ERdj3 regulates BiP occupancy in living cells

• Published in: Journal of cell science Vol. 126; no. Pt 6; pp. 1429 - 1439
• Main Authors: Guo, Feng, Snapp, Erik L
• Format: Journal Article
• Language: English
• Published: England The Company of Biologists 15-03-2013
• Subjects: Animals; Cell Line, Tumor; Dogs; Endoplasmic Reticulum - metabolism; Heat-Shock Proteins - metabolism; HSP40 Heat-Shock Proteins - genetics; HSP40 Heat-Shock Proteins - metabolism; Humans; Madin Darby Canine Kidney Cells; Membrane Proteins - metabolism; Molecular Chaperones - genetics; Molecular Chaperones - metabolism; Mutation - genetics; Protein Binding - genetics; SEC Translocation Channels; Substrate Specificity - genetics; Transgenes - genetics; BiP; FRAP; ERdj; Fluorescence recovery after photobleaching; Superfolder GFP; Quality control
• ISSN: 0021-9533; 1477-9137
• DOI: 10.1242/jcs.118182

Co-chaperones regulate chaperone activities and are likely to impact a protein-folding environment as much as the chaperone itself. As co-chaperones are expressed substoichiometrically, the ability of co-chaperones to encounter a chaperone is crucial for chaperone activity. ERdj3, an abundant soluble endoplasmic reticulum (ER) co-chaperone of the Hsp70 BiP, stimulates the ATPase activity of BiP to increase BiP's affinity for client (or substrate) proteins. We investigated ERdj3 availability, how ERdj3 levels impact BiP availability, and the significance of J proteins for regulating BiP binding of clients in living cells. FRAP analysis revealed that overexpressed ERdj3-sfGFP dramatically decreases BiP-GFP mobility in a client-dependent manner. By contrast, ERdj3-GFP mobility remains low regardless of client protein levels. Native gels and co-immunoprecipitations established that ERdj3 associates with a large complex including Sec61α. Translocon binding probably ensures rapid encounters between emerging nascent peptides and stimulates BiP activity in the crucial early stages of secretory protein folding. Importantly, mutant BiP exhibited significantly increased mobility when it could not interact with any ERdjs. Thus, ERdjs appear to play the dual roles of increasing BiP affinity for clients and regulating delivery of clients to BiP. Our data suggest that BiP engagement of clients is enhanced in ER subdomains enriched in ERdj proteins.