ERdj3 Regulates BiP Occupancy in Living Cells

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 represents a critical parameter for chaperone activity. ERdj3, an abundant soluble endoplasmic reticulum (ER) co-chaperone of the Hsp70 BiP, stimulates BiP's ATPase 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 overexpressed ERdj3-sfGFP dramatically decreases BiP-GFP mobility in a client-dependent manner. In contrast, ERdj3-GFP mobility remains low regardless of client protein levels. Native gels and co-immunoprecipitations established ERdj3 associates with a large complex including Sec61α. Translocon binding likely ensures rapid encounters between emerging nascent peptides and stimulates BiP activity in critical 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 dual roles of increasing BiP affinity for clients and regulating delivery of clients to BiP. Our data suggest BiP engagement of clients will be enhanced in ER subdomains enriched in ERdj proteins.