Abstract
SummaryProtein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor. Hsp40 mediates complex formation between Hsp70 and client proteins prior to interaction with Hsp90. We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR). We found that Hsp40 promotes interactions between the client and Hsp70, and facilitates dimerization of monomeric Hsp70. This dimerization is antiparallel, stabilized by post-translational modifications (PTMs), and maintained in the stable heterohexameric client-loading complex Hsp902Hsp702HopGR identified here. Addition of p23 to this client-loading complex induces transfer of GR onto Hsp90 and leads to expulsion of Hop and Hsp70. Based on these results, we propose that Hsp70 antiparallel dimerization, stabilized by PTMs, positions the client for transfer from Hsp70 to Hsp90.
Highlights
heat shock protein 70 (Hsp70) and Hsp90 are essential and abundant molecular chaperones in the eukaryotic cytosol and are involved in the folding and maturation of a myriad of protein substrates, including many cancer-causing proteins (Brychzy et al, 2003, Taipale et al, 2010)
Protein folding in cells is regulated by networks of chaperones, including the heat shock protein 70 (Hsp70) system, which consists of the Hsp40 cochaperone and a nucleotide exchange factor
We used mass spectrometry (MS) to monitor assemblies formed between eukaryotic Hsp90/Hsp70/Hsp40, Hop, p23, and a client protein, a fragment of the glucocorticoid receptor (GR)
Summary
Hsp and Hsp are essential and abundant molecular chaperones in the eukaryotic cytosol and are involved in the folding and maturation of a myriad of protein substrates, including many cancer-causing proteins (Brychzy et al, 2003, Taipale et al, 2010). The Hsp70/90 system requires a cohort of cochaperones to provide specificity and regulation of the chaperone interactions with their client proteins (Li et al, 2011, Picard et al, 1990, Young et al, 2001). Hsp forms defined binary or ternary complexes with cochaperones to facilitate the maturation of client proteins (reviewed in Prodromou, 2012). It consists of a C-terminal dimerization region, a middle domain, and an N-terminal nucleotidebinding domain (NBD) connected by a charged linker that provides the necessary flexibility for domain rearrangements (Tsutsumi et al, 2012). Hsp binds at its C-terminal MEEVD sequence to tetratricopeptide repeat (TPR) cochaperones, including the ‘‘Hsc70/Hsp organizing protein’’ Hop (Young et al, 1998)
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