Protein science (1992), I , 980-985. Cambridge University Press. Printed in the USA. Copyright 0 1992 The Protein Society

Inhibitory effects of HSP70 chaperones on nascent polypeptides

CHRISTINE RYAN,’ TOM H. STEVENS,2 AND MILTON J. SCHLESINGER’ ‘ Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, St. Louis, Missouri 631 10

Institute of Molecular Biology, University of Oregon, Eugene, Oregon 97403

(RECEIVED January 23, 1992; REVISED MANUSCRIPT RECEIVED March 25, 1992)

Abstract

Several of the major heat shock proteins (HSPs) function normally as molecular chaperones to prevent aggre- gation of immature polypeptides and thereby facilitate folding and oligomerization. To determine their effect on nascent polypeptides, we added purified preparations of different isoforms of HSP7O to in vitro translation re- actions primed by the 26s mRNA of Sindbis virus, which encodes an autoprotease that functions cotranslation- ally, or by the mRNA encoding the yeast vacuolar H+ATPase, which is formed by a novel transpeptidase activity that removes the central region of the initial polypeptide. In the presence of HSP70s both the autoprotease and transpeptidase activities were inhibited, indicating that these chaperones can interact with nascent polypeptides and, in the cases studied here, perturb their normal structures.

Keywords: autoprotease; BiP; HSC70; mRNA translation; protein splicing

A molecular chaperone has been defined as a protein “that mediates the correct assembly of other polypeptides but is not itself a component of the final structure” (Ellis & van der Vies, 1991). The chaperone performs this role by transiently binding to its partner when the latter is ma- turing to its final conformation and thereby preventing the structurally immature polypeptide from misfolding, from interacting with inappropriate metabolites, and from aggregating to irreversibly denatured forms. Among the major molecular chaperones thus far identified are members of two families of heat shock proteins, noted HSP70 and HSP60 or GroEL (Flynn et al., 1989; Pel- ham, 1989; Georgopoulos & Ang, 1990; Hallberg, 1990). The HSP7O family of proteins facilitate the import of proteins into mitochondria and chloroplasts as well as the translocation of polypeptides across membranes (Chiroco et al., 1988; Deshaies et al., 1988; Cheng et al., 1989; Os- termann et al., 1989; Clos et al., 1990; Hemmingsen, 1990). The HSP60 family of chaperones participates in the folding of newly imported proteins into the mitochon- dria (Hemmingsen et al., 1988; Martin et al., 1991; Men- doza et al., 1991) and facilitates a refolding of denatured proteins to their native conformation (Pelham, 1986;

Reprint request to: Milton J. Schlesinger, Department of Molecular Microbiology, Box 8230, Washington University School of Medicine, St. Louis, Missouri 631 10.

Bochkareva et al., 1988; Goulobinoff et al., 1989; Geth- ing & Sambrook, 1990; Buchner et al., 1991). The chap- erones are postulated to bind to interactive surfaces or solvent-exposed hydrophobic domains normally buried within a protein structure (PeIham, 1986; Palleros et at., 1991). These kinds of structures will be present transiently on nascent polypeptides during translation and might in- teract with chaperones. In fact, Rothman (1989) has pro- posed that interactions of this kind may catalyze the folding of polypeptides, and a recent report described the presence of complexes between HSP70 and polyribo- some-associated polypeptides that had been radiolabeled in vivo (Beckmann et al., 1990). Here we show that HSP70 chaperones can interact in vitro with nascent polypeptides and affect their normal activities.

Results

The effect of chaperones on nascent polypeptides was ex- amined by adding purified preparations of several HSP70 isoforms to in vitro translation reactions primed with mRNAs whose immediate polypeptide products con- tained a measurable biological activity. By measuring al- teration in these activities, we could preclude general inhibitory or stimulatory effects by HSP70s on in vitro translation (Mivechi & Oglivie, 1989) that might be arti- factual.

980

Chaperones and nascent polypeptides

Translation of Sindbis virus 26s mRNA

One of the mRNAs we selected was a truncated form of the 26s mRNA that encodes the structural proteins of Sindbis virus (Strauss & Strauss, 1986). The truncated mRNA was prepared by in vitro transcription of a cDNA (see Materials and methods) that encodes the amino acid sequences for the virus capsid, a 33-kDa protein, and an additional 40 amino acids. Translation of the intact poly- cistronic 26s mRNA initiates at a single site near the 5' cap encoding the amino terminus of the capsid (Cancedda et al., 1975); however, the capsid protein is normally re- leased from the growing nascent chain by a cotransla- tional autoproteolytic cleavage (Aliperti & Schlesinger, 1978). The catalytic sites for this autoprotease reside in the capsid protein itself (Hahn et al., 1985); thus any interaction between chaperones and nascent capsid se- quences might be revealed by either enhancement or in- hibition of this protease activity. Inhibition should lead to a run-off polypeptide with a predicted size of 40 kDa.

Translation of this mRNA in a wheat germ lysate led to a 33-kDa protein as the major product of the in vitro reaction (Fig. lA, lane 1). However, when either HSC70, a constitutive form of HSP70 from bovine brain (Sadis et al., 1990), or BiP, a form that is localized to the cell's endoplasmic reticulum (Flynn et al., 1989), was included in the translation reaction, the amount of 33-kDa protein decreased, and a 40-kDa protein appeared (Fig. 1 A, lanes 2, 3). The total amount of these two proteins was about 80% that of the 33-kDa protein formed in the absence of the chaperones; thus, the latter had relatively little effect on the overall efficiency of in vitro translation. The ra- tio of 40 kDa to 33 kDa was 0.2 in reactions containing

A I

4 0 K - CAPSID-

B 2 3 CAPS ID

98 1

the chaperones compared to 0.03 in the absence of chap- erones. Analysis of V-8 protease digests of the 33-kDa and 40-kDa proteins recovered from gels provided evi- dence that the 40-kDa protein was a run-off translation product containing capsid sequences (Fig. 1B). The 40- kDa protein was not formed when the chaperones were added after the in vitro translation had been stopped by cycloheximide (data not shown). This result indicated that the chaperones blocked the nascent capsid-encoded protease cleavage and did not act by inhibiting a post- translational conversion of 40 kDa to 33 kDa. To show that normal termination of translation was not inhibited by chaperones, we tested two Sindbis virus mRNAs that encoded the capsid sequences but had been genetically en- gineered to contain an opal termination codon at a site 40 amino acids upstream from the normal protease cleav- age site of the capsid. One of these mRNAs also had an in-frame deletion, which removed 96 amino acids from the capsid. Release of capsid during translation of these mRNAs occurs by termination of translation and not by autoprotease action. The translation products of these mRNAs in the presence of BiP were of the size predicted for the normal terminations (Fig. lC, lanes 5,6) and were identical to those obtained from in vitro reaction that ei- ther lacked BiP or were supplemented with heat-inacti- vated BiP (data not shown). Samples were inactivated by heating 10 min at 60 "C.

In the reactions described above for the normal 26s mRNA, the most active preparations of BiP and HSC70 were equally effective in forming the 40-kDa protein, and the optimal amount of this protein was detected in trans- lations at 20 "C. At this temperature the ratio of 40 kDa: 33 kDa was 0.5, compared to a value of 0.2 in reactions

C 40K M.W. 4 5 6

-21.5 kD - 43 kD - - -14.3 kD 0- - 29

- 18.4 - 14.5

Fig. 1. Formation of a read-through protein during in vitro translation of Sindbis virus mRNA in the presence of HSC70 or BiP. A: In vitro translation in the presence of BiP or HSC70. Lane I , no HSC70 or BiP added; 2, addition of 7 pg HSC70; 3, addition of 8 pg BiP. B: V8 protease digest patterns of capsid and 40-kDa protein. C: In vitro translation of mutationally al- tered forms of Sindbis virus 26s mRNAs in the presence of BiP. Lane 4, a normal form of the 26s mRNA with a termination site (position of cDNA linearization) 40 amino acids downstream of the protease cleavage site; 5, a form of the 26s mRNA with an opal codon 40 amino acids upstream of the capsid protease site; 6, the 26s mRNA used in lane 5 but also containing an in- frame deletion of % amino acids in the body of the capsid sequences. Reactions contained 7 pg BiP and were incubated at 25 "C for 60 min. A 15% acrylamide gel was used.

982 C. Ryan et a/.

containing heat-inactivated BiP or heat-inactivated HSC70 (see Materials and methods). A dose-response curve showed that the half-maximal level of capsid protease in- hibition was obtained with 2 pg of BiP in the reaction mixture. At this level, we estimate there would be about 300 BiP monomers for each completed polypeptide trans- lated. It is difficult to evaluate the meaning of this high stoichiometry since we do not know how much biologi- cally active BiP was retained during its purification. In fact, two separate preparations of both the bovine brain HSC70 and the rat liver BiP differed in their specific ac- tivity by as much as twofold. Furthermore, the BiP and HSC70 were detected as dimers, tetramers, and hexamers when analyzed by nondenaturing pore-exclusion gel elec- trophoresis (see Materials and methods), and it is un- known which of these might be a more active species. In addition, Freiden et al. (1992) have recently shown that BiP is modified by both phosphorylation and ADP ribo- sylation and suggested that these modifications affect BiP's chaperone function. Possibly, the preparations used in our studies were heterogeneous with respect to these modifications, and this could also have affected biolog- ical activity. To ensure that it was the HSP70 and not an impurity that altered the in vitro translation reactions, we tested five other purified HSP70 isoforms at concentra- tions equivalent to those used for HSC70 and BiP. A bo- vine liver uncoating ATPase (Chappell et al., 1986), an HSP70 from heat-shocked chicken embryo fibroblasts (Kelley & Schlesinger, 1982), and a human HSP70 ex- pressed in Escherichia coli (Wu et al., 1985) were one- quarter to one-half as effective as the BiP and HSC70. The E. coli Dna K (Georgopoulos et al., 1990) prepara- tion and a commercial preparation of HSC70 (StressGen Biotech. Corp., Sidney, British Columbia, Canada) in- hibited the translation system by greater than 90% at pro- tein levels equivalent to the other chaperones. The endogenous level of HSP70 chaperones in the wheat germ lysate used for translations was about 0.1 pg as measured by quantitative immunoblotting with anti-HSP7O anti- bodies.

A purified preparation of HSP90, kindly supplied by Dr. I . Yahara (Tokyo Metropolitan Institute of Medical Science) was tested at levels similar to those used for HSP70, but it had no effect on either total in vitro trans- lation or on formation of the capsid protein. When in- cluded with HSP70 in the reaction, the HSP90 did not interfere with HSP70's block of capsid protease activity.

Translation of yeast TFPl mRNA

Another mRNA that produces in vitro a polypeptide with measurable biological activity encodes the yeast TFPl gene product, a 119-kDa protein, which is a precursor of the 69-kDa subunit of the vacuolar H+ATPase (Kane et al., 1990). The precursor undergoes a novel protein splicing in which the central portion of the sequence is re-

moved to form a 50-kDa spacer polypeptide, whereas the amino and carboxyl portions form the 69-kDa polypep- tide. When the TFPl mRNA was translated in a wheat germ lysate supplemented with heat-inactivated BiP, 119- kDa and 50-kDa proteins were detected among the ma- jor products (Fig. 2, lane 1). Assignment of these bands as the TFPl precursor and spacer, respectively, was based on results of immunoprecipitation with affinity-purified polyclonal rabbit antiserum raised against the 50-kDa protein (Fig. 2, lane 2). The presence of BiP during the in vitro translation led to increased amounts of the 119- kDa precursor and lower levels of the 50-kDa spacer pro- tein (Fig. 2, lane 4), and this accumulation of precur- sor at the expense of the spacer product occurred through- out the in vitro translation (Fig. 3, upper panel). In the presence of heat-inactivated BiP, more of the 50-kDa and less of the 119-kDa were formed throughout the reaction (Fig. 3, lower pane1)-a result consistent with cotransla- tional splicing in this in vitro system.

The HSC70 and BiP were equally effective in blocking 119-kDa splicing at levels equivalent to those inhibiting

1 2 3 4

-92kD

-69kD

-46kD

Fig. 2. Inhibition of TFPl protein splicing by BiP. Lanes 1 and 4, prod- ucts of in vitro translation of mRNA transcribed from pCY59 in the presence of heat-inactivated BiP and BiP, respectively. Lanes 2 and 3, radioimmune precipitation of the reaction products shown in lane 1 using either anti-50-kDa antiserum or Sindbis virus antiserum, respec- tively. Reaction mixtures were similar to those described in the Mate- rials and methods except 5 pg of BiP was used. Incubation was stopped after 30 min at 25 "C with SDS loading buffer and all samples were an- alyzed as described in the Materials and methods. A 7.5% acrylamide gel was used. The 119 kDa precursor and 50 kDa spacer protein are noted by arrows.

Chaperones and nascent polypeptides

600 I

Inactive 400

-0 10 20 30 40 50 Translation Time (min)

Fig. 3. Kinetics of 119-kDa precursor and 50-kDa spacer formation dur- ing in vitro translation of TFPl mRNA in a wheat germ extract. Up- per panel shows active BiP; lower shows heat-inactivated BiP. Reactions are described in the Materials and methods and the legend for Figure 2. W, 119 kDa; 0, 50 kDa.

the virus capsid autoprotease, but the three other HSP70 isoforms noted above were only marginally inhibitory. The Dna K and commercial HSC70 preparation were not tested.

Discussion

These data offer strong support for the hypothesis that the HSP70 class of chaperones can interact with nascent polypeptide chains. However, instead of catalyzing fold- ing and/or promoting the formation of normal nascent polypeptides, denoted here by their proteolytic-like activ- ities, the chaperones were deleterious. These inhibitory effects were not the result of perturbing the general trans- lational machinery, but rather, the chaperones appeared to alter nascent polypeptide structures. Inhibitory effects by HSP70s have been observed in other in vitro systems. For example, a purified HSP70 from reticulocytes inhib- ited the refolding of a precursor of dihydrofolate reduc- tase that had been denatured by urea (Sheffield et al., 1990) and the molecular chaperone SecB from E. coli blocked refolding of a denatured protein (Hardy & Ran- dall, 1991). In vivo data also show that abnormally high levels of HSP70 can be detrimental to normal cellular function (DiDomenico et al., 1982; S. Lindquist, unpubl.).

983

Cells stringently autoregulate HSP70 levels (DiDomenico et al., 1982), and our in vitro experiments can explain why a failure to control levels of HSP70 could adversely affect normal cellular protein synthesis.

Materials and methods

mRNAs

The mRNA encoding Sindbis virus capsid protein was prepared from a cDNA clone kindly provided by Dr. S. Schlesinger (Washington University). The cDNA contains the entire Sindbis virus capsid sequences plus downstream sequences coding for Sindbis virus proteins. The initia- tion codon for capsid is positioned immediately down- stream of the SP6 promoter. The cDNA was linearized with HinD I11 and isolated by phenol-chloroform extrac- tion and ethanol precipitation. Transcription to obtain mRNA was carried out as previously described (Agell et al., 1988). Two additional Sindbis virus mRNAs that contained an opal codon upstream of the normal capsid cleavage site were prepared by G. Li, using a cDNA encod- ing the Sindbis virus genome (Rice et al., 1987). The mRNA encoding the yeast 119-kDa vacuolar H+ATPase was transcribed from the cDNA clone, pCY59 (Kane et al., 1990).

In vitro translation system and analyses of reaction products

An in vitro reaction mixture was prepared with 12.5 pL of wheat germ lysate (Promega), 1.7 p L 1 mM amino acid mixture minus methionine, 0.75 pL RNasin (Pro- mega, 40 units/pL), 0.85 p L 35S-methionine (Amersham, 1,000 Ci/mmol; 15 mCi/mL), 2.95 pL M potassium ac- etate, 5.25 pL water, and 1 pL mRNA (0.5 mg/mL). To 2.5 pL of this mixture was added 1 pL of HSP70 sample and, after 1 h at 23-25 "C, 15 pL of Laemmli sodium do- decyl sulfate (SDS) loading buffer. Samples from in vitro translation reactions were boiled and products separated by SDS-polyacrylamide gel electrophoresis (PAGE) on either 7.5% or 15% polyacrylamide gels. Gels were pro- cessed for fluorography and protein bands excised and counted in a scintillation counter (Agell et al., 1988).

For V-8 protease digestion, the proteins were first sep- arated by SDS-PAGE after in vitro translation as de- scribed above. The gel was washed with water for l h, dried, and autoradiographed. Bands were excised, cut, and incubated with 0.125 M Tris, pH 6.8, 0.1% SDS, 1 mM EDTA for 1 h. Gel pieces and 5 pg Staphylococ- cus aureus V-8 protease (Sigma) were added to wells of a 5-20% gradient polyacrylamide gel containing 1 mM EDTA. Electrophoresis was stopped for 30 min when the dye marker reached the bottom of the stack portion of the gel and then continued until the dye marker reached

C. Ryan et al.

the bottom of the running portion of the gel. The gel was processed for fluorography.

Immunoprecipitation

Methods for immunoprecipitation were described previ- ously (Kelley & Schlesinger, 1982). A polyclonal antisera was obtained by injecting rabbits with a preparation of the 50-kDa spacer protein expressed in E. coli from a re- combinant DNA, and the antibodies were subsequently affinity purified. As a nonspecific antisera, rabbit anti- bodies raised against a Sindbis virus protein were used.

HSP70 chaperones

The constitutive bovine brain HSC70 was kindly provided by S. Sadis and L. Hightower (University of Connecti- cut); BiP from bovine liver microsomes and uncoating ATPase from bovine brain were kindly provided by G. Flynn (Princeton University); HSP70 from heat-shocked chicken embryo fibroblast has been described (Kelley & Schlesinger, 1982), but the preparation was further pu- rified by affinity chromatography on ATP-sepharose (Welch & Feramisco, 1985); recombinant human HSP70 expressed in E. coli was kindly provided by M. Myers and R. Morimoto (Northwestern University); E. coli Dna K was kindly provided by C. Georgopoulos (University of Utah). Most preparations were dialyzed against water and lyophilized to obtain concentrations of 4-8 mg/mL protein. All preparations contained low levels of ATP, measured by the luciferase assay (Sigma), that ranged from 10 to 60 pmol/pL. These amounts were well below the 0.5-1.0 nmol of ATP that could inhibit our in vitro translation systems. The purity and oligomeric states of the HSP70s were examined by Coomassie-blue staining of SDS-PAGE and nondenaturing pore-exclusion gel electrophoresis (Clos et al., 1990). In the denaturing gels, the protein band of 70 kDa or 78 kDa (for BiP) was es- timated to be >98% of the total stained bands. After nondenaturing gel electrophoresis, the HSC70 and BiP preparations displayed a set of discrete bands with esti- mated molecular weights consistent with dimers (the predominant form), tetramers, and hexamers. Virtually all of the Dna K was monomeric and the HSP70 from chicken embryo fibroblasts contained many bands of molecular weights >300 kDa with smaller amounts of HSP70 dimers and monomers. After heat inactivation for 10 min at 60 "C, the oligomeric states of the HSP70s were no longer detected.

Acknowledgments

We are grateful to L. Hightower, S. Sadis, J. Rothman, G. Flynn, M. Morimoto, M. Meyers, and C . Georgopoulos for providing samples of HSP70s and Dna K. This work was sup- ported by a grant to M.S. from the National Science Foundation and to T.S. from the U.S. Public Health Service (GM 38006).

References

Agell, N., Bond, U., & Schlesinger, M.J. (1988). In vitro proteolytic pro- cessing of a diubiquitin and a truncated diubiquitin formed from in vitro-generated mRNAs. Proc. Natl. Acad. Sci. USA 8, 3693-3697.

Aliperti, G. & Schlesinger, M.J. (1978). Evidence for an autoprotease activity of Sindbis virus capsid protein. Virology 90, 366-369.

Beckmann, R.P., Mizzen, L.A., & Welch, W.J. (1990). Interaction of hsp70 with newly synthesized proteins: Implications for protein fold- ing and assembly. Science 248, 850-854.

Bochkareva, E.S., Lissin, N.M., & Girshovich, A S . (1988). Transient association of newly synthesized unfolded proteins with the heat- shock GroEL protein. Nature 336, 254-257.

Buchner, J., Schmidt, M., Fuchs, M., Jaenicke, R., Rudolph, R., Schmid, F.X., & Kiefhaber, F. (1991). GroE facilitates refolding of citrate synthase by suppressing aggregation. Biochemistry 30,

Cancedda, R., Villa-Komaroff, L., Lodish, H.F., & Schlesinger, M . (1975). Initiation sites for translation of Sindbis virus 42s and 26s messenger RNAs. Cell 6 , 215-222.

Chappell, T.G., Welch, J.J., Schlossman, D.M., Palter, K.B., Schlesinger, M.J., & Rothman, J.E. (1986). Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell45, 3-13.

Cheng, M.Y., Hartl, F.-U., Martin, J., Pollock, R.A., Kalousek, F., Neupert, W., Hallberg, E.M., Hallberg, R.L., & Horwich, A.L. (1989). Mitochondrial heat-shock protein hsp60 is essential for as- sembly of proteins imported into yeast mitochondria. Nature 337,

Chiroco, W.J., Waters, M.G., & Blobel, G. (1988). 70K heat shock re- lated proteins stimulate protein translocation into microsomes. Na- ture 332, 805-810.

Clos, J., Westwood, J.T., Becker, P.B., Wilson, S., Lambert, K., & Wu, C. (1990). Molecular cloning and expression of a hexameric drosoph- ila heat shock factor subject to negative regulation. Cell 63, 1085-1097.

Deshaies, R.J., Koch, B.D., Werner-Washburn, M., Craig, E.A., & Schekman, R. (1988). A subfamily of stress proteins facilitates trans- location of secretory and mitochondrial precursor polypeptides. Na- ture 332, 800-805.

DiDomenico, B.J., Bugaisky, G.E., & Lindquist, S. (1982). The heat shock response is self-regulated at both the transcriptional and post transcriptional levels. Cell 31, 593-603.

Ellis, R.J. &van der Vies, S.M. (1991). Molecular chaperones. Annu. Rev. Biochem. 60, 324-347.

Flynn, G.C., Chappell, G.T., & Rothman, J.E. (1989). Peptide bind- ing and release by proteins implicated as catalysts of protein assem- bly. Science 245, 385-390.

Freiden, P.J., Gaut, J.R., & Hendershot, L.M. (1992). Interconversion of three differently modified and assembled forms of BiP. EMBO

Georgopoulos, C. & Ang, D. (1990). The Escherichia coli groE chap- eronins. Semin. Biol. I , 19-25.

Georgopoulos, C., Ang, D., Liberek, K., & Zylicz, M. (1990). Proper- ties of the Escherichia coli heat shock proteins and their role in bac- teriophage growth. In Stress Proteins in Biology and Medicine (Morimoto, R.I., Tissieres, A., & Georgopoulos, C., Eds.), pp. 191-221. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York.

Gething, M.-J. & Sambrook, J.F. (1990). Transport and assembly pro- cesses in the endoplasmic reticulum. Semin. Biol. I , 65-72.

Goulobinoff, P., Gatenby, A.A., & Lorimer, G.H. (1989). GroE heat shock proteins promote assembly of foreign prokaryotic ribulose bis- phosphate carboxylase oligomers in Escherichia coli. Nature 342,

Hahn, C., Strauss, E.G., & Strauss, J.H. (1985). Sequence analysis of three Sindbis virus mutants temperature-sensitive in the capsid pro- tein autoprotease. Proc. Natl. Acad. Sci. USA 82, 4648-4652.

Hallberg, R.L. (1990). A mitochondrial chaperonin: Genetic, biochem- ical, and molecular characteristics. Semin. Biol. I , 37-45.

Hardy, S.J.S. & Randall, L.L. (1991). A kinetic partitioning model Of selective binding of nonnative proteins by the bacterial chaperone SecB. Science 251, 439-443.

Hemmingsen, S.M. (1990). The plastid chaperonin. Semin. Biol. I , 47-54.

1586-1591.

620-625.

J. I l , 63-70.

884-889.

Chaperones and nascent polypeptides 985

Hemmingsen, S.M., Woolford, C.A., van der Vies, S.M., Tilly, K., Dennis, D.T., Georgopoulos, C.P., Hendrix, R.W., & Ellis, R.J. (1988). Homologous plant and bacterial proteins chaperone olig- omeric protein assembly. Nature 333, 330-334.

Kane, P.M., Yamashiro, C.T., Wolczyk, D.F., Neff, N., Goebl, M., & Stevens, T.H. (1990). Protein splicing converts the yeast TFPl gene product to the 69-kDa subunit of the vacuolar H+-adenosine tri- phosphatase. Science 250, 651-657.

Kelley, P.M. & Schlesinger, M.J. (1982). Antibodies to two major chicken heat shock proteins cross-react with similar proteins in widely divergent species. Mol. Cell. Biol. 2, 267-274.

Martin, J . , Langer, T., Boteva, R., Schramel, A., Horwich, A.L., & Hartl, E-U. (1991). Chaperonin-mediated protein folding at the sur- face of groEL through a “molten globule”-like intermediate. Nature

Mendoza, J.A., Rogers, E., Lorimer, G.H., & Horowitz, P.M. (1991). Chaperonins facilitate the in vitro folding of monomeric mitochon- drial rhodanese. J. Biol. Chem. 266, 13044-13049.

Mivechi, N.F. & Oglivie, P.D. (1989). Effects of heat shock proteins (M, 70,000) on protein and DNA synthesis at elevated temperatures in vitro. Cancer Res. 49, 1492-1496.

Ostermann, J., Horwich, A.L., Neupert, W., & Hartl, F.-U. (1989). Pro- tein folding in mitochondria requires complex formation with hsp60 and ATP hydrolysis. Nature 341, 125-130.

Palleros, D.R., Welch, W.J., & Fink, A.L. (1991). Interaction of the hsp7O with unfolded proteins: Effects of temperature and nucleotides on the kinetics of binding. Proc. Nutl. Acad. Sci. USA 88, 5719-5723.

352, 36-42.

Pelham, H.R.B. (1986). Speculations on the functions of the major heat shock and glucose-regulated proteins. Cell 46, 959-961.

Pelham, H.R.B. (1989). Heat shock and the sorting of luminal ER pro- teins. EMBOJ. 8, 3171-3176.

Rice, C.M., Levis, R., Strauss, J.H., & Huang, H.V. (1987). Produc- tion of infectious RNA transcripts from Sindbis virus cDNA clones: Mapping of lethal mutations, rescue of a temperature-sensitive marker and in vitro mutagenesis to generate defined mutants. J. Virol. 61, 3809-3819.

Rothman, J.E. (1989). Polypeptide chain binding proteins: Catalysts of protein folding and related processes in cells. Cell 59, 591-601.

Sadis, S., Raghavendra, K., & Hightower, L.E. (1990). Secondary struc- ture of the mammalian 70-kilodalton heat shock cognate protein an- alyzed by circular dichroism spectroscopy and secondary structure predictions. Biochemistry 29, 8199-8206.

Sheffield, W.P., Shore, G.C., & Randall, S.K. (1990). Mitochondrial precursor protein: Effects of 70-kilodalton heat shock protein on polypeptide folding, aggregation, and import competence. J. Biol. Chem. 265, 11069-1 1076.

Strauss, E.G. & Strauss, J.H. (1986). Structure and replication of the alphavirus genome. In The Toguviridue and Flaviviridue (Schlesinger, S. & Schlesinger, M.J., Eds.), pp. 35-90. Plenum Press, New York.

Welch, W.J. & Feramisco, J.R. (1985). Rapid purification of mamma- lian 70,000-dalton stress proteins: Affinity of the proteins for nu- cleotides. Mol. Cell. Biol. 5 , 1229-1236.

Wu, B.J., Hunt, C., & Morimoto, R.1. (1985). Structure and expres- sion of the human gene encoding a major heat shock protein HSP70. Mol. Cell. Biol. 5. 330-341.