Bag1 functions in vivo as a negative regulator of Hsp70 chaperone activity

Studies on the Hsp70 chaperone machine in eukaryotes have shown that Hsp70 and Hsp40/Hdj1 family proteins are sufficient to prevent protein misfolding and aggregation and to promote refolding of denatured polypeptides. Additi onal protein cofactors include Hip and Bag1, identified in protein interact ion assays, which bind to and modulate Hsp70 chaperone activity in vitro. B ag1, originally identified as an antiapoptotic protein, forms a stoichiomet ric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro pro tein refolding of an unfolded polypeptide. Given its proposed involvement i n multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulato r of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activ ities of Hsp70, as a molecular chaperone essential for stabilization and re folding of a thermally inactivated protein. The levels of Hsp70 and Bag1 we re modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in dif ferent mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denature d luciferase by a factor of 2. This effect was titratable, and higher level s of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refold ing by up to a factor of 5. The negative effects of Bag1 were also observed in a biochemical analysis of Bag1- or Hsp70-overexpressing cells. The abil ity of Hsp70 to maintain thermally denatured firefly luciferase in a solubl e state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observ ed with other cytoplasmic isoforms of Bag1 which have in common the carbony l-terminal Hsp70-binding domain and differ by variable-length amino-termina l extensions. These results provide the first formal evidence that Bag1 fun ctions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.