THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for ’ Biochemistn I and Molecular ’ Biology, Inc.

Vol. 269, No. 22, Issue of June 3, pp. 15718-15723, 1994 Printed in U.S.A.

hsc70 Moderates the Heat Shock (Stress) Response in Xenopus laevis Oocytes and Binds to Denatured Protein Inducers*

(Received for publication, March 4, 1994)

Laura C. Mifflin$ and Robert E. CohenS§n From the $.Department of Chemistry and Biochemistry and The Molecular Biology Institute, University of California, Los Angeles, California 90024 and the $Department of Biochemistry, University of Zowa, Zowa City, Zowa 52242

Injections of hsc70 protein into Xenopus oocytes low- ered the stress response to both a thermal shock and to co-injected protein inducers. Binding of hsc70 to native and modified forms of bovine serum albumin (BSA) were tested by two assays. In one, nitrocellulose-bound proteins were incubated with hsc70, cross-linked with glutaraldehyde, and then bound hsc70 was probed with a monoclonal anti-hsc70 antibody. In the second, per- oxidase-conjugated hsc70 was employed as a more di- rect probe for binding to proteins displayed on nitro- cellulose membranes. hsc70 binding to the BSA derivatives correlated with their abilities to induce a stress response upon microinjection into oocytes. Re- duced and carboxymethylated (rcrn) BSA, the most po- tent stress inducer tested, was bound most tightly by hsc70, whereas hsc70 had moderate affinity for N-methylated rcm-BSA and very little affinity for the native protein. No binding was observed with iodinated BSA The results suggest a mechanism whereby the cell employs hsc70 or other proteins of the hsp70 family both to trigger the response to an environmental stress and to provide a feedback mechanism to attenuate the response.

A wide variety of environmental stresses, and not only heat, can induce the classical heat shock or stress response. A pos- sible mechanism is that denatured proteins, which accumulate as a consequence of the stress, are recognized to trigger the stress response machinery (Hightower, 1980). This idea was supported by experiments in which the microinjection of dena- tured proteins into Xenopus laevis oocytes induced the stress response, whereas introduction of the corresponding native proteins had little effect (Ananthan et al., 1986; Mifflin and Cohen, 1994).

The stress response is characterized by the selective synthe- sis of a specific set of proteins, the heat shock proteins (hsps;’ for reviews, see Lindquist and Craig (19881, Schlesinger (1990), and Hightower (1991)). In addition to their greatly enhanced

* This work was supported in part by United States Public Health Service Grant R01 GM37666 (to R. E. C.) and a fellowship from United States Public Health Service Training Grant GM07185 (to L. C. M.). Preliminary results have been reported in abstract form ((1991) Stress Proteins & The Heat Shock Response, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dept. of Biochemistry, University of Iowa, Iowa City, IA 52242-1109. n To whom correspondence and reprint requests should be addressed:

Tel.: 319-335-8545; Fax: 319-335-9570. The abbreviations used are: hsp, heat shock protein; BSA, bovine

N-methylated; cbm, carbamoylated; rca, reduced and carboxamidom- serum albumin; rcrn, reduced and carboxymethylated; me, reductively

ethylated; I-BSA, iodinated BSA; HSF, heat shock transcription factor; PAGE, polyacrylamide gel electrophoresis.

synthesis upon environmental stress, many hsp’s or their close analogs (hsc proteins) are expressed constitutively under non- stress conditions. Studies of specific hsphsc proteins have re- vealed a chaperone-like involvement in the binding of other, partially unfolded proteins. This may be to catalyze protein folding and assembly, to prevent aggregation or preclude del- eterious interactions with other proteins, to facilitate protein disassembly or turnover, and to assist in the translocation of polypeptides across membranes (reviewed by Lindquist and Craig (19881, Welch (19901, Gething and Sambrook (1992), and Georgopoulos and Welch (1993)). By interacting with the dena- tured or misfolded proteins presumed to arise from a stress condition, hsps may limit irreversible protein aggregation or further denaturation. This behavior suggests a possible link between the recognition of denatured protein and the induction of the stress response.

Reduced expression or mutation of hsp7O can result in the overexpression of other hsps (Solomon et al., 1991; Boorstein and Craig, 1990) and overproduction of an hsp70 protein in yeast suppresses hsp gene expression (Stone and Craig, 1990). From these and other observations that implicated a role for hsp7O in its own and other hsp expression, hsp70 was proposed to be autoregulated and to effect a negative control on the stress response (DiDomenico et al., 1982; Craig and Jacobsen, 1984; Lindquist, 1986; reviewed by Craig and Gross (1991)). hsp7O proteins may limit the stress response by preventing the accumulation of denatured, aggregated proteins which can act as stress response inducers (Ananthan et al., 1986; Mifflin and Cohen, 1994). Another possibility is that hsp70 more directly interacts with the transcription apparatus to down-regulate hsp expression. Genetic studies in yeast (Stone and Craig, 1990) and reports of an association between hsp7O and the human heat shock transcription factor (Abravaya et al., 1992; Baler et al., 1992) support this idea. These two scenarios for the involvement of hsp7O in stress response regulation are not mutually exclusive, and it has been difficult to distinguish them experimentally. A major problem has been to identify molecules that can function in vivo as stress response inducers. Once this is accomplished, it should be possible to test for recognition of inducers by hsp70.

The stress response can be assayed in X. laevis oocytes as the expression of P-galactosidase from a lac2 gene controlled by a hsp70 promoter (Voellmy and Rungger, 1982; Ananthan et al., 1986; Mimin and Cohen, 1994). A characteristic of the proteins found to induce a stress response when injected into oocytes is their propensity to aggregate (Mifflin and Cohen, 1994). Dena- tured proteins produced by an environmental stress might ag- gregate as hydrophobic surfaces normally buried within their native structures are exposed. Protein aggregation in nuclei and, particularly, nucleoli of stressed cells has been described (Pelham, 1984; Lewis and Pelham, 1985; Beckmann et al., 1992), and hsc7O has been found associated with these aggre- gates (e.g. Dubois et al., 1991). Moreover, the nonlinear in-

15718

hsc70 and Induction of the Stress Response crease of the stress response elicited by increasing amounts of injected protein inducers is consistent with the titration of a regulatory factor (Mifflin and Cohen, 1994). An obvious candi- date for this factor is hsc70, an abundant 73-kDa constitutive protein of the hsp70 family that can bind to denatured and newly synthesized proteins (Beckmann et al. (1992) and refer- ences therein).

Microinjection into Xenopus oocytes was employed here to study directly the ability of hsc70 to reduce the stress response in vivo. hsc70 is shown to attenuate the response to stresses from both thermal shock and injections of denatured protein. In addition, by the use of two in vitro binding assays, derivatives of BSA that act as stress response inducers in the Xenopus oocyte system were found to bind preferentially to hsc7O. These results are discussed in terms of a general model for stress response induction.

MATERIALS AND METHODS

Oocyte Injections, Stress Peatments, and Oocyte Extract Preparation

X. laevis oocytes were generously provided by Dr. C. Gunderson (Dept. of Pharmacology, UCLA). Stage V and VI oocytes were selected and prepared for microinjection as described in the accompanying paper (Mifflin and Cohen, 1994). Injection solutions included 1.0 mg/ml plas- mid p622C* in the Injection Buffer (IO m~ sodium HEPES, pH 7.5, 68 mM NaCU with 0.12 mg/ml [metho~y-~HIinulin (DuPont NEN, 0.25 mCi/ ml). Where employed, cytoplasmic injection of hsc70 protein followed a separate nuclear injection of the reporter plasmid p622C*. Typically, batches of 10-20 oocytes were subjected to a given treatment. Injection volumes were determined by the use of Lmetho~y-~Hlinulin as a tracer, and cells were incubated in Barth's solution for 14-17 h at 19 "C fol- lowing injection. For a thermal stress, cells were incubated for 1.5 h at 34 "C before transfer to 19 "C. Extract preparation and P-galactosidase assays were as described (Mifflin and Cohen, 1994). The value for the negative control (plasmid-only injected oocytes) was subtracted, and the corrected @galactosidase activities typically are presented relative to the positive control (plasmid injection, followed by a thermal shock).

Protein Modification Native and modified forms of BSA and a-crystallin were prepared

and characterized as described (Mifflin and Cohen, 1994); bovine a-lact- albumin (Type I, Sigma) was reduced and carboxymethylated by similar procedures.

Preparation of hsc70 Initial samples of bovine hsc70 (the 73-kDa constitutively expressed

"heat-shock cognate" protein) were gifts from Dr. Richard Glickman (StressGen Biotechnologies, Victoria, B.C., Canada) and Drs. Seth Sa- dis and Lawrence Hightower (Univ. of Connecticut, Storrs, CT). Subse- quently, material was isolated from bovine brain essentially as de- scribed by Sadis et al. (1990). Fresh bovine brain from a local slaughterhouse was transported on ice to the laboratory. All of the following purification steps were performed at 4 "C. Homogenization in a Waring blender with ice-cold BufferA(25 mM Tris-HC1, pH 7.0,O.l mM EDTA) containing 1 m~ phenylmethylsulfonyl fluoride was followed by centrifugation for 30 min at 15,000 x g. The supernatant liquid was further centrifuged in a Beckman Ti45 rotor (60 min at 44,000 rpm) and then applied to a 225-m1 column of Q-Sepharose Fast Flow (Pharmacia Biotech, Inc.) at a rate of 1 to 2 mumin. The column was eluted with a 600-ml linear gradient of 0 to 0.6 M KC1 in Buffer A. Fractions contain- ing hsc7O were identified by SDS-PAGE (7.5% acrylamide; Laemmli (1970)), pooled, and dialyzed against >IO volumes of Buffer B (20 mM potassium HEPES, pH 7.0,25 mM KCI, 10 m~ (NH,)$O,, 0.1 mM EDTA, and 1 mM dithiothreitol). The dialyzed fractions were clarified by cen- trifugation at 44,000 rpm for 60 min in a Ti45 rotor prior to the addition of MgfOAc), to 2 mM and loading onto an ATP-agarose column (Sigma, C8 linkage). The column was washed sequentially with several column volumes each of Buffer B, 1 M KC1 in Buffer B, and Buffer B. The hsc70 was then eluted at 1 mumin with Buffer B containing 1 m~ ATP. Frac- tions containing hsc70 were identified by SDS-PAGE and pooled. hsc70 was dialyzed against Injection Buffer, filter-sterilized, and either stored as small aliquots at -80 "C after freezing in liquid nitrogen or stored at 4 "C. Approximately 250 g of tissue yielded from 1 to 5 mg of hsc70 that was greater than 97% pure as determined by SDS-PAGE.

15719

Hsc70 Injected (ng per oocyte)

FIG. 1. Injection of hsc7O lowers the response to a thermal stress in oocytes. hsc7O was injected into the nuclei of oocytes that were then heated for 1.5 h at 37 "C. The stress response was assayed by measuring /%galactosidase expression from the co-injected reporter plasmid p622C*, and activities were determined relative to a negative control and a positive, thermally stressed control, as described under "Materials and Methods." The different symbols represent data from four separate experiments.

Assays for Protein Binding by hsc70 Two methods to evaluate binding of proteins by hsc70 were employed.

In both, proteins in Injection Buffer were applied onto hydrated nitro- cellulose membranes using a slot blot template (Bio-Rad). The mem- branes were then blocked by soaking in Injection Buffer with 5% (wh) BSAfor 1 h at room temperature. Filters were rinsed in Injection Buffer prior to hsc7O binding. The use of BSA as a blocking protein gave acceptable low backgrounds with the protocols outlined below, whereas higher backgrounds were obtained when filters were blocked with casein or nonfat dry milk.

Preparation and Binding of Horseradish Peroxidase-linked hsc70 to BZotted Proteins-Horseradish peroxidase (50 mg/ml; Sigma, Type VI) in Injection Buffer containing 1% (v/v) glutaraldehyde was incubated at room temperature for 18 h (Avrameas et al., 1978). Excess glutaralde- hyde was removed by centrifugation of the mixture through a 1-ml Sephadex G-25 column equilibrated in Injection Buffer (Penefsky, 1979). The glutaraldehyde-modified peroxidase (200 pg) was then coupled to hsc7O (50 pg) by incubation for 24 h at 4 "C in Injection Buffer (0.1 ml total volume) that contained 50 ~ M A D P and 50 p~ MgCI,.

Blotted proteins were incubated overnight a t room temperature with the peroxidase-conjugated hsc7O in 10 ml of Injection Buffer containing 0.1% Nonidet P-40, 1 mM ADP, and 1 mM MgCI,. Filters were rinsed in Injection Buffer, and peroxidase activity was detected by chemilumi- nescence using the ECL Western Blotting Kit (Amersham Corp.) ac- cording to manufacturer's instructions. Films (Kodak X-Omat A R ) were exposed to filters for between 5 s and 10 min, depending upon the sensitivity required, and then developed.

Cross-linking of hsc70 to Blotted Proteins-In this second assay, hsc70 was incubated with the filter-bound proteins in Injection Buffer containing 0.1% Nonidet P-40, 1 mM ADP, and 1 mM MgCl, for 2 to 4 h at 37 "C. Competition studies additionally included a 10-fold excess by weight of a designated protein over the amount of hsc70 in the incuba- tion buffer. The filter then was transferred to a solution of 1% glutar- aldehyde in Injection Buffer and incubated for 30 min at room tempera- ture. The filter was washed extensively in Injection Buffer and then incubated for 1 to 2 h at room temperature in a 1:10,000 to 1:15,000 dilution of a mouse monoclonal anti-human hsp70 antibody (IgG clone 5A5; from Affinity BioReagents, Neshanic Station, NJ) in Injection Buffer. This antibody recognizes an epitope in the ATP-binding domain of hsp70 and cross-reacts with hsc70 and other hsp7O-family proteins from a broad range of species, including bovine hsc7O. After rinsing, the filter was incubated for 1 to 2 h at room temperature with rabbit anti-mouse IgG conjugated to horseradish peroxidase (Calbiochem Corp.) diluted 1:10,000 to 1:15,000 in Injection Buffer. The filter was rinsed, and the presence of peroxidase was detected with the ECL reagent as described above.

RESULTS

hsc70 Injection Lowers the Stress Response to a Thermal Shock-Purified bovine hsc70 was injected into oocytes to de- termine whether it could moderate the stress response. As seen in Fig. 1, nuclear injections of hsc7O attenuated the response to

15720

0.12

hsc70 and Induction of the Stress Response

‘ Exveriment

A C

FIG. 2. Co-injection of hsc70 lowers the stress response of oocytes to rcm-BSA and me-a-crystallin. Injection protocols and assays for P-galactosidase expression from p622C“ are described under ”Materials and Methods.” Upper panel, in two separate experiments (A and B ), rcm-BSA(-lOO ng per oocyte) was injected alone (Light bars) or with hsc7O (-20 ng; black bars). Lowerpanel:A, me-a-crystallin (240 ng per oocyte) was injected alone; B, me-a-crystallin (200 ng per oocyte) co-injected with hsc70 (16 ng); C, me-a-crystallin (300 ng per oocyte) co-injected with hsc70 (25 ng).

a thermal shock. In several independent experiments, from 3 to 10 ng of hsc70 reduced the stress response by 20% to 60%, and increasing the amount of injected hsc70 further reduced the response. The variability in the data in part reflects differences among batches of oocytes, which varied in the magnitude of their stress response as assessed by P-galactosidase expression. Another contribution to the variability of the response among different experiments may have been the condition of the dif- ferent hsc7O protein stocks (see “Materials and Methods”). The injected hsc70 did not adversely affect the survival of the ther- mally stressed oocytes and actually may have protected the cells from the stress.2 In contrast to the results with nuclear injections, cytoplasmic injections of 7, 15, and 22 ng of hsc7O per oocyte in three separate experiments failed to reduce the stress response to a thermal shock (data not shown).

hsc70 Co-injection Lowers the Response to Stress Znducer Proteins-Co-injections of hsc7O with rcm-BSA, a denatured protein that induces the stress response when injected into X. laevis oocytes (Ananthan et al., 1986; Mifflin and Cohen, 1994), significantly reduced the stress response (Fig. 2, upper panel ). As little as 20 ng of hsc70 almost completely eliminated the stress response produced by 100 ng of rcm-BSA. These proteins have approximately the same subunit molecular weight, and,

Oocyte survival was assessed by visual inspection. Loss of pigmen- tation from the animal hemisphere of the cell and/or diffusion of pig- ment into the vegetal hemisphere was indicative of cell distress and death.

therefore, on either a mass or mole basis, a 5-fold excess of rcm-BSA is effectively inactivated as a stress response inducer by hsc70. Because the stress response to denatured protein is nonlinear (Mifflin and Cohen, 1994), small changes in the amount of the inducer rcm-BSA, particularly with as little as 100 ng injected per oocyte, can significantly affect the magni- tude of the response. However, the difference in the responses with and without the co-injection of hsc70 greatly exceeds what would be expected from the effective inactivation of only 20 ng of the 100 ng of rcm-BSA injected.

Co-injection of hsc7O with another stress-inducing protein, me-a-crystallin, also produced a dramatic decrease in the stress response (Fig. 2, lower panel). Although the mole ratio of me-a-crystallin to hsc7O (-40:1, based upon subunit molecular weights) was identical in both injections, injection of the solu- tion with a greater amount of hsc7O protein lowered the re- sponse more. Thus, attenuation of the response to a particular protein inducer may depend less on the ratio of the injected hsc70 and inducer protein than upon the total amount of hsc70 injected.

hsc70 Binding to Stress Response Znducer Proteins-Protein aggregation appears to be an important factor in eliciting a stress response to proteins injected into X. laevis oocytes (Mif- flin and Cohen, 1994). hsc7O is often associated with aggre- gated protein, particularly under conditions of stress (reviewed by Pelham (1990)), and the recognition and solubilization of aggregated protein by hsc7O may play a role in stress response induction. Injection studies had demonstrated a high stress response activity following introduction of rcm-BSA, little re- sponse to me-rcm-BSA, and no response to BSA and I-BSA (Mifflin and Cohen, 1994). I t therefore was of interest to deter- mine whether hsc70 differentially binds to these modified and native forms of BSA.

hsc7O binding to rcm-a-lactalbumin (but not native a-lactal- bumin) was demonstrated by identification of complexes by gel filtration chromatography (Palleros et al., 1991), and native gel electrophoresis has been used to assay hsc7O binding to apocy- tochrome c (Sadis et al., 1990). hsc7O binding to rcm-a-lactal- bumin was repeated successfully here by the use of the Laemmli gel system (Laemmli, 1970) without addition of SDS (data not shown). These experiments were done to verify the activity of the hsc7O preparations used here. However, because BSA (66 kDa) and hsc7O (73 kDa) have similar molecular masses, these methods to assay binding would fail to distin- guish oligomers of one protein from another or from hetero- oligomers that included both. Moreover, under nondenaturing conditions, rcm-BSA appears as a smear of high molecular weight species which could obscure any new complexes. For these reasons, gel chromatography and gel electrophoresis were considered impractical to assess the binding by hsc7O to BSA or its derivatives. Instead, assays were developed based upon binding to proteins absorbed onto nitrocellulose mem- brane filters. Evidence for differential binding of hsc7O to BSA, rcm-BSA, me-rcm-BSA, and I-BSA is described below.

Horseradish Peroxidase-conjugated hsc70 Binds to Nitrocel- lulose-blotted Protein-Peroxidase-conjugated hsc7O bound rcm-BSA preferentially over me-rcm-BSA (Fig. 3). Significant hsc7O binding was evident to as little as 20 ng of rcm-BSA, whereas very little hsc7O bound to 70 ng of blotted me-rcm- BSA, and no binding could be detected with 20 ng of me-rcm- BSA. No binding of peroxidase-conjugated hsc7O to as much as 100 ng of I-BSA could be detected under these conditions. The ability to use BSA as a blocking protein on the nitrocellulose indicates that binding by hsc70 to native BSA is negligible in comparison to rcm-BSA and me-rcm-BSA.

Direct Cross-linking of hsc70 to Nitrocellulose-bound Protein-The preferential binding of hsc7O to rcm-BSA over

hsc70 and Induction of the Stress Response 15721

A B B

14

2 -

3 “

4

FIG. 3. Binding of BSA derivatives to a horseradish peroxi- dase-conjugate of hsc7O. Horseradish peroxidase-linked hsc70 was prepared as described under “Materials and Methods.” Proteins were applied with a slot-blot template to a nitrocellulose filter which was then blocked with BSA and incubated with peroxidase-linked hsc7O overnight at room temperature. Bound peroxidase-hsc70 conjugate was detected with the ECL chemiluminescence reagent. Lanes A and B for rows 1 3 have rcm-BSA and me-rcm-BSA, respectively. Row 1 has 100 ng of protein, row 2 has 70 ng of protein, and row 3 has 20 ng of protein. In row 4, I-BSA was applied at 100 ng (lane A ) and 70 ng (lane B) .

I-BSA was repeated in a different assay in which hsc7O was cross-linked directly with glutaraldehyde to blotted rcm-BSA, once again, no binding to I-BSA was apparent (Fig. 4). In this assay, binding was detected with a monoclonal mouse anti- hsp7O antibody followed by anti-mouse IgG conjugated to horseradish peroxidase. There was no significant binding of hsc7O to BSA, as BSA could be used as a blocking protein without interference in these assays.

Because the glutaraldehyde cross-linking is predominantly through primary amines on lysines, binding to me-rcm-BSA could not be tested directly with this assay. For this reason and also to eliminate any effect that attachment to nitrocellulose could have on the recognition of proteins by hsc70, competition assays were used to evaluate binding preferences of hsc7O for proteins in solution. These experiments confirmed the prefer- ential binding of hsc7O to rcm-BSA over me-rcm-BSA (Fig. 5). A 10-fold excess of rcm-BSA over hsc7O prevented hsc70 binding to blotted rcm-BSA, whereas a 10-fold excess of me-rcm-BSA only partially reduced the amount of hsc7O bound to blotted rcm-BSA.

The binding of hsc7O to modified a-crystallins could not be detected, possibly because dilution (to less than 10% of their injected concentrations) may have affected their aggregation states and binding efficiencies. Other complications with these proteins might have arisen from the ability of a-crystallin itself to serve as a chaperone (Horwitz, 1993; Jakob et al., 1993). Binding of a-crystallin to the blocking proteins on the nitrocel- lulose filters or to the band of rcm-BSAin the competition assay most likely would prevent detection of an a-crystallin:hsc70 interaction. Use of a-crystallin derivatives in the in vitro bind- ing assays therefore was not pursued further.

The results with BSA and modified BSA, however, are con- sistent with the idea that hsc70 binding is a key recognition step in the initiation of the stress response. I-BSA and me-rcm- BSA injected into oocyte nuclei induce no or very little stress response, respectively, whereas rcm-BSA is an effective stress response inducer. Thus, the ability of these proteins to induce a stress response correlates with their binding to hsc70 in vitro.

DISCUSSION Due to the ability of diverse environmental stresses to induce

the stress response, denatured protein, a probable result of the stress condition, was proposed to serve as the common signal for induction of a response (Hightower, 1980). Accordingly, three chemically denatured proteins were found to induce the

2

FIG. 4. hsc70 is cross-linked to nitrocellulose-bound rcm-BSA but not I-BSA. Proteins (100 ng) were applied to a nitrocellulose filter which was then blocked with BSA. hsc70 (30 pg in 1.5 ml) was incu- bated for 4 h at 37 “C with the filter, which was then treated with 1% (vh) glutaraldehyde as detailed under “Materials and Methods.” Bound hsc7O was detected with a monoclonal anti-hsc70 antibody and the ECL reagent system as described under “Materials and Methods.” I , rcm- BSA, 2, I-BSA, 3, hsc70 (positive control).

A B C

1

2

3 L-” A i l

FIG. 5. Competition for hsc7O binding to rcm-BSA. Proteins were applied to nitrocellulose filters which were then blocked with BSA. hsc70 (30 pg in 1.0 ml) was incubated with the filters in the presence of: A, no additional protein; B, rcm-BSA (300 pg); and C, me-rcm-BSA (300 pg). Glutaraldehyde cross-linking and detection of bound hsc70 were as described in Fig. 4. Row 1 had 200 ng of rcm-BSAapplied; row 2,100 ng of rcm-BSA, and row 3, 100 ng of hsc7O (positive control).

stress response when injected into X. laevis oocytes, whereas their native counterparts did not (Ananthan et al., 1986). Ex- tension of this work revealed that denaturation alone was not sufficient to induce a stress response; instead, extensive aggre- gation of the denatured protein correlated with its ability to invoke a stress response (MiMin and Cohen, 1994). The mecha- nism by which the cell selectively recognizes denatured protein, however, is unknown.

hsp70, a protein expressed during the stress response, has been suggested to be involved in the regulation of the stress response (DiDomenico et al., 1982; Craig and Gross, 1991). Many observations support this role for hsp7O. Excess hsp70 can confer thermotolerance (Angelidis et al., 1991), whereas reduced levels can result in constitutive hsp expression (Craig and Jacobsen, 1984; Boorstein and Craig, 1990; Solomon et al., 1991). hsp70 has been found associated with the nuclei of stressed cells (Pelham, 1984; Welch and Suhan, 19861, and its regular cellular functions may include limiting intermolecular interactions among partially folded proteins and assisting in the refolding of unfolded proteins (reviewed by Gething and Sambrook (1992)), the levels of which might increase upon heat shock. hsc70, a member of the hsp7O protein family, can solu- bilize aggregated proteins (Braell et al., 1984; Dubois et al., 1991) and is found associated with aggregated proteins in stressed cells (Welch and Suhan, 1986; Beckmann et al., 1992). The demonstration of hsc7O association with HSF, the heat shock transcription factor (Abravaya et al., 1992; Baler et al., 19921, suggests a direct link to stress response regulation. Fur- thermore, in conjunction with its role in facilitated transport through the nuclear pore (Shi and Thomas, 1992), hsc7O may be responsible for transporting HSF into and out of the nucleus.

15722 hsc70 and Induction of the Stress Response

A model proposed by Abravaya et al. (1992) suggests that bind- ing of HSF by hsc7O modulates the stress response via inacti- vation of HSF. According to the model, competition for hsc70 binding by an increased pool of unfolded proteins produced by the stress condition results in the release and activation of the HSF.

In support of a protective or regulatory role for hsc70, its injection into Xenopus oocytes was observed to reduce the re- sponse to a thermal stress. Similar results were found in co- injection studies of hsc7O with denatured proteins that induce the stress response, rcm-BSA, and me-a-crystallin. The nonlin- ear response reported for the protein injections, in which in- creasing amounts of inducer give a rapid rise in the response only when >lo0 ng of rcm-BSA or >50 ng of me-cy-crystallin is injected per oocyte (Mifflin and Cohen, 19941, may be relevant here. These amounts of protein inducers could reflect a point at which the hsc7O available in the cell is overwhelmed. That such large amounts of denatured protein are required to elicit a stress response is reasonable if, like other chaperone proteins, hsc7O is effective at substoichiometric concentrations (Jakob et al., 1993). Relatively small amounts of hsc70 were sufficient to reverse the ability of rcm-BSA and me-a-crystallin to induce a stress response. This stress response reduction could result equally from an ability of hsc7O to prevent or reverse protein denaturation and aggregation or from hsc7O facilitation of in- ducer protein turnover; these possibilities remain to be distin- guished.

Nuclear injections of as little as 3 ng of hsc7O markedly reduced the response to a thermal shock (Fig. 11, whereas up to 22 ng of hsc7O injected into the cytoplasm had no detectable effect. hsc7O and hsp70 proteins are found in a variety of mam- malian cells distributed throughout the cytoplasm and nucleus. When subjected to a thermal shock, cytoplasmic hsc7O moves to the nucleus and concentrates in nucleoli (Welch and Feramisco, 1984; Pelham, 1984); this nuclear accumulation has been simi- larly observed with Drosophila tissue culture and salivary gland cells (Velazquez et al . , 1980; Arrigo et al . , 1980). Thus, hsc7O injected into the cytoplasm could in principle attenuate the stress response even if the effect requires that the hsc70 be localized in the nucleus. However, because translocation of hsc70 to the nucleus may itself be a consequence of the stress response, only the hsc70 already in the nucleus may effectively mediate the stress. Alternatively, the large size of a Xenopus oocyte and its high concentration of cytoplasmic yolk protein may preclude the efficient transport of cytoplasmically injected hsc70 to the nucleus, thereby preventing an effect on stress response induction.

The production of denatured, aggregated proteins as a con- sequence of stress together with the potential for solubilization of aggregated proteins via hsp7O binding (reviewed by Pelham (1990)) suggests a mechanism that links stress recognition and stress response transcriptional activation. To explore this pos- sible connection, injected proteins were assayed for their bind- ing by hsc70. hsp7O and hsc70 are very similar (Brown et al . , 1993), and it was assumed that their activities would be essen- tially the same in these experiments. hsc70 binding assays revealed a correlation between the abilities of BSA derivatives to induce the stress response and their affinities for hsc7O. hsc7O preferentially bound rcm-BSA (a strong inducer) over me-rcm-BSA (a weak inducer), and me-rcm-BSA was bound better than I-BSA or BSA (non-inducers). Competition assays revealed the same heirarchy of binding. These results support the idea that preferential binding by hsc7O may be a critical regulatory step in stress response induction.

In addition to an involvement in several of the physiological functions of hsc70, protein aggregation correlates with both stress response induction and binding by hsc7O. The extent of

active HSF HSF

aggregate

FIG. 6. A model for regulation of the stress response by hsc70 or hsp70. hsc7O (or hsp7O) molecules are represented by the solid squares; no stoichiometry is implied in the representations of various hsc70 complexes. Denatured proteins, which can arise from an environ- mental stress such as a thermal shock, are bound either directly or after aggregation to hsc7O. Incompletely folded, newly translated polypep- tides may be bound similarly by hsc70 (Beckmann et al., 1992). In this manner, the stress condition depletes the pool of free hsc70, and HSF is activated as less hsc70 is available to bind it. Ultimately, the unfolded and aggregated proteins either are degraded (not shown) or refold, and transcription of new hsps is reduced as hsc7O is again available to bind and inactivate HSF. The events depicted may all be localized to the nucleus; this possibility is discussed in the text.

aggregation may reflect more or tighter interactions between partially unfolded proteins (Mitraki and King, 1989). Thus, the same denatured condition that leads to protein aggregation through specific interactions could be responsible for binding by hsc7O. In principle, the protein aggregate simply could se- quester hsc70 nonspecifically, thereby preventing its function in other solubilization reactions. However, the nature of the binding assays used here to probe hsc7O interactions with in- jected proteins make this latter scenario unlikely.

Preferential binding by hsc7O may be all that distinguishes inducer proteins from non-inducers. This is consistent with the idea that aggregated, denatured proteins compete for hsc7O to promote the release and activation of the HSF. However, a maximum stress response also may require a minimum aggre- gate size (MiMin and Cohen, 1994). This could imply that re- tention of protein aggregates by a physical barrier such as the nuclear envelope enhances stress response induction. In this situation, the role of hsc7O in the transport of proteins out of the nucleus may be critical in addition to its role in aggregate disassembly.

hsc7O (or hsp70) appears to function in the solubilization of aggregated, denatured proteins that can result from a stress condition (Pelham, 1984; Lewis and Pelham, 1985; Beckmann et al . , 1992) and also binds to the HSF (Baler et al., 1992; Abravaya et al., 1992). In this study, the ability of hsc7O to reduce the stress response in vivo has been correlated with specific binding in vitro to protein inducers. These results, how- ever, do not preclude roles for other hsps in stress response induction. For example, other hsps were found to participate in a complex with the Escherichia coli hsc7O analogue, DnaK, in chaperone-mediated protein turnover (Sherman and Goldberg, 1992).

The link established here between specific hsc7O binding to protein inducers of the stress response and hsc70 suppression of the response supports the minimal model outlined in Fig. 6 .

hsc70 and Induction of the Stress Response 15723

As described previously (Abravaya et al., 1992; Baler et al., 19921, a basic feature of this model is the reversible inactiva- tion of HSF by complexation with hsc7O (or hsp7O) and parti- tioning of hsc70 (or hsp7O) among complexes with HSF and stress inducers. Work in this and the accompanying paper (Mif- flin and Cohen, 1994) further suggests that the stress response is elicited most effectively by large aggregates of denatured protein in the nucleus. Aggregation and nuclear localization may serve to slow in vivo degradation of the inducer proteins, thereby prolonging the presence of the pool of denatured pro- teins that can act as a sink to draw hsc7O away from complexes with HSF. hsc70 injection into the nucleus but not into the cytoplasm reduced the oocyte's response to a thermal shock. One interpretation is that critical hsc70:inducer protein inter- actions must occur in the nucleus. A likely alternative is that the key issue again is the in vivo lifetime of the denatured proteins that arise from the thermal shock, and that these proteins are eliminated most efficiently when hsc70 is intro- duced directly into the nucleus.

Acknowledgments-We thank Dr. Cameron Gunderson for providing Xenopus oocytes, Drs. Seth Sadis, Lawrence Hightower, and Richard Glickman for samples of hsc70, Dr. Richard Voellmy for plasmid p622C*, and Dr. Peter Rubenstein for helpful comments.

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