The 70 kDa heat shock family of molecular chaperones is essential to a variety of cellular processes, yet it is unclear how these proteins are regulated in vivo. We present evidence that the protein BAG‐1 is a potential modulator of the molecular chaperones, Hsp70 and Hsc70. BAG‐1 binds to the ATPase domain of Hsp70 and Hsc70, without requirement for their carboxy‐terminal peptide‐binding domain, and can be co‐immunoprecipitated with Hsp/Hsc70 from cell lysates. Purified BAG‐1 and Hsp/Hsc70 efficiently form heteromeric complexes in vitro. BAG‐1 inhibits Hsp/Hsc70‐mediated in vitro refolding of an unfolded protein substrate, whereas BAG‐1 mutants that fail to bind Hsp/Hsc70 do not affect chaperone activity. The binding of BAG‐1 to one of its known cellular targets, Bcl‐2, in cell lysates was found to be dependent on ATP, consistent with the possible involvement of Hsp/Hsc70 in complex formation. Overexpression of BAG‐1 also protected certain cell lines from heat shock‐induced cell death. The identification of Hsp/Hsc70 as a partner protein for BAG‐1 may explain the diverse interactions observed between BAG‐1 and several other proteins, including Raf‐1, steroid hormone receptors and certain tyrosine kinase growth factor receptors. The inhibitory effects of BAG‐1 on Hsp/Hsc70 chaperone activity suggest that BAG‐1 represents a novel type of chaperone regulatory proteins and thus suggest a link between cell signaling, cell death and the stress response.

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Molecular chaperones influence the process of protein folding and, under conditions of stress, recognize non-native proteins to ensure that misfolded proteins neither appear nor accumulate. BAG-1, identified as an Hsp70 associated protein, was shown to have the unique properties of a negative regulator of Hsp70. Here, we demonstrate that BAG-1 inhibits the in vitro protein refolding activity of Hsp70 by forming stable ternary complexes with non-native 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 ternary complex does not aggregate and remains in a soluble intermediate folded state, indistinguishable from the refolding-competent substrate-Hsp70 complex. 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 presence of BAG-1, the conformation of Hsp70 is altered such that the substrate binding domain becomes less accessible to protease digestion, even in the presence of nucleotide and Hdj-1. These results suggest a mechanistic basis for BAG-1 as a negative regulator of the Hsp70-Hdj-1 chaperone cycle.