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W1977793080 abstract "The small heat shock protein of Neurospora crassa, Hsp30, when employed in affinity chromatography, bound two cellular proteins that were identified as Hsp70 and Hsp88. Both Hsp70 and Hsp88 bound to Hsp30 in preference to other proteins, but binding of Hsp88 was more selective for Hsp30, and a direct interaction was observed. Transcripts for Hsp88, a newly characterized protein, are present at normal temperature, but they are strongly induced by heat shock. Its cDNA sequence predicts a protein with homology to mammalian Hsp110 family proteins, which are distantly related to Hsp70. Hsp88 and its homologues show greater similarity to Hsp70 in its N-terminal ATPase domain than in the C-terminal peptide-binding domain, and its ATP-binding motifs are conserved. Nevertheless, the N-terminal domain of Hsp88 (and related proteins) is consistently more hydrophobic and more basic than that of Hsp70 proteins. Within the C-terminal domain, the sequence corresponding to the DnaK α subdomain is conserved in the Hsp88/Hsp110 family proteins, whereas the DnaK β subdomain sequence is not conserved. The interaction between Hsp70 and Hsp30 may reflect their cooperation as cochaperones for denatured proteins, whereas Hsp88 and Hsp30 may form a complex that interacts with potential substrates. The small heat shock protein of Neurospora crassa, Hsp30, when employed in affinity chromatography, bound two cellular proteins that were identified as Hsp70 and Hsp88. Both Hsp70 and Hsp88 bound to Hsp30 in preference to other proteins, but binding of Hsp88 was more selective for Hsp30, and a direct interaction was observed. Transcripts for Hsp88, a newly characterized protein, are present at normal temperature, but they are strongly induced by heat shock. Its cDNA sequence predicts a protein with homology to mammalian Hsp110 family proteins, which are distantly related to Hsp70. Hsp88 and its homologues show greater similarity to Hsp70 in its N-terminal ATPase domain than in the C-terminal peptide-binding domain, and its ATP-binding motifs are conserved. Nevertheless, the N-terminal domain of Hsp88 (and related proteins) is consistently more hydrophobic and more basic than that of Hsp70 proteins. Within the C-terminal domain, the sequence corresponding to the DnaK α subdomain is conserved in the Hsp88/Hsp110 family proteins, whereas the DnaK β subdomain sequence is not conserved. The interaction between Hsp70 and Hsp30 may reflect their cooperation as cochaperones for denatured proteins, whereas Hsp88 and Hsp30 may form a complex that interacts with potential substrates. Exposure to high temperature induces all organisms to synthesize a distinct group of proteins, the heat shock proteins (hsp), 1The abbreviations used are: hsp, heat shock protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; bp, base pair(s). 1The abbreviations used are: hsp, heat shock protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; bp, base pair(s). many of which act as chaperones that assist in the folding or unfolding of other proteins (1Hendrick J.P. Hartl F.-U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). These activities are especially useful during cell exposure to high temperatures that denature proteins and can lead to protein aggregation, but they are also required under conditions of normal growth. The chaperone functions of prominent high molecular weight hsps, such as Hsp70, Hsp104, and Hsp60, depend on cycles of ATP binding and hydrolysis; and cochaperones are required for optimal activity.We have been interested in understanding the roles of small hsps, which are strongly expressed in response to high temperature. These proteins were first characterized by their homology to eye lens α-crystallin (2Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (673) Google Scholar), which is also a heat shock-induced protein in non-lens tissue (3Klemenz R. Frohli E. Steiger R. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar). Multiple small hsps are produced by several organisms, includingDrosophila melanogaster and Caenorhabditis elegans (4Plesofsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar), and they are especially abundant in plants, which have organellar as well as cytosolic forms of these proteins. The small hsps of diverse organisms show only limited sequence conservation, and their activities appear to be dispensable at normal temperature. However, overexpression of these proteins increased the resistance of mammalian cells to high temperature and toxic chemicals (5Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar). Furthermore, disruption of the single copy Hsp30 gene ofNeurospora crassa showed that this small hsp dramatically improves cell survival at high temperature, under conditions of glucose limitation (6Plesofsky-Vig N. Brambl R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5032-5036Crossref PubMed Scopus (67) Google Scholar). Like other hsps, the small hsps appear to be chaperones that in vitro prevent thermal aggregation of substrate proteins (7Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar), but the targets of their chaperone activity in vivo and the nature of the requirements for this activity, including cooperation with cochaperones, are not yet known.Hsp30 is the sole α-crystallin-related small hsp of N. crassa (9Plesofsky-Vig N. Brambl R. J. Biol. Chem. 1990; 265: 15432-15440Abstract Full Text PDF PubMed Google Scholar), and it is strongly induced by high temperature (45 °C). Under inducing conditions Hsp30 associates with membranes, chiefly with mitochondrial membranes, but it dissociates, becoming a soluble cytosolic protein at normal temperature (9Plesofsky-Vig N. Brambl R. J. Biol. Chem. 1990; 265: 15432-15440Abstract Full Text PDF PubMed Google Scholar). To understand better the cellular and molecular roles played by Hsp30 during heat shock, we have attempted to identify cellular proteins that interact with Hsp30. We report here that two proteins, identified as Hsp70 and Hsp88, which is a previously uncharacterized protein, bind specifically to Hsp30 linked to a matrix in affinity chromatography. Furthermore, purified Hsp30 binds to an immobilized Hsp88-containing fusion protein. We determined the cDNA sequence of Hsp88 and aligned its predicted amino acid sequence with that of five related proteins, mammalian Hsp110 (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Hsp70RY (11Fathallah D.M. Cherif D. Dellagi K. Arnaout M.A. J. Immunol. 1993; 151: 810-813PubMed Google Scholar), Hsp87 of C. elegans (12Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fraser A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kerswaw J. Kirsten J. Laisster N. Latreille P. Lightning J. Lloyd C. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Sims M. Smaldon N. Smith A. Smith M. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterson R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar), Hsp79 (Sse2) of Saccharomyces cerevisiae (13Mukai H. Takayoshi K. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst .). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar), andArabidopsis thaliana Hsp91 (14Storozhenko S. De Pauw P. Kushnir S. Van Montagu M. Inze D. FEBS Lett. 1996; 390: 113-118Crossref PubMed Scopus (29) Google Scholar). These proteins were also aligned with Hsp70, to which they are distantly related; and properties of these two classes of proteins were compared. This is the first report of a specific interaction between a small hsp and cellular proteins, whose identities suggest that they may be cochaperones.DISCUSSIONTo identify cellular proteins that interact with Hsp30, the small hsp of N. crassa, we analyzed proteins in a cellular extract that were retained by an Hsp30 affinity resin. We found that Hsp70 and Hsp88 specifically bound to immobilized Hsp30. We confirmed the identity of Hsp70 by N-terminal amino acid sequencing. Hsp88 is a previously uncharacterized protein, whose cDNA we isolated and sequenced. Hsp88 appears to be a normal cellular constituent, whose expression increases severalfold in response to high temperature stress. Distantly related to Hsp70, Hsp88 is homologous to several recently characterized proteins, which are also reported to be induced by heat shock. Mammalian Hsp110 is one of the most strongly induced hsps in heat-stressed Chinese hamster ovary cells (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Murine Osp94, in addition to being induced by heat shock, is strongly synthesized in response to hyperosmotic stress (35Kojima R. Randall J. Brenner B.M. Gullans S.R. J. Biol. Chem. 1996; 271: 12327-12332Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These proteins localize chiefly to the cytosol, although Hsp110 is also peripherally associated with nucleoli (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Hsp105 with nuclei (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). N. crassa Hsp88 is also predominantly cytosolic with a minor portion being membrane-associated. Unlike Hsp70 and small hsps, these proteins do not appear to change their location in response to high temperature (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).There are consistent differences between the Hsp110 family proteins and Hsp70. The two Neurospora proteins have only 18% identity in their C-terminal putative peptide-binding domains, indicating extensive sequence divergence. In contrast their putative ATPase domains have 38% identity, and the recognized ATP-binding motifs are conserved in Hsp88. In our alignment of Hsp88-related and Hsp70 proteins, we found 49 identical and 67 similar residues conserved within the N-terminal domain and only 7 and 9, respectively, within the C-terminal domain. Nevertheless, properties of the N-terminal ATPase domains display surprising contrasts. This domain is more hydrophobic in Hsp88-related proteins than in Hsp70, due particularly to regions within three separate subdomains of the Hsc70 crystal structure, IA, IIA, and IIB (29Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar). Furthermore, the Hsp88/Hsp110 ATPase domain is slightly basic, whereas this domain in Hsp70 is acidic. These properties would be expected to affect binding and hydrolysis of ATP by Hsp88 and related proteins. We found that binding of Hsp88 to ATP-agarose does not occur in the presence of Mg2+ but does occur when Mn2+ is the divalent cation, as has also been reported for purified Hsp90/Hsp83 (34Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4943-4950Abstract Full Text PDF PubMed Google Scholar). Hsp105 was also reported not to bind Mg2+ATP-agarose (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Except for Hsp70, other N. crassa proteins in addition to Hsp88, such as Hsp83 and Hsp98, also bind more strongly to Mn2+ATP- than to Mg2+ATP-agarose, with binding to Ca2+ATP-agarose being intermediate. The distinctive properties of the ATPase domain of Hsp88-related proteins are likely to influence the function of these proteins. In S. cerevisiae, the Ssb subgroup of Hsp70 is required for growth at low temperature, and domain swapping experiments showed that the N-terminal ATPase domain of Ssb1 is responsible for conferring this function (37James P. Pfund C. Craig E.A. Science. 1997; 275: 387-389Crossref PubMed Scopus (184) Google Scholar).The C-terminal domains of the Hsp88-related proteins and Hsp70 include a region of conserved sequence (Hsp88: 551–653) that corresponds to the α-helical region within the peptide-binding domain of E. coli DnaK, an Hsp70 homologue (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The preceding region of β structure in the peptide-binding domain is not conserved in Hsp110 family proteins, although it is more strongly conserved than the α-helical region among Hsp70s (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The β strands comprise the actual peptide-binding subdomain, and the α helices apparently form a lid over the peptide bound within the β sandwich. Movement of the longest α helix relative to the β sandwich, seen in an alternate crystal structure, may reflect nucleotide-induced conformational changes within DnaK (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). Sequence conservation of the α subdomain suggests that the Hsp88-related proteins retain the Hsp70 mechanism of nucleotide-regulated peptide-binding and release. The lack of conservation of the β subdomain suggests that the actual peptides bound by these proteins differ.Several interactions between small hsps and purified protein substrates have been reported. For example, the small hsp of avian cells was found to inhibit actin polymerization in vitro (39Miron T. Vancompernolle K. Vandekerckhove J. Wilchek M. Geiger B. J. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (387) Google Scholar). Murine Hsp25 (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar) and pea Hsp18.1 (40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar) form complexes with unfolded citrate synthase and other model substrates, and α-crystallin binds to denatured proteins, including β- and γ-crystallins (41Wang K. Spector A. J. Biol. Chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar). The interaction that we have detected between Hsp30 and Hsp88 (and Hsp70) is the first report of complex formation between a small hsp and a protein selected from a cellular extract, and it implies that this association likely occurs in vivo. Hsp30 and Hsp88 appear to interact directly, since the purified proteins bind to one another in the absence of other components. This is also the first report that a member of the Hsp110 protein family associates with a specific protein.Hsp70 was the first hsp proposed to act as a chaperone (42Lewis M.J. Pelham H.R.B. EMBO J. 1985; 4: 3137-3143Crossref PubMed Scopus (238) Google Scholar), and its effect on protein unfolding is well established (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The chaperone activity of small hsps is only beginning to be understood; they appear to bind to unfolded proteins and prevent their aggregation (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar, 40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar). Under moderate conditions of heat stress, small hsps may be sufficient to reactivate substrate enzymes. We found that α-crystallin protected hexokinase activity from thermal inactivation, 3N. Plesofsky-Vig and R. Brambl, manuscript in preparation. and pea small hsps were reported to protect citrate synthase from being inactivated (7Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Under more stringent conditions, however, in addition to murine Hsp25, reactivation of citrate synthase required Hsp70 and ATP, which apparently dissociated the Hsp25-citrate synthase complex (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). This suggests cooperation between Hsp25 and Hsp70. Small hsps were proposed to complex with unfolded proteins early in heat shock and to be released from substrate proteins by ATP-dependent chaperones, such as Hsp70, when ATP-generating processes recover in stressed cells (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). The activity of most chaperones either requires or increases in cooperation with cochaperones. E. coli DnaK, for example, is aided by DnaJ, which enhances its ATPase activity, and by the nucleotide exchange factor GrpE (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The multi-protein complexes formed by Hsp90 with Hsp70, p60, immunophilins, and p23 are required for Hsp90 to bind to and activate unliganded steroid receptors (44Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar).The affinity binding we report between Hsp70 and Hsp30 may reflect the cooperation between these two classes of chaperone in reactivating a substrate enzyme in vitro (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). In contrast to Hsp70, the binding of Hsp88 to Hsp30 is more selective and is not increased by a reaction temperature which denatures proteins in vitro(Table I). Furthermore, the interaction between purified Hsp88 and Hsp30 occurs in the absence of substrate proteins, implying that Hsp88 and Hsp30 may together form a chaperone complex before interacting with substrate proteins. An additional possibility is that the temperature-dependent dissociation of Hsp30 from membranes depends on Hsp88, which is located predominantly in the cytosol. Exposure to high temperature induces all organisms to synthesize a distinct group of proteins, the heat shock proteins (hsp), 1The abbreviations used are: hsp, heat shock protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; bp, base pair(s). 1The abbreviations used are: hsp, heat shock protein; GST, glutathione S-transferase; PCR, polymerase chain reaction; bp, base pair(s). many of which act as chaperones that assist in the folding or unfolding of other proteins (1Hendrick J.P. Hartl F.-U. Annu. Rev. Biochem. 1993; 62: 349-384Crossref PubMed Scopus (1461) Google Scholar). These activities are especially useful during cell exposure to high temperatures that denature proteins and can lead to protein aggregation, but they are also required under conditions of normal growth. The chaperone functions of prominent high molecular weight hsps, such as Hsp70, Hsp104, and Hsp60, depend on cycles of ATP binding and hydrolysis; and cochaperones are required for optimal activity. We have been interested in understanding the roles of small hsps, which are strongly expressed in response to high temperature. These proteins were first characterized by their homology to eye lens α-crystallin (2Ingolia T.D. Craig E.A. Proc. Natl. Acad. Sci. U. S. A. 1982; 79: 2360-2364Crossref PubMed Scopus (673) Google Scholar), which is also a heat shock-induced protein in non-lens tissue (3Klemenz R. Frohli E. Steiger R. Schafer R. Aoyama A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3652-3656Crossref PubMed Scopus (478) Google Scholar). Multiple small hsps are produced by several organisms, includingDrosophila melanogaster and Caenorhabditis elegans (4Plesofsky-Vig N. Vig J. Brambl R. J. Mol. Evol. 1992; 35: 537-545Crossref PubMed Scopus (59) Google Scholar), and they are especially abundant in plants, which have organellar as well as cytosolic forms of these proteins. The small hsps of diverse organisms show only limited sequence conservation, and their activities appear to be dispensable at normal temperature. However, overexpression of these proteins increased the resistance of mammalian cells to high temperature and toxic chemicals (5Lavoie J.N. Gingras-Breton G. Tanguay R.M. Landry J. J. Biol. Chem. 1993; 268: 3420-3429Abstract Full Text PDF PubMed Google Scholar). Furthermore, disruption of the single copy Hsp30 gene ofNeurospora crassa showed that this small hsp dramatically improves cell survival at high temperature, under conditions of glucose limitation (6Plesofsky-Vig N. Brambl R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5032-5036Crossref PubMed Scopus (67) Google Scholar). Like other hsps, the small hsps appear to be chaperones that in vitro prevent thermal aggregation of substrate proteins (7Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar, 8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar), but the targets of their chaperone activity in vivo and the nature of the requirements for this activity, including cooperation with cochaperones, are not yet known. Hsp30 is the sole α-crystallin-related small hsp of N. crassa (9Plesofsky-Vig N. Brambl R. J. Biol. Chem. 1990; 265: 15432-15440Abstract Full Text PDF PubMed Google Scholar), and it is strongly induced by high temperature (45 °C). Under inducing conditions Hsp30 associates with membranes, chiefly with mitochondrial membranes, but it dissociates, becoming a soluble cytosolic protein at normal temperature (9Plesofsky-Vig N. Brambl R. J. Biol. Chem. 1990; 265: 15432-15440Abstract Full Text PDF PubMed Google Scholar). To understand better the cellular and molecular roles played by Hsp30 during heat shock, we have attempted to identify cellular proteins that interact with Hsp30. We report here that two proteins, identified as Hsp70 and Hsp88, which is a previously uncharacterized protein, bind specifically to Hsp30 linked to a matrix in affinity chromatography. Furthermore, purified Hsp30 binds to an immobilized Hsp88-containing fusion protein. We determined the cDNA sequence of Hsp88 and aligned its predicted amino acid sequence with that of five related proteins, mammalian Hsp110 (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Hsp70RY (11Fathallah D.M. Cherif D. Dellagi K. Arnaout M.A. J. Immunol. 1993; 151: 810-813PubMed Google Scholar), Hsp87 of C. elegans (12Wilson R. Ainscough R. Anderson K. Baynes C. Berks M. Bonfield J. Burton J. Connell M. Copsey T. Cooper J. Coulson A. Craxton M. Dear S. Du Z. Durbin R. Favello A. Fraser A. Fulton L. Gardner A. Green P. Hawkins T. Hillier L. Jier M. Johnston L. Jones M. Kerswaw J. Kirsten J. Laisster N. Latreille P. Lightning J. Lloyd C. Mortimore B. O'Callaghan M. Parsons J. Percy C. Rifken L. Roopra A. Saunders D. Shownkeen R. Sims M. Smaldon N. Smith A. Smith M. Sonnhammer E. Staden R. Sulston J. Thierry-Mieg J. Thomas K. Vaudin M. Vaughan K. Waterson R. Watson A. Weinstock L. Wilkinson-Sproat J. Wohldman P. Nature. 1994; 368: 32-38Crossref PubMed Scopus (1439) Google Scholar), Hsp79 (Sse2) of Saccharomyces cerevisiae (13Mukai H. Takayoshi K. Tanaka H. Hirata D. Miyakawa T. Tanaka C. Gene (Amst .). 1993; 132: 57-66Crossref PubMed Scopus (101) Google Scholar), andArabidopsis thaliana Hsp91 (14Storozhenko S. De Pauw P. Kushnir S. Van Montagu M. Inze D. FEBS Lett. 1996; 390: 113-118Crossref PubMed Scopus (29) Google Scholar). These proteins were also aligned with Hsp70, to which they are distantly related; and properties of these two classes of proteins were compared. This is the first report of a specific interaction between a small hsp and cellular proteins, whose identities suggest that they may be cochaperones. DISCUSSIONTo identify cellular proteins that interact with Hsp30, the small hsp of N. crassa, we analyzed proteins in a cellular extract that were retained by an Hsp30 affinity resin. We found that Hsp70 and Hsp88 specifically bound to immobilized Hsp30. We confirmed the identity of Hsp70 by N-terminal amino acid sequencing. Hsp88 is a previously uncharacterized protein, whose cDNA we isolated and sequenced. Hsp88 appears to be a normal cellular constituent, whose expression increases severalfold in response to high temperature stress. Distantly related to Hsp70, Hsp88 is homologous to several recently characterized proteins, which are also reported to be induced by heat shock. Mammalian Hsp110 is one of the most strongly induced hsps in heat-stressed Chinese hamster ovary cells (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Murine Osp94, in addition to being induced by heat shock, is strongly synthesized in response to hyperosmotic stress (35Kojima R. Randall J. Brenner B.M. Gullans S.R. J. Biol. Chem. 1996; 271: 12327-12332Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These proteins localize chiefly to the cytosol, although Hsp110 is also peripherally associated with nucleoli (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Hsp105 with nuclei (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). N. crassa Hsp88 is also predominantly cytosolic with a minor portion being membrane-associated. Unlike Hsp70 and small hsps, these proteins do not appear to change their location in response to high temperature (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar).There are consistent differences between the Hsp110 family proteins and Hsp70. The two Neurospora proteins have only 18% identity in their C-terminal putative peptide-binding domains, indicating extensive sequence divergence. In contrast their putative ATPase domains have 38% identity, and the recognized ATP-binding motifs are conserved in Hsp88. In our alignment of Hsp88-related and Hsp70 proteins, we found 49 identical and 67 similar residues conserved within the N-terminal domain and only 7 and 9, respectively, within the C-terminal domain. Nevertheless, properties of the N-terminal ATPase domains display surprising contrasts. This domain is more hydrophobic in Hsp88-related proteins than in Hsp70, due particularly to regions within three separate subdomains of the Hsc70 crystal structure, IA, IIA, and IIB (29Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar). Furthermore, the Hsp88/Hsp110 ATPase domain is slightly basic, whereas this domain in Hsp70 is acidic. These properties would be expected to affect binding and hydrolysis of ATP by Hsp88 and related proteins. We found that binding of Hsp88 to ATP-agarose does not occur in the presence of Mg2+ but does occur when Mn2+ is the divalent cation, as has also been reported for purified Hsp90/Hsp83 (34Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4943-4950Abstract Full Text PDF PubMed Google Scholar). Hsp105 was also reported not to bind Mg2+ATP-agarose (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Except for Hsp70, other N. crassa proteins in addition to Hsp88, such as Hsp83 and Hsp98, also bind more strongly to Mn2+ATP- than to Mg2+ATP-agarose, with binding to Ca2+ATP-agarose being intermediate. The distinctive properties of the ATPase domain of Hsp88-related proteins are likely to influence the function of these proteins. In S. cerevisiae, the Ssb subgroup of Hsp70 is required for growth at low temperature, and domain swapping experiments showed that the N-terminal ATPase domain of Ssb1 is responsible for conferring this function (37James P. Pfund C. Craig E.A. Science. 1997; 275: 387-389Crossref PubMed Scopus (184) Google Scholar).The C-terminal domains of the Hsp88-related proteins and Hsp70 include a region of conserved sequence (Hsp88: 551–653) that corresponds to the α-helical region within the peptide-binding domain of E. coli DnaK, an Hsp70 homologue (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The preceding region of β structure in the peptide-binding domain is not conserved in Hsp110 family proteins, although it is more strongly conserved than the α-helical region among Hsp70s (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The β strands comprise the actual peptide-binding subdomain, and the α helices apparently form a lid over the peptide bound within the β sandwich. Movement of the longest α helix relative to the β sandwich, seen in an alternate crystal structure, may reflect nucleotide-induced conformational changes within DnaK (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). Sequence conservation of the α subdomain suggests that the Hsp88-related proteins retain the Hsp70 mechanism of nucleotide-regulated peptide-binding and release. The lack of conservation of the β subdomain suggests that the actual peptides bound by these proteins differ.Several interactions between small hsps and purified protein substrates have been reported. For example, the small hsp of avian cells was found to inhibit actin polymerization in vitro (39Miron T. Vancompernolle K. Vandekerckhove J. Wilchek M. Geiger B. J. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (387) Google Scholar). Murine Hsp25 (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar) and pea Hsp18.1 (40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar) form complexes with unfolded citrate synthase and other model substrates, and α-crystallin binds to denatured proteins, including β- and γ-crystallins (41Wang K. Spector A. J. Biol. Chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar). The interaction that we have detected between Hsp30 and Hsp88 (and Hsp70) is the first report of complex formation between a small hsp and a protein selected from a cellular extract, and it implies that this association likely occurs in vivo. Hsp30 and Hsp88 appear to interact directly, since the purified proteins bind to one another in the absence of other components. This is also the first report that a member of the Hsp110 protein family associates with a specific protein.Hsp70 was the first hsp proposed to act as a chaperone (42Lewis M.J. Pelham H.R.B. EMBO J. 1985; 4: 3137-3143Crossref PubMed Scopus (238) Google Scholar), and its effect on protein unfolding is well established (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The chaperone activity of small hsps is only beginning to be understood; they appear to bind to unfolded proteins and prevent their aggregation (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar, 40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar). Under moderate conditions of heat stress, small hsps may be sufficient to reactivate substrate enzymes. We found that α-crystallin protected hexokinase activity from thermal inactivation, 3N. Plesofsky-Vig and R. Brambl, manuscript in preparation. and pea small hsps were reported to protect citrate synthase from being inactivated (7Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Under more stringent conditions, however, in addition to murine Hsp25, reactivation of citrate synthase required Hsp70 and ATP, which apparently dissociated the Hsp25-citrate synthase complex (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). This suggests cooperation between Hsp25 and Hsp70. Small hsps were proposed to complex with unfolded proteins early in heat shock and to be released from substrate proteins by ATP-dependent chaperones, such as Hsp70, when ATP-generating processes recover in stressed cells (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). The activity of most chaperones either requires or increases in cooperation with cochaperones. E. coli DnaK, for example, is aided by DnaJ, which enhances its ATPase activity, and by the nucleotide exchange factor GrpE (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The multi-protein complexes formed by Hsp90 with Hsp70, p60, immunophilins, and p23 are required for Hsp90 to bind to and activate unliganded steroid receptors (44Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar).The affinity binding we report between Hsp70 and Hsp30 may reflect the cooperation between these two classes of chaperone in reactivating a substrate enzyme in vitro (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). In contrast to Hsp70, the binding of Hsp88 to Hsp30 is more selective and is not increased by a reaction temperature which denatures proteins in vitro(Table I). Furthermore, the interaction between purified Hsp88 and Hsp30 occurs in the absence of substrate proteins, implying that Hsp88 and Hsp30 may together form a chaperone complex before interacting with substrate proteins. An additional possibility is that the temperature-dependent dissociation of Hsp30 from membranes depends on Hsp88, which is located predominantly in the cytosol. To identify cellular proteins that interact with Hsp30, the small hsp of N. crassa, we analyzed proteins in a cellular extract that were retained by an Hsp30 affinity resin. We found that Hsp70 and Hsp88 specifically bound to immobilized Hsp30. We confirmed the identity of Hsp70 by N-terminal amino acid sequencing. Hsp88 is a previously uncharacterized protein, whose cDNA we isolated and sequenced. Hsp88 appears to be a normal cellular constituent, whose expression increases severalfold in response to high temperature stress. Distantly related to Hsp70, Hsp88 is homologous to several recently characterized proteins, which are also reported to be induced by heat shock. Mammalian Hsp110 is one of the most strongly induced hsps in heat-stressed Chinese hamster ovary cells (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). Murine Osp94, in addition to being induced by heat shock, is strongly synthesized in response to hyperosmotic stress (35Kojima R. Randall J. Brenner B.M. Gullans S.R. J. Biol. Chem. 1996; 271: 12327-12332Abstract Full Text Full Text PDF PubMed Scopus (95) Google Scholar). These proteins localize chiefly to the cytosol, although Hsp110 is also peripherally associated with nucleoli (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar) and Hsp105 with nuclei (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). N. crassa Hsp88 is also predominantly cytosolic with a minor portion being membrane-associated. Unlike Hsp70 and small hsps, these proteins do not appear to change their location in response to high temperature (10Lee-Yoon D. Easton D. Murawski M. Burd R. Subjeck J.R. J. Biol. Chem. 1995; 270: 15725-15733Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). There are consistent differences between the Hsp110 family proteins and Hsp70. The two Neurospora proteins have only 18% identity in their C-terminal putative peptide-binding domains, indicating extensive sequence divergence. In contrast their putative ATPase domains have 38% identity, and the recognized ATP-binding motifs are conserved in Hsp88. In our alignment of Hsp88-related and Hsp70 proteins, we found 49 identical and 67 similar residues conserved within the N-terminal domain and only 7 and 9, respectively, within the C-terminal domain. Nevertheless, properties of the N-terminal ATPase domains display surprising contrasts. This domain is more hydrophobic in Hsp88-related proteins than in Hsp70, due particularly to regions within three separate subdomains of the Hsc70 crystal structure, IA, IIA, and IIB (29Flaherty K.M. DeLuca-Flaherty C. McKay D.B. Nature. 1990; 346: 623-628Crossref PubMed Scopus (823) Google Scholar). Furthermore, the Hsp88/Hsp110 ATPase domain is slightly basic, whereas this domain in Hsp70 is acidic. These properties would be expected to affect binding and hydrolysis of ATP by Hsp88 and related proteins. We found that binding of Hsp88 to ATP-agarose does not occur in the presence of Mg2+ but does occur when Mn2+ is the divalent cation, as has also been reported for purified Hsp90/Hsp83 (34Csermely P. Kahn C.R. J. Biol. Chem. 1991; 266: 4943-4950Abstract Full Text PDF PubMed Google Scholar). Hsp105 was also reported not to bind Mg2+ATP-agarose (36Yasuda K. Nakai A. Hatayama T. Nagata K. J. Biol. Chem. 1995; 270: 29718-29723Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar). Except for Hsp70, other N. crassa proteins in addition to Hsp88, such as Hsp83 and Hsp98, also bind more strongly to Mn2+ATP- than to Mg2+ATP-agarose, with binding to Ca2+ATP-agarose being intermediate. The distinctive properties of the ATPase domain of Hsp88-related proteins are likely to influence the function of these proteins. In S. cerevisiae, the Ssb subgroup of Hsp70 is required for growth at low temperature, and domain swapping experiments showed that the N-terminal ATPase domain of Ssb1 is responsible for conferring this function (37James P. Pfund C. Craig E.A. Science. 1997; 275: 387-389Crossref PubMed Scopus (184) Google Scholar). The C-terminal domains of the Hsp88-related proteins and Hsp70 include a region of conserved sequence (Hsp88: 551–653) that corresponds to the α-helical region within the peptide-binding domain of E. coli DnaK, an Hsp70 homologue (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The preceding region of β structure in the peptide-binding domain is not conserved in Hsp110 family proteins, although it is more strongly conserved than the α-helical region among Hsp70s (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). The β strands comprise the actual peptide-binding subdomain, and the α helices apparently form a lid over the peptide bound within the β sandwich. Movement of the longest α helix relative to the β sandwich, seen in an alternate crystal structure, may reflect nucleotide-induced conformational changes within DnaK (38Zhu X. Zhao X. Burkholder W.F. Gragerov A. Ogata C.M. Gottesman M.E. Hendrickson W.A. Science. 1996; 272: 1606-1614Crossref PubMed Scopus (1046) Google Scholar). Sequence conservation of the α subdomain suggests that the Hsp88-related proteins retain the Hsp70 mechanism of nucleotide-regulated peptide-binding and release. The lack of conservation of the β subdomain suggests that the actual peptides bound by these proteins differ. Several interactions between small hsps and purified protein substrates have been reported. For example, the small hsp of avian cells was found to inhibit actin polymerization in vitro (39Miron T. Vancompernolle K. Vandekerckhove J. Wilchek M. Geiger B. J. Cell Biol. 1991; 114: 255-261Crossref PubMed Scopus (387) Google Scholar). Murine Hsp25 (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar) and pea Hsp18.1 (40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar) form complexes with unfolded citrate synthase and other model substrates, and α-crystallin binds to denatured proteins, including β- and γ-crystallins (41Wang K. Spector A. J. Biol. Chem. 1994; 269: 13601-13608Abstract Full Text PDF PubMed Google Scholar). The interaction that we have detected between Hsp30 and Hsp88 (and Hsp70) is the first report of complex formation between a small hsp and a protein selected from a cellular extract, and it implies that this association likely occurs in vivo. Hsp30 and Hsp88 appear to interact directly, since the purified proteins bind to one another in the absence of other components. This is also the first report that a member of the Hsp110 protein family associates with a specific protein. Hsp70 was the first hsp proposed to act as a chaperone (42Lewis M.J. Pelham H.R.B. EMBO J. 1985; 4: 3137-3143Crossref PubMed Scopus (238) Google Scholar), and its effect on protein unfolding is well established (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The chaperone activity of small hsps is only beginning to be understood; they appear to bind to unfolded proteins and prevent their aggregation (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar, 40Lee G.J. Roseman A.M. Saibil H.R. Vierling E. EMBO J. 1997; 16: 659-671Crossref PubMed Scopus (653) Google Scholar). Under moderate conditions of heat stress, small hsps may be sufficient to reactivate substrate enzymes. We found that α-crystallin protected hexokinase activity from thermal inactivation, 3N. Plesofsky-Vig and R. Brambl, manuscript in preparation. and pea small hsps were reported to protect citrate synthase from being inactivated (7Lee G.J. Pokala N. Vierling E. J. Biol. Chem. 1995; 270: 10432-10438Abstract Full Text Full Text PDF PubMed Scopus (271) Google Scholar). Under more stringent conditions, however, in addition to murine Hsp25, reactivation of citrate synthase required Hsp70 and ATP, which apparently dissociated the Hsp25-citrate synthase complex (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). This suggests cooperation between Hsp25 and Hsp70. Small hsps were proposed to complex with unfolded proteins early in heat shock and to be released from substrate proteins by ATP-dependent chaperones, such as Hsp70, when ATP-generating processes recover in stressed cells (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). The activity of most chaperones either requires or increases in cooperation with cochaperones. E. coli DnaK, for example, is aided by DnaJ, which enhances its ATPase activity, and by the nucleotide exchange factor GrpE (43Hartl F.U. Nature. 1996; 381: 571-580Crossref PubMed Scopus (3088) Google Scholar). The multi-protein complexes formed by Hsp90 with Hsp70, p60, immunophilins, and p23 are required for Hsp90 to bind to and activate unliganded steroid receptors (44Owens-Grillo J.K. Czar M.J. Hutchison K.A. Hoffmann K. Perdew G.H. Pratt W.B. J. Biol. Chem. 1996; 271: 13468-13475Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar). The affinity binding we report between Hsp70 and Hsp30 may reflect the cooperation between these two classes of chaperone in reactivating a substrate enzyme in vitro (8Ehrnsperger M. Graber S. Gaestel M. Buchner J. EMBO J. 1997; 16: 221-229Crossref PubMed Scopus (630) Google Scholar). In contrast to Hsp70, the binding of Hsp88 to Hsp30 is more selective and is not increased by a reaction temperature which denatures proteins in vitro(Table I). Furthermore, the interaction between purified Hsp88 and Hsp30 occurs in the absence of substrate proteins, implying that Hsp88 and Hsp30 may together form a chaperone complex before interacting with substrate proteins. An additional possibility is that the temperature-dependent dissociation of Hsp30 from membranes depends on Hsp88, which is located predominantly in the cytosol. We thank Dr. Ben Madden of the Mayo Clinic (Rochester, MN) for assistance with peptide sequencing." @default.
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W1977793080 title "Characterization of an 88-kDa Heat Shock Protein of Neurospora crassa That Interacts with Hsp30" @default.
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