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Molecular and Cellular Biology, February 2000, p. 1083-1088, Vol. 20, No. 3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Bag1 Functions In Vivo as a Negative Regulator of Hsp70 Chaperone Activity

Ellen A. A. Nollen,1 Jeanette F. Brunsting,1 Jaewhan Song,2 Harm H. Kampinga,1 and Richard I. Morimoto2,*

Department of Radiobiology, Faculty of Medical Sciences, University of Groningen, Groningen, The Netherlands,1 and Department of Biochemistry, Molecular Biology and Cell Biology, Rice Institute for Biomedical Research, Northwestern University, Evanston, Illinois 602082

Received 23 September 1999/Returned for modification 26 October 1999/Accepted 1 November 1999

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. Additional protein cofactors include Hip and Bag1, identified in protein interaction assays, which bind to and modulate Hsp70 chaperone activity in vitro. Bag1, originally identified as an antiapoptotic protein, forms a stoichiometric complex with Hsp70 and inhibits completely Hsp70-dependent in vitro protein refolding of an unfolded polypeptide. Given its proposed involvement in multiple cell signaling events as a regulator of Raf1, Bcl2, or androgen receptor, we wondered whether Bag1 functions in vivo as a negative regulator of Hsp70. In this study, we demonstrate that Bag1, expressed in mammalian tissue culture cells, has pronounced effects on one of the principal activities of Hsp70, as a molecular chaperone essential for stabilization and refolding of a thermally inactivated protein. The levels of Hsp70 and Bag1 were modulated either by transient transfection or conditional expression in stably transfected lines to achieve levels within the range detected in different mammalian tissue culture cell lines. For example, a twofold increase in the concentration of Bag1 reduced Hsp70-dependent refolding of denatured luciferase by a factor of 2. This effect was titratable, and higher levels of wild-type but not a mutant form of Bag1 further inhibited Hsp70 refolding 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 ability of Hsp70 to maintain thermally denatured firefly luciferase in a soluble state was reversed by Bag1, thus providing an explanation for the in vivo chaperone-inhibitory effects of Bag1. Similar effects on Hsp70 were observed with other cytoplasmic isoforms of Bag1 which have in common the carboxyl-terminal Hsp70-binding domain and differ by variable-length amino-terminal extensions. These results provide the first formal evidence that Bag1 functions in vivo as a regulator of Hsp70 and suggest an intriguing complexity for Hsp70-regulatory events.

* Corresponding author. Mailing address: Department of Biochemistry, Molecular Biology and Cell Biology, Northwestern University, 2153 North Campus Dr., Evanston, IL 60208. Phone: (847) 491-3340. Fax: (847) 491-4461. E-mail: r-morimoto{at}nwu.edu.

Molecular and Cellular Biology, February 2000, p. 1083-1088, Vol. 20, No. 3
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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