(Received for publication, April 17, 1995; and in revised form, May 30, 1995)
From the
The heat shock proteins hsp90 and hsp70 have been immunopurified from rabbit reticulocyte lysate in a multiprotein complex that acts as a self-sufficient protein folding machine. This immunopurified ``foldosome'' directs the assembly of the glucocorticoid receptor-hsp90 complex and refolds the receptor to the steroid binding state (Hutchison, K. A., Dittmar, K. D., and Pratt, W. B.(1994) J. Biol. Chem. 269, 27894-27899). Extensive washing of the immunoadsorbed foldosome eliminates a weakly bound component required for receptor heterocomplex assembly and folding. This protein factor is contained in a Centricon C-100 filtrate of lysate which reconstitutes the receptor activating activity of the washed foldosome. This hsp90-associated protein folding system is present in both animal and plant cells, and the Centricon C-100 fraction of rabbit reticulocyte lysate potentiates receptor folding directed by wheat germ lysate. We have used this ability to stimulate wheat germ lysate-directed folding of the glucocorticoid receptor as a rapid assay for the factor. We demonstrate that the activity segregates with the 23-kDa acidic protein component of the hsp90 foldosome when rabbit reticulocyte lysate is fractionated by ammonium sulfate precipitation and ion exchange chromatography. Immunoadsorption of the Centricon C-100 filtrate with a monoclonal antibody against p23 eliminates its ability to stimulate the wheat germ heterocomplex assembly/receptor folding system, and the activity is replaced by purified, bacterially expressed p23. Immunodepletion of p23 also eliminates the ability of the Centricon C-100 filtrate to reconstitute receptor activating activity of the washed foldosome and addition of purified, bacterially expressed p23 restores its activity, confirming that p23 is the weakly bound component of the foldosome complex required for refolding of the receptor to the steroid binding conformation.
In cytosols prepared from hormone-free mammalian cells, steroid
receptors exist in multiprotein complexes containing hsp90, ()hsp70, two immunophilin chaperones (hsp56 and CyP-40), and
a 23-kDa acidic protein, p23 (see (1) and (2) for
review). These multiprotein receptor-hsp90 heterocomplexes can be
formed by incubating immunoadsorbed, hormone-free receptors (pretreated
with salt to strip them free of associated proteins) with rabbit
reticulocyte lysate(3, 4) . Some of the steroid
receptors, for example the glucocorticoid receptor (GR), must be bound
to hsp90 for the hormone binding domain to be in a steroid binding
conformation(5) , and incubation of the immunoadsorbed GR with
reticulocyte lysate not only forms the receptor heterocomplex but
restores the receptor to the steroid binding state(4) . Until
recently, all of the experiments involving cell-free assembly of
receptor-hsp90 heterocomplexes have been carried out with the protein
folding system in rabbit reticulocyte lysate, but this system appears
to be ubiquitous in that concentrated lysates of human, monkey, mouse,
insect (Sf9), and plant (wheat germ) cells fold the GR into a
functional (i.e. steroid binding) heterocomplex with hsp90. (
)A number of requirements for the heterocomplex
assembly/receptor folding system of reticulocyte lysate have been
defined. Formation of the receptor-hsp90 complex is an
ATP/Mg
-dependent process(6, 7) that
requires the presence of a monovalent cation, such as K
or NH
(7) . hsp70 is required
for the binding of hsp90 to steroid receptors (6, 8) and for simultaneous conversion of the GR to
the steroid binding conformation (8) .
In reticulocyte
lysate, as well as in a variety of mammalian cell lysates, hsp90 and
hsp70 exist in multiprotein complexes containing the immunophilins
hsp56 (FKBP52) or CyP-40(9, 10) , ()a
60-kDa stress-related protein(11) , the p50 protein that was
originally identified as a member of the
pp60
-hsp90 complex(12, 13) ,
and the conserved and widely distributed p23(14) . When these
multiprotein complexes are immunoadsorbed from reticulocyte lysate,
they retain protein chaperoning activity as assayed by renaturation of
thermally denatured luciferase (15) and by formation of a
functional GR-hsp90 heterocomplex (16) . Thus, the multiprotein
hsp90 heterocomplex acts as a self-sufficient protein folding machine
that we refer to as a ``foldosome''(16) .
The
foldosome can be immunoadsorbed to protein A-Sepharose from rabbit
reticulocyte lysate using the 3G3 monoclonal IgM directed against
hsp90. When this immunopellet is incubated at 30 °C with
immunoadsorbed GR, ATP/Mg, and KCl, the receptor is
bound to hsp90 and restored to the steroid binding
conformation(16) . But, if the immunoadsorbed multiprotein
hsp90 complex is washed extensively with a low salt buffer, it loses a
weakly bound protein (not hsp90 or hsp70) that is required for receptor
activation to the steroid binding state. This factor is contained in a
Centricon C-100 filtrate of lysate that reconstitutes the receptor
activating activity of the washed foldosome(16) .
In this work we show that the Centricon C-100 filtrate of rabbit reticulocyte lysate also stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate. Using this stimulation of the wheat germ heterocomplex assembly system as a simple and rapid assay for the factor, we show by ammonium sulfate fractionation and ion exchange chromatography of reticulocyte lysate that the activity segregates with p23 and not with any other component of the foldosome (i.e. hsp90, hsp70, p60, p50, hsp56, or CyP-40). Johnson and Toft (10) have shown recently that immunoadsorption of reticulocyte lysate with the JJ3 monoclonal antibody against p23 reduces its ability to form a progesterone receptor-hsp90 heterocomplex. Here we show that immunoadsorption of the Centricon C-100 filtrate of reticulocyte lysate with the similar JJ5 monoclonal antibody eliminates its ability to reconstitute the receptor activating activity of the washed foldosome, and addition of purified bacterially expressed p23 restores the ability of the system to refold the receptor to the steroid binding conformation. This work provides the first assay of p23 activity in a substantially purified hsp90-associated chaperone system, providing an important step toward the long term goal of reconstituting a biologically active foldosome from purified protein components.
Figure 1:
The Centricon C-100 filtrate of
reticulocyte lysate stimulates reconstitution of the glucocorticoid
receptor-hsp90 heterocomplex by both the washed 3G3-immunoadsorbed
rabbit hsp complex and wheat germ lysate. Glucocorticoid receptors were
immunoadsorbed from aliquots of L cell cytosol to protein A-Sepharose
prebound with BuGR or nonimmune IgG. Receptor-associated proteins were
stripped from the immunopellets with salt and incubated for 20 min at
30 °C with the additions noted under the indicated conditions.
Receptor, hsp90, and hsp70 were assayed in each sample by Western
blotting, and a portion of the immunopellet was incubated with
[H]triamcinolone acetonide to determine steroid
binding activity. A, reconstitution of GR-hsp90
heterocomplexes with the washed 3G3-immunoadsorbed hsp complex. B, reconstitution of GR-hsp90 heterocomplexes with wheat germ
lysate. In both panels condition 1 is a single lane showing
the stripped glucocorticoid receptor, and condition 2 is
divided into two lanes of stripped nonimmune (no receptor) and immune
(GR-containing) pellets incubated with whole rabbit reticulocyte
lysate. Condition 3, stripped nonimmune and immune pellets
incubated with the proteins released from the washed 3G3 immunopellet
of reticulocyte lysate with 100 mM KCl (A) or with
whole wheat germ lysate (B). Condition 4 is condition 3 plus the Centricon C-100 filtrate of reticulocyte
lysate. Condition 5, stripped nonimmune and immune pellets
incubated with only the Centricon C-100 filtrate. The solid bars represent the steroid binding for the samples presented in the upper panel.
Figure 5: DE52 fractionation of activity from reticulocyte lysate. A, rabbit reticulocyte lysate (20 ml) was chromatographed on a column of DE52 as described under ``Methods'' (solid line, absorbance at 280 nm; dotted line, KCl gradient). Fractions were pooled (A-D) as indicated by the bars under the graph, concentrated to the original volume of lysate and dialyzed against 10 mM HEPES, 25 mM KCl, and 2 mM DTT for further analysis. Aliquots of each fraction pool were Western blotted for the proteins in the hsp heterocomplex (shown below graph). B, each fraction was assayed for its ability to stimulate the GR-hsp90 reconstituting activity of wheat germ lysate. Condition 1, stripped receptor; condition 2, stripped nonimmune and immune pellets, respectively, incubated with wheat germ lysate; condition 3, stripped nonimmune and immune pellets incubated with wheat germ lysate and 1 µg of purified, bacterially expressed p23; conditions 4-7, stripped receptors incubated with wheat germ lysate and DE52 fractions A-D, respectively.
Fig.1B shows the effect of the same Centricon C-100 filtrate on assembly in wheat germ lysate of a complex between the GR and plant hsp90. As shown in condition 3, incubation of stripped receptors with wheat germ lysate produces a small amount of complex with the plant hsp90. The plant hsp90 is the band migrating slightly faster than the rabbit hsp90, which is shown in condition 2. Addition of the Centricon C-100 filtrate to the wheat germ lysate yields more complexes between the GR and plant hsp90 and is accompanied by substantial reactivation of steroid binding activity. The same amount of wheat hsp70 binds to the receptor with (condition 4) and without (condition 3) the Centricon C-100 filtrate. As we have noted previously(8) , the binding of hsp90 is hsp70-dependent, but it is only GR-associated hsp90 (not hsp70) that is associated with steroid binding activity. As shown in Fig.2, the factor in the Centricon C-100 filtrate produces a concentration-dependent increase in both the amount of GR-plant hsp90 complex that is recovered and steroid binding activity.
Figure 2:
The Centricon C-100 filtrate of
reticulocyte lysate produces a concentration-dependent stimulation of
glucocorticoid receptor-plant hsp90 heterocomplex assembly by wheat
germ lyate. Immunoadsorbed, stripped receptors were incubated with
wheat germ lysate in the presence of the indicated concentrations of
Centricon C-100 filtrate of reticulocyte lysate. After the 20-min
incubation, the immunopellets were washed and assayed for steroid
binding activity () and receptor-associated plant hsp90 (Western
blot). Lanes 1 and 2, stripped nonimmune and immune
pellet, respectively, incubated with wheat germ lysate alone; lane
3, nonimmune pellet incubated with wheat germ lysate and a 10-fold
concentration of Centricon C-100 filtrate; lanes 4-7,
immune pellets containing stripped GR incubated with wheat germ lysate
and 1-, 2.5-, 5-, and 10-fold concentrated Centricon C-100 filtrate,
respectively.
We have
shown previously that GR-plant hsp90 complexes formed in wheat germ
lysate are less stable than GR-rabbit hsp90 complexes formed in
reticulocyte lysate. A rapid dissociation of hsp90 from receptors
during the incubation with wheat germ lysate at 30 °C would account
for the fact that fewer GR-hsp90 complexes are recovered from wheat
germ lysate than reticulocyte lysate and that there is very little
steroid binding activity in the immunopellet after lysate removal. It
is possible, however, to trap the steroid binding state by having
[H]triamcinolone acetonide present during the
20-min incubation with wheat germ lysate at 30 °C. This effect is
shown in Fig.3A. When [
H]steroid
is present during the incubation with wheat germ lysate (condition
1, open bar), the amount of steroid binding is about two-thirds
that seen with reticulocyte lysate (condition 1, solid bar).
However, if the wheat germ reconstitution mix is simply cooled on ice
after the 20-min incubation at 30 °C and
[
H]steroid is added to the cooled mixture, then
there is very little steroid binding activity (condition 2, open
bar). Essentially the same steroid binding activity is seen under
the two conditions with rabbit reticulocyte lysate. As shown by the slashed bars, the presence of the Centricon C-100 filtrate
does not affect the amount of steroid binding activity seen under condition 1, but it increased it under condition 2.
The GR that is reactivated in the wheat germ lysate in the presence of
the Centricon C-100 filtrate can be washed free of lysate with both
retention of steroid binding activity (Fig.3A, condition
3) and the expected increased yield of GR-hsp90 complex (Fig.3B, cf. lanes 7 and 3).
Figure 3:
The Centricon C-100 filtrate of
reticulocyte lysate stabilizes the GR-hsp90 complex during the 30
°C incubation with wheat germ lysate. A, effect of C-100
filtrate on steroid binding activity. Immunoadsorbed, stripped
receptors were incubated with rabbit reticulocyte lysate (solid
bars), wheat germ lysate (open bars), or wheat germ
lysate plus Centricon C-100 filtrate of reticulocyte lysate (hatched bars), and steroid binding was assayed under three
conditions. Condition 1,
[H]triamcinolone acetonide was present during the
20 min reconstitution at 30 °C, samples were centrifuged, washed,
and radioactivity in the immunopellet was assayed. Condition
2, following the 20-min reconstitution, the entire reconstitution
mix was placed on ice for 20 min,
[
H]triamcinolone acetonide was then added and the
mix was incubated overnight at 0 °C. Condition 3, after
the 20-min reconstitution, samples were washed once with 1 ml of cold
TEGM buffer and then incubated overnight at 0 °C with
[
H]triamcinolone acetonide. B, effect of
C-100 filtrate on the amount of plant hsp90 recovered with the GR in
the same experiment. Lane 1, stripped receptors; lanes 2 and 3, stripped nonimmune and immune pellet,
respectively, incubated with wheat germ lysate; lanes 4 and 5, stripped nonimmune and immune pellets incubated with the
Centricon C-100 filtrate of reticulocyte lysate; lanes 6 and 7, stripped nonimmune and immune pellets incubated with both
wheat germ lysate and Centricon C-100 filtrate. The conditions of lanes 3 and 7 in B are the same as the open bar and hatched bar, respectively, shown in
section 3 of A.
Fig.4A shows
the fractional precipitation of rabbit reticulocyte lysate with
ammonium sulfate and the distribution of the hsp heterocomplex proteins
in each fraction. Fig.4B shows the ability of each
fraction to stimulate formation of a GR-hsp90 complex when added to
wheat germ lysate. As we have reported previously (16, 19) , the ammonium sulfate fractions of
reticulocyte lysate do not form complexes between the GR and rabbit
hsp90. However, the 60-85% fraction of reticulocyte lysate
stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate, and a
small amount of activity is present in the 45-60% fraction (Fig.4B, lanes 8 and 7,
respectively). Two-thirds of the hsp90 and 90% of the hsp70 are
precipitated below 60% ammonium sulfate, with the remainder of each
being present in the 60-85% fraction (Fig.4A).
In addition, the 60-85% fraction contains a trace amount of p60
and almost all of the p23 (a small amount of p23 that can be seen in
the 45-60% fraction in the original blot is not seen in the
photograph).
Figure 4: Ammonium sulfate fractionation of activity from reticulocyte lysate. Rabbit reticulocyte lysate was sequentially fractionated with ammonium sulfate at 30, 45, 60, and 85% of saturation, and each fraction was dialyzed and contracted to one-half the original lysate volume. A, aliquots of each fraction were resolved by SDS-PAGE and either stained with Coomassie blue (left) or immunoblotted with antibodies (right) to detect the components of the hsp heterocomplex. B, each fraction was then assayed for its ability to stimulate GR-hsp90 reconstituting activity by wheat germ lysate. Lane 1, stripped receptor; lane 2, stripped receptor plus rabbit reticulocyte lysate; lane 3, stripped receptor plus wheat germ lysate; lane 4, stripped receptor plus wheat germ lysate and Centricon C-100 filtrate; lane 5, stripped receptor plus wheat germ lysate plus 0-30% ammonium sulfate fraction; lane 6, plus lysate and 30-45% fraction; lane 7, plus lysate and 45-60% fraction; lane 8, plus lysate and 60-85% fraction; lane 9, plus lysate and 85% supernatant.
p23 is more acidic than the other components of the hsp heterocomplex and can be separated from them by chromatography on DE52. As with ammonium sulfate fractionation, the GR-hsp90 assembly/receptor activating activity of reticulocyte lysate is destroyed by ion exchange chromatography. However, a fraction obtained by DE52 chromatography has the ability to stimulate GR-hsp90 heterocomplex assembly by wheat germ lysate. In the experiment shown in Fig.5A, reticulocyte lysate was fractionated on a DE52 column, each fraction was assayed for each member of the hsp heterocomplex by Western blotting (not shown), and fractions were combined into four pools designated A-D indicated by the bars under the figure. As shown in Fig.5B, addition of fraction C to wheat germ lysate (condition 6) stimulates GR-hsp90 heterocomplex assembly by wheat germ lysate. The only component of the hsp heterocomplex in fraction C is p23 (Fig.5A), and the rabbit p23 is recovered with the GR-plant hsp90 complex (Fig.5B, condition 6) formed in the wheat germ lysate.
Because the ability to stimulate GR-hsp90 heterocomplex assembly by wheat germ lysate segregated with p23 on fractionation of reticulocyte lysate, we asked if purified, bacterially expressed human p23 would stimulate GR-hsp90 heterocomplex formation. As shown in condition 3 of Fig.5B, the purified p23 stimulated formation of GR-plant hsp90 heterocomplexes by wheat germ lysate and it is a component of receptor heterocomplexes formed in wheat germ lysate. The bacterially expressed p23 is greater than 90% pure by Coomassie Blue staining of a gel-resolved sample.
Figure 6: p23 accounts for the wheat germ stimulating activity of the Centricon C-100 filtrate. A, p23 is present in the Centricon C-100 filtrate. One ml of reticulocyte lysate was centrifuged in a Centricon C-100 concentrator, both the retentate (0.3 ml) and the filtrate (0.7 ml) were diluted to 1 ml with buffer, and a portion of the filtrate was concentrated 10-fold. Aliquots of each fraction were resolved by SDS-PAGE, and hsp90 and p23 were assayed by Western blotting. Lane 1, whole reticulocyte lysate; lane 2, Centricon C-100 filtrate, lane 3, Centricon C-100 retentate; lane 4, 10-fold concentrated Centricon C-100 filtrate. B, depletion of p23 from Centricon C-100 filtrate with the JJ5 antibody. Aliquots of 10-fold concentrated Centricon C-100 filtrate were immunoadsorbed with JJ5 or nonimmune IgG prebound to protein A-Sepharose, and p23 was assayed in the supernatant by Western blotting. Lane 1, Centricon C-100 filtrate; lane 2, Centricon C-100 filtrate extracted with nonimmune IgG; lane 3, Centricon C-100 filtrate extracted with JJ5. C. Activity of p23-depleted Centricon C-100 filtrate. The Centricon C-100 filtrate preparations shown in B were assayed for their ability to stimulate GR-hsp90 reconstitution in wheat germ lysate. Stripped nonimmune and immune pellets (left and right lanes, respectively) were incubated with wheat germ lysate in all conditions. Condition 1, wheat germ lysate alone; condition 2, lysate plus C-100 filtrate; condition 3, lysate plus 1 µg purified p23; condition 4, lysate plus C-100 filtrate extracted with nonimmune IgG; condition 5, lysate plus C-100 filtrate extracted with JJ5; condition 6, lysate plus JJ5-extracted C-100 filtrate and 1 µg purified p23.
Although more than 90% of the p23 is retained by the filter (Fig.6A, lane 3), it seems that a sufficient amount passes through it to account for the activity of the filtrate. Extraction of the Centricon C-100 filtrate with the JJ5 antibody depletes it of p23 (Fig.6B, cf. lanes 1 and 3). As shown in Fig. 6C, Centricon C-100 filtrate that is depleted of p23 does not stimulate GR-hsp90 heterocomplex formation by wheat germ lysate (cf. condition 5 with condition 1) and addition of purified, bacterially expressed p23 to the p23 depleted lysate restores activity (condition 6). The effect of p23 on receptor heterocomplex assembly by wheat germ lysate is concentration-dependent. As shown in Fig.7, peak activity is achieved with addition of 0.5-1 µg of purified p23 to the 56 µl wheat germ reconstitution mix. At higher concentrations, there is less activity, and an addition of 20 µg p23 yields no stimulation of the wheat germ lysate.
Figure 7: Concentration dependence of p23 effect in wheat germ lysate. Immunoadsorbed, stripped receptors were incubated with wheat germ lysate plus various concentrations of p23 and steroid binding was assayed. Lane 1, stripped receptor plus wheat germ lysate alone; lanes 2-6, receptors plus lysate and 0.1 µg (lane 2), 0.5 µg (lane 3), 1 µg (lane 4), 2.5 µg (lane 5), 10 µg (lane 6), 20 µg (lane 7) of purified bacterially expressed p23.
Figure 8: p23 accounts for the ability of the Centricon C-100 filtrate to restore receptor activating activity to the washed foldosome. A, depletion of p23 inactivates the Centricon C-100 filtrate. Aliquots of Centricon C-100 filtrates were immunoadsorbed with JJ5 or nonimmune IgG prebound to protein A-Sepharose, and samples were incubated with stripped receptors and the washed 3G3 immunopellet of reticulocyte lysate. Lane 1, stripped receptor; lane 2, stripped receptor plus Centricon C-100 filtrate alone; lane 3, stripped receptor plus washed 8D3 immunopellet of reticulocyte lysate; lanes 4-6, condition of lane 3 plus Centricon C-100 filtrate (lane 4), filtrate extracted with nonimmune IgG (lane 5), filtrate extracted with JJ5 (lane 6). B, addition of purified human p23 to the washed foldosome increases receptor activating activity. Stripped receptors (lane 1) were incubated with the washed 8D3 immunopellet of reticulocyte lysate plus filtrate extracted with JJ5 (lane 2) or these conditions plus 0.1 µg (lane 3), 0.3 µg (lane 4), 1 µg (lane 5), or 3 µg (lane 6) of purified human p23.
The fact that human p23 interacts with the components of the
wheat germ lysate to stimulate receptor heterocomplex assembly and
folding attests to the conservation of function and ubiquitous
distribution of the hsp heterocomplex chaperone system. Although the
anti-p23 antibodies recognize a 23-kDa protein in a broad range of
species from yeast to man, they do not react with a similar protein in
wheat germ. The fact that rabbit and human p23s stimulate the wheat
germ system could reflect either the lack of an animal p23 homolog in
the plant lysate, or it could reflect different properties between the
animal p23s and an as yet undetectible plant homolog.
The effect of p23 in the wheat germ lysate might involve either acceleration of GR-hsp90 heterocomplex assembly or stabilization of newly assembled complexes to dissociation during the reconstitution incubation at 30 °C (Fig.3). The effect of p23 is such that the number of GR-hsp90 complexes present in wheat germ lysate at the end of 20 min is higher (Fig.3, condition 2), but the total number of GR-hsp90 complexes generated (i.e. those that can be ``trapped'' by steroid binding during the 20 min incubation) remains the same (Fig.3, condition 1). At the moment, it seems quite reasonable to assume that p23 is required for receptor-hsp90 heterocomplex assembly and its presence in the receptor heterocomplex may contribute to, or even be necessary for, maintaining the particularly high affinity interaction between the steroid receptors and hsp90. In both the wheat germ system (Fig.7) and the washed foldosome (Fig.8B), p23 is less active when present at high concentrations. It is possible that p23 aggregates at high concentration, but it is also possible that the stoichiometry between p23 and hsp90 and/or other components of the system must be optimal for efficient receptor heterocomplex assembly.
The
identification of p23 as an essential component that can restore
GR-activating activity to the washed hsp heterocomplex is an important
step toward total reconstitution of a functional foldosome. We now have
an immunopurified hsp90 heterocomplex (the washed foldosome) to which
purified p23 and the required dialyzable components of the receptor
heterocomplex assembly system, ATP/Mg and
K
(6, 7) , can be added. The
essential elements of the washed foldosome must now be identified and
reconstituted into a functional chaperone system. The washed 3G3
immunopellet contains hsp90, hsp70, p60, hsp56, p50, and CyP-40. The
peptidylprolyl isomerase activity of the two immunophilins, hsp56 and
CyP-40 is apparently not required for GR-hsp90 heterocomplex assembly,
because assembly is not affected by the inhibitors FK506 and
cyclosporin A(20) .
Also, hsp56 can be removed from
the system without affecting assembly activity.
hsp70 is
required for receptor binding to hsp90 (6, 8) , but
hsp70 and hsp90 do not form a complex with each other unless they are
incubated with a third factor (or factors) from reticulocyte lysate and
ATP/Mg
(9) . It seems this third factor must
be required for generating an active foldosome, and it may or may not
be present in the hsp90 complexes we have immunoadsorbed with the 3G3
antibody. Given the possibility that the immunophilins are not required
for assembly of a receptor-hsp90 complex, it is reasonable to focus on
potential requirements for p60 and p50 in promoting association of
hsp90 and hsp70 and in reconstitution of a core foldosome complex that
is active at receptor heterocomplex assembly when it is supplemented
with ATP/Mg
, K
, and p23.
Although the Centricon C-100 filtrate and bacterially expressed p23 facilitate reconstitution of the GR-hsp90 complex, the addition of either preparation never gives more than about 55% of the binding activity seen with reticulocyte lysate. It is possible that factors other than those discussed above are required to achieve the full activity of the reticulocyte lysate. As we have noted before(16) , eukaryotic homologs of the bacterial DnaJ and GrpE proteins may be required for full folding activity.