Wolfgang M.J. Obermann

Heat shock protein 90 (Hsp90), an abundant molecular chaperone in the eukaryotic cytosol, is involved in the folding of a set of cell regulatory proteins and in the re-folding of stress-denatured polypeptides. The basic mechanism of action of Hsp90 is not yet understood. In particular, it has been debated whether Hsp90 function is ATP dependent. A recent crystal structure of the NH2-terminal domain of yeast Hsp90 established the presence of a conserved nucleotide binding site that is identical with the binding site of geldanamycin, a specific inhibitor of Hsp90. The functional significance of nucleotide binding by Hsp90 has remained unclear. Here we present evidence for a slow but clearly detectable ATPase activity in purified Hsp90. Based on a new crystal structure of the NH2-terminal domain of human Hsp90 with bound ADP-Mg and on the structural homology of this domain with the ATPase domain of Escherichia coli DNA gyrase, the residues of Hsp90 critical in ATP binding (D93) and ATP hydrolysis (E47) were identified. The corresponding mutations were made in the yeast Hsp90 homologue, Hsp82, and tested for their ability to functionally replace wild-type Hsp82. Our results show that both ATP binding and hydrolysis are required for Hsp82 function in vivo. The mutant Hsp90 proteins tested are defective in the binding and ATP hydrolysis-dependent cycling of the co-chaperone p23, which is thought to regulate the binding and release of substrate polypeptide from Hsp90. Remarkably, the complete Hsp90 protein is required for ATPase activity and for the interaction with p23, suggesting an intricate allosteric communication between the domains of the Hsp90 dimer. Our results establish Hsp90 as an ATP-dependent chaperone.

The ATP-dependent molecular chaperone Hsp90 is an essential and abundant stress protein in the eukaryotic cytosol that cooperates with a cohort of cofactors/cochaperones to fulfill its cellular tasks. We have identified Aha1 (activator of Hsp90 ATPase) and its relative Hch1 (high copy Hsp90 suppressor) as binding partners of Hsp90 in Saccharomyces cerevisiae. By using genetic and biochemical approaches, the middle domain of Hsp90 (amino acids 272-617) was found to mediate the interaction with Aha1 and Hch1. Data base searches revealed that homologues of Aha1 are conserved from yeast to man, whereas Hch1 was found to be restricted to lower eukaryotes like S. cerevisiae and Candida albicans. In experiments with purified proteins, Aha1 but not Hch1 stimulated the intrinsic ATPase activity of Hsp90 5-fold. To establish their cellular role further, we deleted the genes encoding Aha1 and Hch1 in S. cerevisiae. In vivo experiments demonstrated that Aha1 and Hch1 contributed to efficient activation of the heterologous Hsp90 client protein v-Src. Moreover, Aha1 and Hch1 became crucial for cell viability under non-optimal growth conditions when Hsp90 levels are limiting. Thus, our results identify a novel type of cofactor involved in the regulation of the molecular chaperone Hsp90.

The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90α (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the ∼25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains. The molecular chaperone hsp90 in the eukaryotic cytosol interacts with a variety of protein cofactors. Several of these cofactors have protein domains containing tetratricopeptide repeat (TPR) motifs, which mediate binding to hsp90. Using a yeast two-hybrid screen, the 12-kDa C-terminal domain of human hsp90α (C90) was found to mediate the interaction of hsp90 with TPR-containing sequences from the hsp90 cofactors FKBP51/54 and FKBP52. In addition, the mitochondrial outer membrane protein hTOM34p was identified as a TPR-containing putative partner protein of hsp90. In experiments with purified proteins, the TPR-containing cofactor p60 (Hop) was shown to form stable complexes with hsp90. A deletion mutant of hsp90 lacking the C90 domain was unable to bind p60, whereas deletion of the ∼25-kDa N-terminal domain of hsp90 did not affect complex formation. Both p60 and FKBP52 bound specifically to the C90 domain fused to glutathione S-transferase and competed with each other for binding. In reticulocyte lysate, the C90 fusion protein recognized the TPR proteins p60, FKBP52, and Cyp40. Thus, our results identify the C90 domain as the specific binding site for a set of hsp90 cofactors having TPR domains. The 90-kDa heat shock protein (hsp90) 1The abbreviations used are: hsp, heat shock protein; Cyp, cyclophilin; C90, hsp90 C-terminal domain; FKBP, FK506-binding protein; GST, glutathione S-transferase; N90, hsp90 N-terminal domain; PAGE, polyacrylamide gel electrophoresis; SD, synthetic dropout medium; TPR, tetratricopeptide repeat. 1The abbreviations used are: hsp, heat shock protein; Cyp, cyclophilin; C90, hsp90 C-terminal domain; FKBP, FK506-binding protein; GST, glutathione S-transferase; N90, hsp90 N-terminal domain; PAGE, polyacrylamide gel electrophoresis; SD, synthetic dropout medium; TPR, tetratricopeptide repeat. is a highly conserved molecular chaperone in the eukaryotic cytosol. In vitro, hsp90 can prevent the aggregation of unfolded proteins and cooperate with the hsp70/hsp40 chaperone system in the ATP-dependent refolding of unfolded model proteins (1Wiech H. Buchner J. Zimmermann R. Jacob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (420) Google Scholar, 2Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 3Freeman B.C. Morimoto R.I. EMBO J. 1996; 15: 2969-2979Crossref PubMed Scopus (380) Google Scholar, 4Young J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). However, in vivo studies indicate a more restricted role of hsp90 in the conformational regulation of certain signal transduction molecules, including steroid hormone receptors and proto-oncogenic kinases (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 6Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (311) Google Scholar, 8Pratt W.B. 1997; PubMed Scopus Google Scholar). hsp90 role in the refolding of certain proteins D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (311) Google Scholar, S. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In these hsp90 interacts with a of protein cofactors as the FKBP52 and and (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 8Pratt W.B. 1997; PubMed Scopus Google Scholar, S. D.F. 1996; PubMed Scopus Google Scholar). The of hsp90 is specifically certain which have S. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google cofactors of the tetratricopeptide repeat (TPR) (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, S. D.F. 1996; PubMed Scopus Google Scholar, W.B. 1997; PubMed Google a in Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). The TPR-containing domains of FKBP52, and protein mediate binding of these proteins to hsp90 C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (155) Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of p60 to hsp90 a of p60 TPR (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, D.F. 1996; PubMed Google Scholar). The of the TPR domain EMBO J. PubMed Scopus Google the of interaction with hsp90 is as a specific of TPR-containing proteins, with TPR motifs, can bind hsp90. the cofactor protein is not to bind to TPR J. Hartl F.U. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, D.F. 1996; Google Scholar, H. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cofactors as and TPR the hsp90 was domains Schneider C. Hartl F.U. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The ∼25-kDa N-terminal domain was identified as the binding site for the Schneider C. Hartl F.U. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google and for C. R. Cell. 1997; 90: Full Text Full Text PDF PubMed Scopus Google Scholar). Both the domain and 12-kDa C-terminal domain of hsp90 (C90) shown to chaperone in J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). In addition, the C-terminal of including the C90 domain and of the is in hsp90 J. Biochem. 1995; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). yeast two-hybrid and was used to identify the C90 domain as the binding site for the TPR-containing cofactor of human hsp90α (C90) was used as the in a yeast two-hybrid with a of identified shown to specifically with the C90 for a of the FKBP51/54 S. D.F. Cell. Biol. 1997; PubMed Scopus Google other FKBP52 Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google The and for hTOM34p a mitochondrial outer membrane protein to in protein J. and J. the with C90 of proteins containing TPR domains the with hsp90 TPR C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (155) Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google our results identify the C-terminal domain of hsp90 as a binding site for these the two-hybrid was not the of hTOM34p as a putative protein the of TPR proteins recognized hsp90 the mitochondrial outer membrane protein in specific of proteins for is to bind hsp90 in W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). these to a role of hsp90 in mitochondrial protein was not the C90 domain in the two-hybrid The for to the C-terminal of hsp90 and is in the C90 domain J. Biochem. 1995; PubMed Scopus Google the C-terminal domain of hsp90 the of hsp90 interacts with TPR the hsp90 protein a of hsp90 the N-terminal and domains and the C-terminal domain (C90) in the two-hybrid system for binding to the TPR protein The hsp90 in the yeast as and C90 binding to FKBP52, and did not In these two-hybrid indicate binding of TPR proteins to hsp90 is the C-terminal domain of binding to and the results of the two-hybrid purified hsp90 and deletion of hsp90 for to with purified p60 hsp90 and p60 gel with the of hsp90 in and p60 in and The molecular of the hsp90 of is in with J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). p60 as a a containing of hsp90 and p60 was gel hsp90 and the of p60 in with the of a stable complex the The binding of p60 to hsp90 was of was and was a of C-terminal domain of hsp90 is to form stable complexes with hsp90 and domain deletion with p60 and gel a and of proteins shown and purified purified hsp90 with with with binding of TPR proteins to the C-terminal domain of hsp90. p60 and with hsp90 domains fused to and protein with was and of purified p60 and of purified FKBP52. p60 bound to and The with in is protein with and is to FKBP52 binding to in the of p60 and in the of p60 a to FKBP52 of and The of FKBP52 bound with p60 is as a of the bound in the of was with hsp90 and with hsp90 and a and and the of p60 in each was and to the of of hsp90 and proteins of reticulocyte bound to with and with bound with identified with specific and the of proteins with in is a deletion mutant of hsp90 the and C90 the sequences in hsp90 J. Biochem. 1995; PubMed Scopus Google 1996; PubMed Scopus Google and was to form to in a with molecular p60 and and p60 with in a the and domains of hsp90 in a with the of the deletion mutant In the of p60 gel in to the of p60 The of was in the of p60 is unable to bind the can form complexes with the and with hsp90. of C90 to p60 is not shown in complexes containing C90 and p60 not from protein results purified FKBP52 in of p60 Thus, the C90, not the N90, domain is for binding of these TPR proteins, in with the results from the two-hybrid of p60 and FKBP52 for binding to the and C-terminal hsp90 domains fused to glutathione S-transferase and of p60 with each of the purified fusion proteins to and proteins with p60 was not with and bound to FKBP52 was with the fusion protein the TPR proteins with hsp90 for binding to the C-terminal hsp90 was the binding of FKBP52 to in the of of p60 in of and to FKBP52 the of p60 more p60 and FKBP52 with and of in the of a of p60, binding of FKBP52 was of in the of The of p60 is in these experiments the of the TPR proteins is of of the is TPR domain proteins for binding to the C-terminal domain of hsp90. Thus, hsp90 with specific TPR protein a the interaction the C90 domain and TPR proteins is with hsp90 for binding to TPR of hsp90 and p60 with a of and gel as in The of p60 in is shown in The of p60 in a the of and with a complex containing p60 and A p60 complexes in the of to and p60 in p60 and indicate and hsp90 for the binding site the C-terminal domain of hsp90 the specific TPR interaction site as the hsp90 the of the C90 was with a reticulocyte and with complexes hsp90 and p60 FKBP52 in proteins bound to from the with a A set of proteins in the from the protein of the with molecular of and identified as p60, FKBP52, and Thus, the C90 domain specifically in a cytosol the TPR-containing cofactors with the of hsp90. for and a molecular to a proteins bound to the the and with the TPR-containing protein which to the domain of J. Hartl F.U. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, H. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. D.F. Cell. Biol. 1996; PubMed Scopus Google not in the bound Thus, for with a of TPR-containing hsp90 was not bound to the TPR-containing cofactors recognized the C-terminal hsp90 In with our interaction of hsp90 with shown to of the C90 domain J. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in the TPR proteins cooperate with results in identify the C-terminal domain of hsp90 as and for interaction of hsp90 with TPR-containing proteins domain is the for TPR binding the C90 domain found to as a chaperone in the aggregation of unfolded proteins as and binding the J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). TPR protein the of C90 to as a molecular chaperone J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google the C90 domain bind to a specific TPR protein and to unfolded The 90-kDa heat shock protein (hsp90) 1The abbreviations used are: hsp, heat shock protein; Cyp, cyclophilin; C90, hsp90 C-terminal domain; FKBP, FK506-binding protein; GST, glutathione S-transferase; N90, hsp90 N-terminal domain; PAGE, polyacrylamide gel electrophoresis; SD, synthetic dropout medium; TPR, tetratricopeptide repeat. 1The abbreviations used are: hsp, heat shock protein; Cyp, cyclophilin; C90, hsp90 C-terminal domain; FKBP, FK506-binding protein; GST, glutathione S-transferase; N90, hsp90 N-terminal domain; PAGE, polyacrylamide gel electrophoresis; SD, synthetic dropout medium; TPR, tetratricopeptide repeat. is a highly conserved molecular chaperone in the eukaryotic cytosol. In vitro, hsp90 can prevent the aggregation of unfolded proteins and cooperate with the hsp70/hsp40 chaperone system in the ATP-dependent refolding of unfolded model proteins (1Wiech H. Buchner J. Zimmermann R. Jacob U. Nature. 1992; 358: 169-170Crossref PubMed Scopus (420) Google Scholar, 2Jakob U. Lilie H. Meyer I. Buchner J. J. Biol. Chem. 1995; 270: 7288-7294Abstract Full Text Full Text PDF PubMed Scopus (318) Google Scholar, 3Freeman B.C. Morimoto R.I. EMBO J. 1996; 15: 2969-2979Crossref PubMed Scopus (380) Google Scholar, 4Young J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). However, in vivo studies indicate a more restricted role of hsp90 in the conformational regulation of certain signal transduction molecules, including steroid hormone receptors and proto-oncogenic kinases (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 6Johnson J.L. Craig E.A. Cell. 1997; 90: 201-204Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar, 7Nathan D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (311) Google Scholar, 8Pratt W.B. 1997; PubMed Scopus Google Scholar). hsp90 role in the refolding of certain proteins D.F. Vos M.H. Lindquist S. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12949-12956Crossref PubMed Scopus (311) Google Scholar, S. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar). In these hsp90 interacts with a of protein cofactors as the FKBP52 and and (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, 8Pratt W.B. 1997; PubMed Scopus Google Scholar, S. D.F. 1996; PubMed Scopus Google Scholar). The of hsp90 is specifically certain which have S. Hartl F.U. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Several cofactors of the tetratricopeptide repeat (TPR) (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, S. D.F. 1996; PubMed Scopus Google Scholar, W.B. 1997; PubMed Google a in Trends Biochem. Sci. Full Text PDF PubMed Scopus Google Scholar). The TPR-containing domains of FKBP52, and protein mediate binding of these proteins to hsp90 C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (155) Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). The binding of p60 to hsp90 a of p60 TPR (5Frydman J. Höhfeld J. Trends Biochem. Sci. 1997; 22: 87-92Abstract Full Text PDF PubMed Scopus (255) Google Scholar, D.F. 1996; PubMed Google Scholar). The of the TPR domain EMBO J. PubMed Scopus Google the of interaction with hsp90 is as a specific of TPR-containing proteins, with TPR motifs, can bind hsp90. the cofactor protein is not to bind to TPR J. Hartl F.U. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, D.F. 1996; Google Scholar, H. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). cofactors as and TPR In the hsp90 was domains Schneider C. Hartl F.U. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). The ∼25-kDa N-terminal domain was identified as the binding site for the Schneider C. Hartl F.U. Cell. 1997; Full Text Full Text PDF PubMed Scopus Google and for C. R. Cell. 1997; 90: Full Text Full Text PDF PubMed Scopus Google Scholar). Both the domain and 12-kDa C-terminal domain of hsp90 (C90) shown to chaperone in J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). In addition, the C-terminal of including the C90 domain and of the is in hsp90 J. Biochem. 1995; PubMed Scopus Google Scholar, 1996; PubMed Scopus Google Scholar). yeast two-hybrid and was used to identify the C90 domain as the binding site for the TPR-containing cofactor of human hsp90α (C90) was used as the in a yeast two-hybrid with a of identified shown to specifically with the C90 for a of the FKBP51/54 S. D.F. Cell. Biol. 1997; PubMed Scopus Google other FKBP52 Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google The and for hTOM34p a mitochondrial outer membrane protein to in protein J. and J. the with C90 of proteins containing TPR domains the with hsp90 TPR C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (155) Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google our results identify the C-terminal domain of hsp90 as a binding site for these the two-hybrid was not the of hTOM34p as a putative protein the of TPR proteins recognized hsp90 the mitochondrial outer membrane protein in specific of proteins for is to bind hsp90 in W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). these to a role of hsp90 in mitochondrial protein was not the C90 domain in the two-hybrid The for to the C-terminal of hsp90 and is in the C90 domain J. Biochem. 1995; PubMed Scopus Google the C-terminal domain of hsp90 the of hsp90 interacts with TPR the hsp90 protein a of hsp90 the N-terminal and domains and the C-terminal domain (C90) in the two-hybrid system for binding to the TPR protein The hsp90 in the yeast as and C90 binding to FKBP52, and did not In these two-hybrid indicate binding of TPR proteins to hsp90 is the C-terminal domain of binding to and the results of the two-hybrid purified hsp90 and deletion of hsp90 for to with purified p60 hsp90 and p60 gel with the of hsp90 in and p60 in and The molecular of the hsp90 of is in with J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). p60 as a a containing of hsp90 and p60 was gel hsp90 and the of p60 in with the of a stable complex the The binding of p60 to hsp90 was of was and was a of binding of TPR proteins to the C-terminal domain of hsp90. p60 and with hsp90 domains fused to and protein with was and of purified p60 and of purified FKBP52. p60 bound to and The with in is protein with and is to FKBP52 binding to in the of p60 and in the of p60 a to FKBP52 of and The of FKBP52 bound with p60 is as a of the bound in the of was with hsp90 and with hsp90 and a and and the of p60 in each was and to the of of hsp90 and proteins of reticulocyte bound to with and with bound with identified with specific and the of proteins with in is a deletion mutant of hsp90 the and C90 the sequences in hsp90 J. Biochem. 1995; PubMed Scopus Google 1996; PubMed Scopus Google and was to form to in a with molecular p60 and and p60 with in a the and domains of hsp90 in a with the of the deletion mutant In the of p60 gel in to the of p60 The of was in the of p60 is unable to bind the can form complexes with the and with hsp90. of C90 to p60 is not shown in complexes containing C90 and p60 not from protein results purified FKBP52 in of p60 Thus, the C90, not the N90, domain is for binding of these TPR proteins, in with the results from the two-hybrid of p60 and FKBP52 for binding to the and C-terminal hsp90 domains fused to glutathione S-transferase and of p60 with each of the purified fusion proteins to and proteins with p60 was not with and bound to FKBP52 was with the fusion protein the TPR proteins with hsp90 for binding to the C-terminal hsp90 was the binding of FKBP52 to in the of of p60 in of and to FKBP52 the of p60 more p60 and FKBP52 with and of in the of a of p60, binding of FKBP52 was of in the of The of p60 is in these experiments the of the TPR proteins is of of the is TPR domain proteins for binding to the C-terminal domain of hsp90. Thus, hsp90 with specific TPR protein a the interaction the C90 domain and TPR proteins is with hsp90 for binding to TPR of hsp90 and p60 with a of and gel as in The of p60 in is shown in The of p60 in a the of and with a complex containing p60 and A p60 complexes in the of to and p60 in p60 and indicate and hsp90 for the binding site the C-terminal domain of hsp90 the specific TPR interaction site as the hsp90 the of the C90 was with a reticulocyte and with complexes hsp90 and p60 FKBP52 in proteins bound to from the with a A set of proteins in the from the protein of the with molecular of and identified as p60, FKBP52, and Thus, the C90 domain specifically in a cytosol the TPR-containing cofactors with the of hsp90. for and a molecular to a proteins bound to the the and with the TPR-containing protein which to the domain of J. Hartl F.U. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, H. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. D.F. Cell. Biol. 1996; PubMed Scopus Google not in the bound Thus, for with a of TPR-containing hsp90 was not bound to the TPR-containing cofactors recognized the C-terminal hsp90 In with our interaction of hsp90 with shown to of the C90 domain J. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in the TPR proteins cooperate with results in identify the C-terminal domain of hsp90 as and for interaction of hsp90 with TPR-containing proteins domain is the for TPR binding the C90 domain found to as a chaperone in the aggregation of unfolded proteins as and binding the J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). TPR protein the of C90 to as a molecular chaperone J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google the C90 domain bind to a specific TPR protein and to unfolded The of human hsp90α (C90) was used as the in a yeast two-hybrid with a of identified shown to specifically with the C90 for a of the FKBP51/54 S. D.F. Cell. Biol. 1997; PubMed Scopus Google other FKBP52 Proc. Natl. Acad. Sci. U. S. A. 1992; PubMed Scopus Google The and for hTOM34p a mitochondrial outer membrane protein to in protein J. and J. the with C90 of proteins containing TPR domains the with hsp90 TPR C. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus (155) Google Scholar, A. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google our results identify the C-terminal domain of hsp90 as a binding site for these the two-hybrid was not the of hTOM34p as a putative protein the of TPR proteins recognized hsp90 the mitochondrial outer membrane protein in specific of proteins for is to bind hsp90 in W.B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). these to a role of hsp90 in mitochondrial protein hsp90 was not the C90 domain in the two-hybrid The for to the C-terminal of hsp90 and is in the C90 domain J. Biochem. 1995; PubMed Scopus Google Scholar). the C-terminal domain of hsp90 the of hsp90 interacts with TPR the hsp90 protein a of hsp90 the N-terminal and domains and the C-terminal domain (C90) in the two-hybrid system for binding to the TPR protein The hsp90 in the yeast as and C90 binding to FKBP52, and did not In these two-hybrid indicate binding of TPR proteins to hsp90 is the C-terminal domain of hsp90. binding to and the results of the two-hybrid purified hsp90 and deletion of hsp90 for to with purified p60 hsp90 and p60 gel with the of hsp90 in and p60 in and The molecular of the hsp90 of is in with J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar, J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). p60 as a a containing of hsp90 and p60 was gel hsp90 and the of p60 in with the of a stable complex the The binding of p60 to hsp90 was of was and was a of a deletion mutant of hsp90 the and C90 the sequences in hsp90 J. Biochem. 1995; PubMed Scopus Google 1996; PubMed Scopus Google and was to form to in a with molecular p60 and and p60 with in a the and domains of hsp90 in a with the of the deletion mutant In the of p60 gel in to the of p60 The of was in the of p60 is unable to bind the can form complexes with the and with hsp90. of C90 to p60 is not shown in complexes containing C90 and p60 not from protein results purified FKBP52 in of p60 Thus, the C90, not the N90, domain is for binding of these TPR proteins, in with the results from the two-hybrid of p60 and FKBP52 for binding to the and C-terminal hsp90 domains fused to glutathione S-transferase and of p60 with each of the purified fusion proteins to and proteins with p60 was not with and bound to FKBP52 was with the fusion protein the TPR proteins with hsp90 for binding to the C-terminal hsp90 was the binding of FKBP52 to in the of of p60 in of and to FKBP52 the of p60 more p60 and FKBP52 with and of in the of a of p60, binding of FKBP52 was of in the of The of p60 is in these experiments the of the TPR proteins is of of the is TPR domain proteins for binding to the C-terminal domain of hsp90. Thus, hsp90 with specific TPR protein a the interaction the C90 domain and TPR proteins is with hsp90 for binding to TPR of hsp90 and p60 with a of and gel as in The of p60 in is shown in The of p60 in a the of and with a complex containing p60 and A p60 complexes in the of to and p60 in p60 and indicate and hsp90 for the binding site the C-terminal domain of hsp90 the specific TPR interaction site as the hsp90 the of the C90 was with a reticulocyte and with complexes hsp90 and p60 FKBP52 in proteins bound to from the with a A set of proteins in the from the protein of the with molecular of and identified as p60, FKBP52, and Thus, the C90 domain specifically in a cytosol the TPR-containing cofactors with the of hsp90. for and a molecular to a proteins bound to the the and with the TPR-containing protein which to the domain of J. Hartl F.U. Cell. 1995; Full Text PDF PubMed Scopus Google Scholar, H. J. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, S. A. D.F. Cell. Biol. 1996; PubMed Scopus Google not in the bound Thus, for with a of TPR-containing hsp90 was not bound to the TPR-containing cofactors recognized the C-terminal hsp90 In with our interaction of hsp90 with shown to of the C90 domain J. H. J. Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar). in the TPR proteins cooperate with hsp90. The results in identify the C-terminal domain of hsp90 as and for interaction of hsp90 with TPR-containing proteins domain is the for TPR binding the C90 domain found to as a chaperone in the aggregation of unfolded proteins as and binding the J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google Scholar). TPR protein the of C90 to as a molecular chaperone J.C. Schneider C. Hartl F.U. FEBS Lett. 1997; 418: 139-143Crossref PubMed Scopus (133) Google the C90 domain bind to a specific TPR protein and to unfolded for the FKBP52 and and for the

The M band of vertebrate cross-striated myofibrils has remained an enigmatic structure. In addition to myosin thick filaments, two major structural proteins, myomesin and M-protein, have been localized to the M band. Also, titin is expected to be anchored in this structure. To begin to understand the molecular layout of these three proteins, a panel of 16 polyclonal and monoclonal antibodies directed against unique epitopes of defined sequence was assembled, and immunoelectron microscopy was used to locate the position of the epitopes at the sarcomere level. The results allow the localization and orientation of defined domains of titin, myomesin, and M-protein at high resolution. The 250-kD carboxy-terminal region of titin clearly enters the M band with the kinase domain situated approximately 52 nm from the central M1-line. The positions of three additional epitopes are compatible with the view that the titin molecule reaches approximately 60 nm into the opposite sarcomere half. Myomesin also seems to bridge the central M1-line and is oriented parallel to the long axis of the myofibril. The neighboring molecules are oriented in an antiparallel and staggered fashion. The amino-terminal portion of the protein, known to contain a myosin binding site, seems to adopt a specific three-dimensional arrangement. While myomesin is present in both slow and fast fibers, M-protein is restricted to fast fibers. It appears to be organized in a fundamentally different manner: the central portion of the polypeptide is around the M1-line, while the terminal epitopes seem to be arranged along thick filaments. This orientation fits the conspicuously stronger M1-lines in fast twitch fibers. Obvious implications of this model are discussed.