Mutational Analysis of the hsp70-Interacting Protein Hip

Abstract

The hsp70-interacting protein Hip participates in the assembly pathway for progesterone receptor complexes. During assembly, Hip appears at early assembly stages in a transient manner that parallels hsp70 interactions. In this study, a cDNA for human Hip was used to develop various mutant Hip forms in the initial mapping of functions to particular Hip structural elements. Hip regions targeted for deletion and/or truncation included the C-terminal region (which has some limited homology with Saccharomyces cerevisiae Sti1 and its vertebrate homolog p60), a glycine-glycine-methionine-proline (GGMP) tandem repeat, and a tetratricopeptide repeat (TPR). Binding of Hip to hsp70's ATPase domain was lost with deletions from the TPR and from an adjoining highly charged region; correspondingly, these Hip mutant forms were not recovered in receptor complexes. Truncation of Hip's Sti1-related C terminus resulted in Hip binding to hsp70 in a manner suggestive of a misfolded peptide substrate; this hsp70 binding was localized to the GGMP tandem repeat. Mutants lacking either the C terminus or the GGMP tandem repeat were still recovered in receptor complexes. Truncations from Hip's N terminus resulted in an apparent loss of Hip homo-oligomerization, but these mutants retained association with hsp70 and were recovered in receptor complexes. This mutational analysis indicates that Hip's TPR is required for binding of Hip with hsp70's ATPase domain. In addition, some data suggest that hsp70's peptide-binding domain may alternately or concomitantly bind to Hip's GGMP repeat in a manner regulated by Sti1-related sequences.

Heat shock cognate protein 70 chaperone-binding site in the co-chaperone murine stress-inducible protein 1 maps to within three consecutive tetratricopeptide repeat motifs

“…Unlike PP5, Hip and the immunophilins [12][13][14]19,20], extensive regions flanking the mSTI1 TPR domains are not imperative for mSTI1-hsp90 and mSTI1-hsc70 interactions. These results might support the classification of TPR proteins involved in chaperone-cochaperone interactions into two different groups ( Figure 5) encompassing those that do not require extensive TPR flanking regions (less than 20 residues) and those that require more extensive flanking regions (more than 20 residues).…”

“…This class includes mSTI1/Hop [3,15]. Class II represents the group of TPR co-chaperones in which extensive TPR flanking regions (more than 20 residues) are necessary for interaction with heat shock protein, including the immunophilins (CyP40, FKBP51 and FKBP52) [10,12,13], Hip [19,20] and PP5 [14]. The TPR motifs in each of the TPR proteins are shaded in grey.…”

“…It is thought that these regions are similarly necessary in FKBP51 and FKBP52. In Hip, removal of the Nterminus to within 14 residues of the TPR domain allowed hsp70 recognition, albeit at lower levels than full-length Hip [20]. Additional N-terminal flanking regions might be necessary for hsp70 recognition [19], as indicated by the underlying dotted line.…”

“…Competitive binding studies between the immunophilins, PP5 and Hop suggest that they bind to an overlapping TPR acceptor site on hsp90 [16][17][18]. TPR domains of both mSTI1\Hop and Hip mediate their interactions with hsp70 [2,3,15,19,20]. However, these cochaperones do not share a common TPR acceptor site on hsp70 [21,22].…”

“…The proposed TPR acceptor sites for Hip and Hop are the N-terminal ATPase domain and C-terminal domain of hsp70 respectively, suggesting that their mechanism of protein recognition differs. In addition to the TPR domain itself, the interaction of Hip with hsp70 requires the participation of an acidic, positively charged α-helix after the TPR domain [19,20].…”