Abstract
The homo-oligomeric Hip protein cooperates with the 70-kDa heat shock cognate Hsc70 in the folding of newly synthesized polypeptide chains and in the conformational regulation of signaling molecules known to interact with Hsc70 and Hsp90. In order to further assess the role of Hip during protein biogenesis, a structure-function analysis of the Hip protein was initiated. By employing the yeast two-hybrid system, the Hsc70-binding site of Hip was mapped to a domain comprising multiple tetratricopeptide repeats and flanking charged alpha-helices. Affinity chromatography confirmed direct interaction of isolated Hip fragments and 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 alpha-helix following the tetratricopeptide repeats. Furthermore, a domain required for homo-oligomerization was identified at the extreme amino terminus of Hip.
Highlights
Molecular chaperones of the 70-kDa Hsps1 play a key role in intracellular protein biogenesis due to their ability to stabilize nonnative protein conformations [1,2,3]
In Escherichia coli, cycling of the Hsp70 homologue DnaK is regulated by the DnaJ protein, which promotes the formation of DnaK-substrate complexes by stimulating DnaK’s ATPase activity and by the nucleotide exchange factor GrpE required for complex dissociation (4 – 6)
By Employing the Yeast Two-hybrid System, the Hsc70-binding Site of Hip Was Mapped to a Domain Comprising Multiple TPR Repeats and Flanking Charged ␣-Helices—To identify structural elements required for binding of Hip to the ATPase
Summary
Molecular chaperones of the 70-kDa Hsps play a key role in intracellular protein biogenesis due to their ability to stabilize nonnative protein conformations [1,2,3]. The ATP dependence of the reaction confers the means of extensive regulation through the function of cooperating proteins that influence the nucleotide state of Hsp and modulate its affinity for polypeptide substrate. Hsc activity in the eukaryotic cytosol is regulated through a Hip-mediated stabilization of chaperone-substrate complexes, in contrast to the GrpE-mediated dissociation characteristic of the prokaryotic reaction cycle [11]. By presenting the receptor in a conformation that can be recognized by Hsp, Hip in concert with Hsc and a DnaJ homologue appear to mediate early stages of complex assembly (16 –20). A functional characterization of individual domains of the Hip protein was initiated, resulting in the localization of the Hsc70-binding site of Hip and the identification of a domain required for homo-oligomerization These findings are discussed with respect to the function of Hip in Hsc70/Hsp90-mediated protein biogenesis
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