Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells

Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-den atured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Oh tsuka, K,, Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272, 332 83-33289), We now provide further evidence for a functional interaction bet ween Hsp70 and the J-domain of Hsp40 with denatured luciferase resulting in reactivation of heat-denatured luciferase within living mammalian cells. T he stimulating effect of Hsp40 on the Hsp70-mediated refolding is lost when the proteins cannot interact as accomplished by their expression in differ ent intracellular compartments, Likewise, the cooperation between Hsp40 and Hsp70 is lost by introduction of a point mutation in the conserved HPD mot if of the Hsp40 J domain or by deletion of the four C-terminal amino acids of Hsp70 (EEVD motif), Most strikingly, co-expression of a truncated protei n restricted to the J-domain of Hsp40 had a dominant negative effect on Hsp 70-facilitated luciferase reactivation. Taken together, these experiments i ndicate for the first time that the Hsp70/Hsp40 chaperones functionally int eract with a heat-denatured protein within mammalian cells. The dominant ne gative effect of the Hsp40 J-domain on the activity of Hsp70 demonstrates t he importance of J-domain-containing proteins in Hsp70-dependent processes.