title: Heat shock protein (Hsp) 40 mutants inhibit Hsp70 in mammalian cells.
abstract: Heat shock protein (Hsp) 70 and Hsp40 expressed in mammalian cells had been previously shown to cooperate in accelerating the reactivation of heat-denatured firefly luciferase (Michels, A. A., Kanon, B., Konings, A. W. T., Ohtsuka, K., Bensaude, O., and Kampinga, H. H. (1997) J. Biol. Chem. 272, 33283-33289). We now provide further evidence for a functional interaction between Hsp70 and the J-domain of Hsp40 with denatured luciferase resulting in reactivation of heat-denatured luciferase within living mammalian cells. The stimulating effect of Hsp40 on the Hsp70-mediated refolding is lost when the proteins cannot interact as accomplished by their expression in different intracellular compartments. Likewise, the cooperation between Hsp40 and Hsp70 is lost by introduction of a point mutation in the conserved HPD motif 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 protein restricted to the J-domain of Hsp40 had a dominant negative effect on Hsp70-facilitated luciferase reactivation. Taken together, these experiments indicate for the first time that the Hsp70/Hsp40 chaperones functionally interact with a heat-denatured protein within mammalian cells. The dominant negative effect of the Hsp40 J-domain on the activity of Hsp70 demonstrates the importance of J-domain-containing proteins in Hsp70-dependent processes.
authors.affiliation: Department of Radiobiology, Faculty of Medical Sciences, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands.
authors.forename: A A
authors.initials: AA
authors.lastname: Michels
authors.forename: B
authors.initials: B
authors.lastname: Kanon
authors.forename: O
authors.initials: O
authors.lastname: Bensaude
authors.forename: H H
authors.initials: HH
authors.lastname: Kampinga
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D006224:Cricetinae; D005347:Fibroblasts; D005786:Gene Expression Regulation; D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D015398:Signal Transduction
chemical_list: D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins