BAG-1, a negative regulator of Hsp70 chaperone activity, uncouples nucleotide hydrolysis from substrate release

Molecular chaperones influence the process of protein folding and, under co nditions of stress, recognize nonnative proteins to ensure that misfolded p roteins neither appear nor accumulate, BAG-1, identified as an Hsp70 associ ated protein, was shown to have the unique properties of a negative regulat or of Hsp70. Here, we demonstrate that BAG-1 inhibits the in vitro protein refolding activity of Hsp70 by forming stable ternary complexes with non-na tive substrates that do not release even in the presence of nucleotide and the co-chaperone, Hdj-1. However, the substrate in the BAG-1-containing ter nary complex does not aggregate and remains in a soluble intermediate folde d state, indistinguishable from the refolding-competent substrate-Hsp70 com plex. BAG-1 neither inhibits the Hsp70 ATPase, nor has the properties of a nucleotide exchange factor; instead, it stimulates ATPase activity, similar to that observed for Hdj-1, but with opposite consequences, In the presenc e of BAG-I, the conformation of Hsp70 is altered such that the substrate bi nding domain becomes less accessible to protease digestion, even in the pre sence of nucleotide and Hdj-1. These results suggest a mechanistic basis fo r BAG-1 as a negative regulator of the Hsp70-Hdj-1 chaperone cycle.