CHARACTERIZATION OF FUNCTIONAL DOMAINS OF THE EUKARYOTIC CO-CHAPERONEHIP

The homo-oligomeric Rip protein cooperates with the 70-kDa heat shock cognate Hsc70 in the folding of newly synthesized polypeptide chains a nd in the conformational regulation of signaling molecules known to in teract with Hsc70 and Hsp90. In order to further assess the role of Hi p during protein biogenesis, a structure-function analysis of the Hip protein was initiated. By employing the yeast two-hybrid system, the H sc70-binding site of Rip was mapped to a domain comprising multiple te tratricopeptide repeats and flanking charged alpha-helices. Affinity c hromatography confirmed direct interaction of isolated Rip fragments a nd protein fusions bearing this region with the ATPase domain of Hsc70 in an ATP- and salt-dependent manner. Contact of Hip with the ATPase domain appears to be mediated primarily by the positively charged alph a-helix following the tetratricopeptide repeats. Furthermore, a domain required for homo-oligomerization was identified at the extreme amino terminus of Hip.