ABSTRACT
Osp94 (also known as HSPA4L or HSPH3), a member of the Hsp110/Sse1 family of heat-shock proteins, has a longer C-terminus than found in Hsc70/Hsp70 family proteins, composed of the loop region with a partial substrate-binding domain (SBD) ß (L), and the SBDα and the C-terminal extension (H), but the functions of these domains are poorly understood. Here, we found that Osp94 suppressed heat-induced aggregation of luciferase (Luc). Osp94-bound heat-inactivated Luc was reactivated in the presence of rabbit reticulocyte lysate (RRL) and/or a combination of Hsc70 and Hsp40 (also known as HSPA8 and DNAJB1, respectively). Targeted deletion mutagenesis revealed that the SBDß and H domains of Osp94 are critical for protein disaggregation and RRL-mediated refolding. Reactivation of Hsp90-bound heat-inactivated Luc was abolished in the absence of RRL but compensated for by PA28α (also known as PSME1), a proteasome activator. Interestingly, the LH domain also reactivated heat-inactivated Luc, independently of PA28α. Biotin-tag cross-linking experiments indicated that the LH domain and PA28α interact with Luc bound by Hsp90 during refolding. A chimeric protein in which the H domain was exchanged for PA28α also mediated disaggregation and reactivation of heat-inactivated Luc. These results indicate that Osp94 acts as a holdase, and that the C-terminal region plays a PA28α-like role in the refolding of unfolded proteins.
