope (DYKDDDDK)-tagged TPR domain of
rat PP5 (Chen et al., 1996), at a multiplicity of infection of 3, and
then incubated for 48 h at 27°C. Cells were harvested by
scraping into Earle's balanced saline, washed once, suspended in 1×
volume of HKD buffer (10 mM Hepes, pH 7.4, 25 mM KCl, and 2 mM DTT), and ruptured by Dounce
homogenization. Homogenates were centrifuged for 15 min at
12,000g, and the supernatant was stored at 70°C.
p23 Immunoadsorption
The JJ3 antibody was prebound to protein A-Sepharose pellets by
incubating 40 µL of a 20% slurry of protein A-Sepharose with 8 µL
of ascites in 300 µL of HEG buffer (10 mM Hepes, pH 7.4, 1 mM EDTA, and 10% glycerol) for 1 h at 4°C,
followed by centrifugation and washing with additional HEG buffer. p23
was immunoadsorbed from 20 µL of purified p23 stock (1 mg/mL) by
rotation for 1 h at 4°C with JJ3-protein A-Sepharose pellets.
Immunoadsorbed p23 was washed once with 1 mL of HEG buffer prior to
incubation with wheat germ lysate. The nonimmune protein A-Sepharose
pellets were prepared similarly and incubated with p23 as described for
JJ3 immunopellets.
Mammalian p23-Plant hsp90 Heterocomplex Formation
The JJ3 immunopellets containing p23 were incubated with 50 µL
(or as indicated in the figure legends) of wheat germ lysate at 30°C
for 30 min with resuspension of the pellets every 5 min. The
immunopellets were washed three times by suspension and centrifugation in 1 mL of HEG buffer containing 100 mM KCl.
In experiments in which geldanamycin was added, wheat germ lysate was
preincubated at 30°C for 10 min with DMSO or with varying concentrations of geldanamycin dissolved in DMSO, as described in the
legend to Figure 4. The geldanamycin-containing lysate was then added
to the immunoadsorbed p23-protein A-Sepharose pellets and incubated
again for 30 min at 30°C. For examination of the effect of
geldanamycin at 0°C, both preincubation and incubation were carried
out at this temperature.
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| Figure 4.
Inhibition of p23-hsp90 heterocomplex formation by
geldanamycin in a concentration-dependent manner. A, Protein-antibody
complex visualized using an enhanced chemiluminescence detection
system. Lane 1, Commercial wheat germ lysate (0.25 µL). Lanes 2 through 7, Wheat germ lysate (30 µL) preincubated for 10 min at
30°C with 6 µL of DMSO (lane 2) or with geldanamycin at a
concentration of 0.5 µg/mL (lane 3), 1.0 µg/mL (lane 4), 1.5 µg/mL (lane 5), 2.0 µg/mL (lane 6), or 2.5 µg/mL (lane 7) in a
final volume of 6 µL of DMSO. The samples were then incubated with
immunoadsorbed p23 for 30 min at 30°C. After the pellets were washed,
proteins were extracted in 60 µL of SDS sample buffer, and an aliquot
of 20 µL was separated on a 11% SDS-polyacrylamide gel. Following
transfer to nitrocellulose membrane, hsp90 and FKBP73 were detected by
western blotting. Lanes 8 and 9 are results of a similar experiment in
which incubation of immunoadsorbed p23 with geldanamycin (10 µg/mL)
containing wheat germ lysate was at either 30°C (lane 8) or 0°C
(lane 9). B, Proteins visualized by silver staining. Lanes 1 and 2 are
same as lanes 8 and 9, respectively, in A.
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In experiments in which binding of immunophilins to hsp90 was competed
for by PP5 TPR motifs, wheat germ lysate was preincubated at
30°C for 10 min with insect cell lysate containing the expressed TPR
fragment or with an equal amount of wild-type lysate (without the TPR
domain). The mixture of lysates was then added to immunoadsorbed p23-protein A-Sepharose pellets and the entire sample was incubated at
30°C for 30 min.
Gel Electrophoresis and Western Blotting
The immunoadsorbed protein A-Sepharose pellets, after three
washes with HEG buffer containing 100 mM KCl, were heated
in SDS sample buffer with 10% 
-mercaptoethanol. The released
proteins were resolved on either 7.5% or 12.5% SDS-polyacrylamide
gels (Laemmli, 1970) and transferred to nitrocellulose membranes.
The membranes were probed for 2 h with the R2 antibody at a
dilution factor of 1:5000 to detect hsp90, JJ3 antibody at a
dilution of 1:5000 to detect p23, anti-FKBP73 antibody at a dilution of
1:10,000, and anti-FKBP77 antibody at a dilution of 1:1000 to detect
FKBP73 and FKBP77, respectively. The immunoblots were incubated
with the appropriate horseradish peroxidase-conjugated secondary
antibody, and the protein-antibody complex was visualized using an
enhanced chemiluminescence detection system. For the experiment
described in Figure 5, the membrane was probed with anti-FKBP77
antibody, incubated in the strip buffer (100 mM
-mercaptoethanol, 2% SDS, and 62.5 mM Tris-HCl, pH 6.8)
at 50°C for 30 min, washed twice in the same buffer, and then
reprobed with the R2 antibody to detect hsp90.
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| Figure 5.
FKBP77 is also a component of the mammalian
p23-plant hsp90 heterocomplex. Protein A-Sepharose (50 µL) was coated
with 10 µL of JJ3 ascites followed by adsorption of p23.
Immunoadsorbed p23 was incubated with 50 µL of wheat germ lysate.
After the pellets were washed, proteins were extracted in SDS sample
buffer and separated on a 7.5% SDS-polyacrylamide gel. FKBP77 was
detected by western blotting. Lane 1, Wheat germ lysate; lane 2, nonimmune IgG-bound protein A-Sepharose incubated first with p23 and
then with wheat germ lysate; lane 3, JJ3-bound protein A-Sepharose
incubated directly with wheat germ lsyate; lane 4, JJ3-bound-protein
A-Sepharose incubated first with p23 and then with wheat germ lysate;
lane 5, immunoadsorbed p23 incubated with wheat germ lysate mixed with
15 µL of insect cell lysate without the TPR domain; lane 6, immunoadsorbed p23 incubated with wheat germ lysate mixed with 15 µL
of insect cell lysate containing the TPR domain; lane 7, immunoadsorbed
p23 incubated with wheat germ lysate in the presence of geldanamycin
(10 µg/mL) at 30°C; lane 8, same as lane 7 but incubation was at
0°C.
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| |
RESULTS |
Identification of a Wheat Immunophilin Coimmunoadsorbed with Human
p23-Wheat hsp90 Complex
The highly acidic p23 protein is a component of
steroid-receptor-hsp90 heterocomplexes (Pratt and Toft, 1997). Johnson
and Toft (1994) demonstrated that immunoadsorption of p23 from rabbit reticulocyte lysate results in coisolation of hsp90 and immunophilins, including FKBP52, FKBP54, and Cyp40. Subsequently, it was shown that
p23 binds directly to hsp90 through a process that requires ATP
(Johnson et al., 1996). Immunophilins were shown, using purified proteins, to bind directly to a TPR-binding domain on hsp90
(Owens-Grillo et al., 1995). Although a p23 homolog has not yet been
identified in plants, purified human p23 was demonstrated to form a
complex with plant hsp90 following incubation with wheat germ lysate
(Owens-Grillo et al., 1996b). This complex was found to contain an
immunophilin(s) of the FKBP class. At that time the immunophilin(s) in
the complex could not be identified any further, due in part to the
unavailability of antibodies specific to plant immunophilins.
Recently, a cDNA encoding a high-Mr FKBP,
FKBP73, was isolated from wheat, and a polyclonal antibody was raised
to its recombinant form (Blecher et al., 1996). Since antibodies
against plant hsp90 and FKBPs are not efficient for immunoadsorption,
we used the previously published (Owens-Grillo et al., 1996b) indirect
strategy of adding immunoadsorbed p23 to wheat germ lysate to isolate a human p23-plant hsp90 complex, and we investigated the presence of
FKBP73 in this complex by immunoblotting. Incubation for 30 min at
30°C allowed complex formation between wheat hsp90 and human p23
(Fig. 1, lane 4), and the resulting
complex also contained the wheat FKBP73 (Fig. 1, lane 4). Neither hsp90
nor FKBP73 were detected in the absence of p23 (Fig. 1, lane 3) or when
immunoadsorption was carried out with nonimmune IgG-bound protein
A-Sepharose pellets (Fig. 1, lane 2).
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| Figure 1.
Mammalian p23-plant hsp90-plant FKBP73
heterocomplex formation. Protein A-Sepharose (40 µL) was coated with
8 µL of JJ3 ascites followed by immunoadsorption of p23. The
immunoadsorbed p23 was incubated with 30 µL of wheat germ lysate for
30 min at 30°C. After the pellets were washed, proteins were
extracted in 50 µL of SDS sample buffer. Proteins (30 µL of the
sample) were separated on a 7.5% SDS-polyacrylamide gel and then
transferred to a nitrocellulose membrane by electroblotting. hsp90 and
FKBP73 were detected using R2 and anti-wheat FKBP73
antisera, respectively. An aliquot (7 µL) of the sample containing
extracted proteins was run on a 12.5% SDS-polyacrylamide gel. After
transfer to a nitrocellulose membrane, p23 was detected using the JJ3
antibody. Lane 1, Wheat germ lysate; lane 2, nonimmune IgG-bound
protein A-Sepharose incubated first with p23 and then with wheat germ
lysate; lane 3, JJ3-bound protein A-Sepharose incubated directly with
wheat germ lsyate; lane 4, JJ3-bound-protein A-Sepharose incubated
first with p23 and then with wheat germ lysate.
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FKBP73 Binds to hsp90 via Its TPR Domain
FKBP73 contains TPR motifs (Blecher et al., 1996); therefore, we
wondered whether it binds to hsp90 via its TPR motifs. Previously, Silverstein et al. (1997) showed that rat PP5 binds directly to mammalian hsp90 via TPR motifs. The TPR domain of PP5 is closely related to TPR domains of several high-Mr
immunophilins known to associate with hsp90 (Chinkers, 1994). We
therefore added lysate of insect cells expressing the TPR domain of PP5
to wheat germ lysate before incubating it with immunoadsorbed p23.
Figure 2 shows that the addition of
increasing amounts of insect cell lysate containing the TPR domain did
not affect the presence of hsp90 in the heterocomplex but eliminated
FKBP73. Addition of equivalent amounts of wild-type insect cell lysate
(without the TPR domain) to wheat germ lysate did not affect the amount
of either hsp90 or FKBP73 in the complex (data not shown). Lanes 8 and
9 of Figure 2 represent results of a similar experiment, in which it
can also be seen that the addition of 20 µL of insect cell lysate
without the TPR domain to wheat germ lysate had no effect on the
amounts of hsp90, FKBP73, or p23 in the immune pellet (compare lane 8 of Fig. 2 with lane 4 of Fig. 1; the experiments were carried out at the same time), but the addition of lysate with the
TPR domain eliminated the binding of FKBP73 to hsp90 due to
competition by TPR domain (Fig. 2, lane 9). From these data we conclude
that wheat FKPB73 binds to wheat hsp90 via its TPR domain.
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| Figure 2.
Rat PP5 TPR domain competes for wheat FKBP73
binding to wheat hsp90. A, Protein A-Sepharose beads (40 µL) were
coated with 8 µL of JJ3 ascites followed by adsorption of p23. The
immunoadsorbed p23 was incubated with wheat germ lysate either without
addition of insect cell lysate or with addition of insect cell lysate
containing the expressed rat PP5 TPR domain. After the pellets were
washed, proteins were extracted in SDS sample buffer, resolved on a
7.5% polyacrylamide gel, transferred to nitrocellulose membrane, and
analyzed for hsp90 and FKBP73 by western blotting. Lane 1, Wheat germ
lysate; lane 2, immunoadsorbed p23 incubated with 30 µL of wheat germ
lysate; lanes 3 to 7, immunoadsorbed p23 incubated with wheat germ
lysate mixed with 2, 4, 6, 8, and 10 µL, respectively, of insect cell
lysate containing the expressed TPR domain; lane 8, immunoadsorbed p23
incubated with 50 µL of wheat germ lysate mixed with 20 µL of
wild-type insect cell lysate without TPR domain; lane 9, immunoadsorbed
p23 incubated with wheat germ lysate mixed with 20 µL of insect cell
lysate containing the TPR domain. B, Insect cell lysates (0.5, 1.0, 2.0, and 3.0 µL) without the TPR domain (wild type [WT]) and with
the TPR domain were subjected to electrophoresis on a 12.5%
SDS-polyacrylamide gel. Proteins were visualized by Coomassie blue
staining. The arrow indicates the expressed TPR domain of PP5 only in
the lysates of cells infected with the recombinant baculovirus.
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Detection of Wheat Proteins Coimmunoprecipitating with Human
p23 by Silver Staining of the SDS-Polyacrylamide Gel
Under the experimental conditions used in the present study, two
major proteins coimmunoprecipitated with p23 (Fig.
3, lane 4). These proteins were not
detected in the two control lanes (Fig. 3, lanes 2 and 3), suggesting
that their presence was due to specific interactions within the
complex. Based on their mobility on the SDS-polyacrylamide gel, these
proteins were identified as hsp90 and FKBP73. Further evidence that the
lower band is FKBP73 is that it was lost from the complex in the
presence of insect cell lysate containing the TPR domain (Fig. 3, lane
6) but not in the presence of wild-type lysate (Fig. 3, lane 5). It is
possible that additional proteins were present in this complex but were either masked by the antibody heavy chain or were not present in the
quantity required for detection by silver staining.
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| Figure 3.
Wheat proteins coimmunoadsorbed with human p23.
Samples were purified from 50 µL of wheat germ lysate using p23
immunoadsorbed with JJ3 IgG prebound to 60 µL of protein A-Sepharose.
Proteins were extracted in SDS sample buffer, separated on a 11%
SDS-polyacrylamide gel, and visualized by silver staining of the gel.
Lane 1, One microliter of wheat germ lysate; lane 2, nonimmune
IgG-bound protein A-Sepharose incubated with p23 and then with wheat
germ lysate; lane 3, JJ3-bound protein A-Sepharose incubated directly
with wheat germ lysate; lane 4, JJ3-bound protein A-Sepharose incubated
first with p23 and then with wheat germ lysate; lanes 5 and 6 are the
same as lane 4 with the exception that the wheat germ lysate was mixed
with 20 µL of insect cell lysate without PP5 TPR domain (lane 5) or
with lysate containing the expressed TPR domain (lane 6). HC, Heavy
chain; LC, light chain.
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Geldanamycin Eliminates hsp90 Binding to p23, Which Is Accompanied
by the Loss of FKBP73 from the Complex
Geldanamycin, a benzoquinione ansamycin, disrupts both mammalian
p23-hsp90 complexes (Johnson and Toft, 1995) and mammalian p23-plant
hsp90 complexes (Owens-Grillo et al., 1996b). In the latter case, the
blockage of heterocomplex formation was temperature-dependent and took
place after preincubation of wheat germ lysate with geldamamycin or
after subsequent incubation with p23 at 30°C. Previously, it was
observed that inhibition of hsp90 binding to p23 in the presence of
geldanamycin was paralleled by decline of
[3H]FK506 binding to the immune pellet
(Owens-Grillo et al., 1996b), suggesting that the unidentified FKBP was
bound to the complex through interaction with hsp90. We wanted to know
whether FKBP73 would conform to this behavior. Figure
4A, lane 5, shows that geldanamycin
at a concentration of 1.5 µg/mL completely inhibited the binding
of hsp90 to p23. Like hsp90, FKBP73 was not detected in this sample.
In a previous study (Owens-Grillo et al., 1996b), 5 µg/mL of
geldanamycin was observed to completely disrupt p23-hsp90 complexes. Since differences were observed in the immunoadsorption and
western-blotting techniques in the two studies, variation in what may
be the most effective concentration of geldanamycin in blocking the
complex formation was not unexpected. Lanes 8 and 9 of Figure 4A
represent data from a similar experiment. When the preincubation and
incubation were at 0°C, hsp90 and FKBP73 were not completely
eliminated from the complex (compare Fig. 4, lane 9, to Fig. 1, lane 4;
the experiments were carried out at the same time), but at 30°C
neither hsp90 nor FKBP73 was detected in the immune pellet (Fig. 4,
lane 8). The results obtained by western blotting were further
confirmed by silver staining of proteins present in the immune pellet
(Fig. 4B). These data are consistent with a previous report by
Owens-Grillo et al. (1996b) in that the effect of geldanamycin on plant
hsp90 was temperature-dependent.
The Heat-Regulated Wheat FKBP77 Is a Component of the p23-hsp90
Heterocomplex
Recently, FKBP77 was identified in wheat as an immunophilin whose
synthesis is enhanced severalfold by high-temperature stress (Kurek et
al., 1999). In the absence of high temperature, the protein was present
in root and shoot tips and soaked embryos of wheat, but the levels were
very low. The availability of an antibody to FKBP77 allowed us to check
for its presence in the p23-hsp90 heterocomplex. FKBP77 was detected in
wheat germ lysate (Fig. 5, lane 1) and
its presence in the p23 immune pellet paralleled that of hsp90 (Fig. 5,
lane 4). FKBP77 also binds to hsp90 via its TPR domain, because the
insect cell lysate with the PP5 TPR domain could successfully compete
for its binding to hsp90 (Fig. 5, lane 6), but the wild-type insect
cell lysate was ineffective in eliminating its binding to hsp90 (Fig.
5, lane 5). The effect of geldanamycin on hsp90 and indirectly on
FKBP77 was similar to that observed for FKBP73 (Fig. 5, lanes 7 and 8).
| |
DISCUSSION |
In an earlier study the presence of FKBPs in the human p23-plant
hsp90 complex was demonstrated using the radiolabeled FK506 drug-binding assay (Owens-Grillo et al., 1996b). The recent cloning of
two high-Mr wheat FKBPs has allowed the
generation of antibodies specific to these proteins (Blecher et al.,
1997; Kurek et al., 1999). Using these antibodies in immunoblot assays,
we demonstrate here that wheat FKBP73 and FKBP77 are components of the
wheat hsp90 heterocomplex. Since p23 interacts specifically with hsp90 (Johnson and Toft, 1995) and does not affect the binding of
immunophilins to hsp90 (W.B. Pratt, personal communication), the
present data unequivocally establish that hsp90-immunophilin complexes
do exist in plant cells, as has already been demonstrated for animal
and yeast cells.
The high-Mr immunophilins were first
discovered as part of steroid-receptor multiprotein heterocomplexes in
animals (Pratt and Toft, 1997). These studies also revealed that the
chaperone components of these heterocomplexes exist in cytosols as
heterocomplexes independent of their association with steroid receptors
and that each component has a unique function in the assembly and
maturation process of steroid receptors to the steroid-binding state.
The first step in the assembly process is the formation of a
steroid-receptor-hsp90-p60-hsp70 complex. The p60 cycles out by an
unknown mechanism, and p23 enters the complex dynamically and helps to
stabilize the receptor-hsp90 heterocomplex (Pratt, 1997). Once p60
dissociates from hsp90, the immunophilin can bind to the TPR-binding
domain, which p60 has vacated.
Although immunophilins were detected in steroid-receptor
heterocomplexes some time ago, their specific function in these
complexes remains unclear. One view is that immunophilins may play a
role in targeting receptor movement to the nucleus. In support of this view, an antibody raised to a negatively charged region of FKBP52, which is electrostatically complimentary to the receptor's
nuclear-localization signal, inhibited the movement of the receptor to
the nucleus (Czar et al., 1995). FKBP52 has also been localized by
indirect immunofluorescence to the nucleus (Owens-Grillo et al.,
1996a), supporting the idea that immunophilins participate in the
targeted movement of the complexes.
The other view is that immunophilins take part in the protein-folding
process. Recently, it was shown through in vitro protein-folding assays
that FKBP52 can efficiently suppress the aggregation of citrate
synthase and promote reactivation of unfolded citrate synthase by
stabilizing folding-competent intermediates (Bose et al., 1996). These
data define FKBP52 as a molecular chaperone. However, a chaperone
function for immunophilins in the assembly process of the
steroid-receptor heterocomplex has not yet been described. In fact,
receptor heterocomplexes have been assembled with purified proteins in
the absence of immunophilins (Dittmar et al., 1996). Although
immunophilins appear to be linked with protein targeting in receptor
heterocomplexes, the possibility that they have a specific role in
protein folding cannot be ruled out at this point. The observations
that the abundance of ROF1 (the Arabidopsis homolog of
FKBP59) mRNA is increased severalfold under stress conditions such as
wounding or exposure to high salt concentrations (Vucich and Gasser,
1996), that wheat FKBP73 is expressed at higher levels in young
tissues (Blecher et al., 1996), and that wheat FKBP77 accumulates to
higher levels in response to high-temperature stress (I. Kurek, K. Aviezer, N. Erel, E. Herman, and A. Breiman, unpublished data) together
suggest that high-Mr FKBPs may have a role
in protein folding.
The identification of FKBP73 and FKBP77 in hsp90 heterocomplexes has
produced a number of questions: What are the other components of these
heterocomplexes? What are the plant proteins analogous to steroid
receptors that are chaperoned by hsp90-immunophilin heterocomplexes?
What are the specific functions of immunophilins within these
complexes?
Prior to demonstrating the presence of hsp90-immunophilin complexes in
plant cells (Owens-Grillo et al., 1996b; present study), studies of
cell-free assembly of receptor-hsp90 complexes revealed that the
assembly process is conserved in animal and plant cell lysates
(Stancato et al., 1996), that purified plant hsp90 and hsp70 proteins
have the conserved ability to interact functionally with chaperone
proteins of the animal kingdom (Stancato et al., 1996), and that
purified human p23 added to wheat germ lysate can stabilize the
receptor-plant hsp90 heterocomplex formed in wheat germ lysate
(Hutchison et al., 1995). All of these findings attest to the conserved
nature of the chaperone complex in animals and plants. On the basis of
these findings it can be speculated that functions of immunophilins, in
a general sense, may also be conserved in animal and plant cells.
A p23 homolog has not yet been identified in plants; however, a
stress-inducible gene encoding a protein with high homology to p60 has
been isolated from soybean (Torres et al., 1995). Whether the
plant p60 homolog occupies the same site on hsp90 as the immunophilins remains to be determined. The identification of FKBP73 and FKBP77 as
components of the hsp90 heterocomplex will allow such questions to be
answered in the future through reconstitution of heterocomplexes with
purified proteins.
We have shown that in the presence of geldanamycin, which disrupts the
hsp90-p23 heterocomplex, FKBP73 and FKBP77 were eliminated from the
heterocomplex. Recently, geldanamycin was demonstrated to inhibit
steroid-dependent translocation of the glucocorticoid receptor from the
cytoplasm to the nucleus (Czar et al., 1997). Because geldanamycin
blocks the assembly of the receptor-hsp90 heterocomplex at an
intermediate state, this suggests that dynamic association of receptors
with hsp90 is required for receptor translocation from the cytoplasm to
the nucleus. According to the model proposed by Owens-Grillo et al.
(1996a), the immunophilin component that binds to the complex via hsp90
would be absent, resulting in the inability of the receptor to
translocate to the nucleus. The fact that geldanamycin interacts with
plant hsp90 in a manner similar to that of animal hsp90 suggests that
it will prove useful in the future for dissecting the functions of
plant chaperone proteins within the heterocomplex.
| |
FOOTNOTES |
1
This work was supported by a research grant to
P.K. by the National Science and Engineering Research Council of
Canada, by a research grant to A.B. from the Israeli National Academy
of Science, and by a National Institutes of Health grant to M.C. (no.
HL47063).
*
Corresponding author; e-mail pkrishna{at}julian.uwo.ca; fax
1-519-661-3935.
Received April 17, 1998;
accepted September 8, 1998.
| |
ABBREVIATIONS |
Abbreviations:
FKBP, FK506-binding protein(s).
hsp, heat-shock
protein.
PP5, protein phosphatase 5.
TPR, tetratricopeptide repeat.
| |
ACKNOWLEDGMENTS |
We thank Dr. W.B. Pratt for many useful discussions and for
providing geldanamycin and Dr. D.O. Toft for providing
Escherichia coli-expressing human p23 and JJ3 antibody.
| |
LITERATURE CITED |
Blecher O,
Erel N,
Callebaut I,
Aviezer K,
Breiman A
(1996)
A novel plant peptidylprolyl-cis-trans-isomerase (PPIase): cDNA cloning, structural analysis, enzymatic activity and expression.
Plant Mol Biol
32:
493-504
[CrossRef][ISI][Medline]
Bose S,
Weikl T,
Bugl H,
Buchner J
(1996)
Chaperone function of hsp90-associated proteins.
Science
274:
1715-1717
[Abstract/Free Full Text]
Boston RS,
Viitanen PV,
Vierling E
(1996)
Molecular chaperones and protein folding in plants.
Plant Mol Biol
32:
191-222
[CrossRef][ISI][Medline]
Callebaut I,
Renoir JM,
Lebeau MC,
Massol N,
Burny A,
Baulieu EE,
Mornon JP
(1992)
An immunophilin that binds Mr 90,000 heat shock protein: main structural features of a mammalian p59 protein.
Proc Natl Acad Sci USA
89:
6270-6274
[Abstract/Free Full Text]
Chen M-S,
Silverstein AM,
Pratt WB,
Chinkers M
(1996)
The tetratricopeptide repeat domain of protein phosphatase 5 mediates binding to glucocorticoid receptor heterocomplexes and acts as a dominant negative mutant.
J Biol Chem
271:
32315-32320
[Abstract/Free Full Text]
Chinkers M
(1994)
Targeting of a distinctive protein-serine phosphatase to the protein kinase-like domain of the atrial natriuretic peptide receptor.
Proc Natl Acad Sci USA
91:
11075-11079
[Abstract/Free Full Text]
Clipstone NA,
Crabtree GR
(1992)
Identification of calcineurin as a key signalling enzyme in T-lymphocyte activation.
Nature
357:
695-697
[CrossRef][Medline]
Czar MJ,
Galigniana MD,
Silverstein AM,
Pratt WB
(1997)
Geldanamycin, a heat shock protein 90-binding benzoquinone ansamycin, inhibits steroid-dependent translocation of the glucocorticoid receptor from the cytoplasm to the nucleus.
Biochemistry
36:
7776-7785
[CrossRef][Medline]
Czar MJ,
Lyons RH,
Welsh MJ,
Renoir JM,
Pratt WB
(1995)
Evidence that the FK506-binding immunophilin heat shock protein 56 is required for trafficking of the glucocorticoid receptor from the cytoplasm to the nucleus.
Mol Endocrinol
9:
1549-1560
[Abstract]
Dittmar KD,
Hutchison KA,
Owens-Grillo JK,
Pratt WB
(1996)
Reconstitution of the steroid receptor-hsp90 heterocomplex assembly system of rabbit reticulocyte lysate.
J Biol Chem
271:
12833-12839
[Abstract/Free Full Text]
Freeman BC,
Toft DO,
Morimoto RI
(1996)
Molecular chaperone machines: chaperone activities of the cyclophilin Cyp-40 and the steroid aporeceptor-associated protein p23.
Science
274:
1718-1720
[Abstract/Free Full Text]
Gasser CS,
Gunning DA,
Budelier KA,
Brown SM
(1990)
Structure and expression of cytosolic cyclophilin/peptidyl-prolyl cis-trans isomerase of higher plants and production of active tomato cyclophilin in Escherichia coli.
Proc Natl Acad Sci USA
87:
9519-9523
[Abstract/Free Full Text]
Hayman GT,
Miernyk JA
(1994)
The nucleotide and deduced amino acid sequences of a peptidyl-prolyl cis-trans isomerase from Arabidopsis thaliana.
Biochim Biophys Acta
1219:
536-538
[Medline]
Hutchison KA,
Scherrer LC,
Czar MJ,
Ning Y,
Sanchez ER,
Leach KL,
Diebel MR,
Pratt WB
(1993)
FK506 binding to the 56-kilodalton immunophilin (hsp56) in the glucocorticoid receptor heterocomplex has no effect on receptor folding or function.
Biochemistry
32:
3953-3957
[CrossRef][Medline]
Hutchison KA,
Stancato LF,
Owens-Grillo JK,
Johnson JL,
Krishna P,
Toft DO,
Pratt WB
(1995)
The 23-kDa acidic protein in reticulocyte lysate is the weakly bound component of the hsp foldosome that is required for assembly of the glucocorticoid receptor into a functional heterocomplex with hsp90.
J Biol Chem
270:
18841-18847
[Abstract/Free Full Text]
Johnson J,
Corbisier R,
Stensgard B,
Toft DO
(1996)
The involvement of p23, hsp90, and immunophilins in the assembly of progesterone receptor complexes.
J Steroid Biochem Mol Biol
56:
31-37
[CrossRef][Medline]
Johnson JL,
Beito TG,
Krco CJ,
Toft DO
(1994)
Characterization of a novel 23- kilodalton protein of unactive progesterone receptor complexes.
Mol Cell Biol
14:
1956-1963
[Abstract/Free Full Text]
Johnson JL,
Toft DO
(1994)
A novel chaperone complex for steroid receptors involving heat shock proteins, immunophilins, and p23.
J Biol Chem
269:
24989-24993
[Abstract/Free Full Text]
Johnson JL,
Toft DO
(1995)
Binding of p23 and hsp90 during assembly with the progesterone receptor.
Mol Endocrinol
9:
670-678
[Abstract]
Krishna P,
Reddy RK,
Sacco M,
Frappier JRH,
Felsheim RF
(1997)
Analysis of the native forms of the 90 kDa heat shock protein (hsp90) in plant cytosolic extracts.
Plant Mol Biol
33:
457-466
[CrossRef][Medline]
Kurek I, Aviezer K, Erel N, Herman E, Breiman A (1999) The wheat
peptidyl prolyl cis-trans isomerase FKBP77 is heat induced
and developmentally regulated. Plant Physiol 119: (in press)
Laemmli UK
(1970)
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:
680-685
[CrossRef][Medline]
Liu J,
Farmer JD,
Lane WS,
Friedman J,
Weissman I,
Schreiber SL
(1991)
Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes.
Cell
66:
807-815
[CrossRef][ISI][Medline]
Lodish HF,
Kong N
(1991)
Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum.
J Biol Chem
266:
14835-14838
[Abstract/Free Full Text]
Luan S,
Albers MW,
Schreiber SL
(1994)
Light-regulated, tissue-specific immunophilins in higher plants.
Proc Natl Acad Sci USA
91:
984-988
[Abstract/Free Full Text]
Owens-Grillo JK,
Czar MJ,
Hutchison KA,
Hoffmann K,
Perdew GH,
Pratt WB
(1996a)
A model of protein targeting mediated by immunophilins and other proteins that bind to hsp90 via tetratricopeptide repeat (TPR) domains.
J Biol Chem
271:
13468-13475
[Abstract/Free Full Text]
Owens-Grillo JK,
Hoffmann K,
Hutchison KA,
Yem AW,
Deibel MR,
Handschumacher RE,
Pratt WB
(1995)
The cyclosporin A-binding immunophilin CyP-40 and the FK506-binding immunophilin hsp56 bind to a common site on hsp90 and exist in independent cytosolic heterocomplexes with the untransformed glucocorticoid receptor.
J Biol Chem
270:
20479-20484
[Abstract/Free Full Text]
Owens-Grillo JK,
Stancato LF,
Hoffmann K,
Pratt WB,
Krishna P
(1996b)
Binding of immunophilins to the 90 kDa heat shock protein (hsp90) via a tetratricopeptide repeat domain is a conserved protein interaction in plants.
Biochemistry
35:
15249-15255
[CrossRef][Medline]
Pratt WB
(1997)
The role of the hsp90-based chaperone system in signal transduction by nuclear receptors and receptors signaling via MAP kinase.
Annu Rev Pharmacol Toxicol
37:
297-326
[CrossRef][ISI][Medline]
Pratt WB,
Toft DO
(1997)
Steroid receptor interactions with heat shock protein and immunophilin chaperones.
Endocrine Rev
18:
306-360
[Abstract/Free Full Text]
Radanyi C,
Chambraud B,
Baulieu EE
(1994)
The ability of the immunophilin FKBP59-HB1 to interact with the 90-kDa heat shock protein is encoded by its tetratricopeptide repeat domain.
Proc Natl Acad Sci USA
91:
11197-11201
[Abstract/Free Full Text]
Schmid FX
(1993)
Prolyl isomerase: enzymatic catalysis of slow protein folding reactions.
Annu Rev Biophys Biomol Struct
22:
123-143
[ISI][Medline]
Schreiber SL
(1991)
Chemistry and biology of the immunophilins and their immunosuppressive ligands.
Science
251:
283-287
[Abstract/Free Full Text]
Sikorski RS,
Boguski MS,
Goebl M,
Hieter P
(1990)
A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis.
Cell
60:
307-317
[CrossRef][ISI][Medline]
Silverstein AM,
Galigniana MD,
Chen M-S,
Owens-Grillo JK,
Chinkers M,
Pratt WB
(1997)
Protein phosphatase 5 is a major component of glucocorticoid receptor.hsp90 complexes with properties of an FK506-binding immunophilin.
J Biol Chem
272:
16224-16230
[Abstract/Free Full Text]
Stancato LF,
Hutchison KA,
Krishna P,
Pratt WB
(1996)
Animal and plant cell lysates share a conserved chaperone system that assembles the glucocorticoid receptor into a functional heterocomplex with hsp90.
Biochemistry
35:
554-561
[CrossRef][Medline]
Steinmann B,
Bruckner P,
Superti-Furga A
(1991)
Cyclosporin A slows collagen triple-helix formation in vivo: indirect evidence for a physiologic role of peptidyl-prolyl cis-trans-isomerase.
J Biol Chem
266:
1299-1303
[Abstract/Free Full Text]
Torres JH,
Chantellard P,
Stutz E
(1995)
Isolation and characterization of gmsti, a stress-inducible gene from soybean (Glycine max) coding for a protein belonging to the TPR (tetratricopeptide repeats) family.
Plant Mol Biol
27:
1221-1226
[CrossRef][ISI][Medline]
Vucich VA,
Gasser CS
(1996)
Novel structure of a high molecular weight FK506 binding protein from Arabidopsis thaliana.
Mol Gen Genet
252:
510-517
[Medline]
Walsh CT,
Zydowsky LD,
McKeon FD
(1992)
Cyclosporin A, the cyclophilin class of peptidylprolyl isomerases, and blockade of T cell signal transduction.
J Biol Chem
267:
13115-13118
[Abstract/Free Full Text]
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Abstract
Introduction