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-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.
Michels
A A
AA
Department of Radiobiology, Faculty of Medical Sciences, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands.
Kanon
B
B
Bensaude
O
O
Kampinga
H H
HH
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
D050956:HSP40 Heat-Shock Proteins; D018840:HSP70 Heat-Shock Proteins; D006360:Heat-Shock Proteins