HSP70 AND HSP40 CHAPERONE ACTIVITIES IN THE CYTOPLASM AND THE NUCLEUSOF MAMMALIAN-CELLS

The existence and function of a Hsp40-Hsp70 chaperone machinery in mam malian cells in vivo was investigated, The rate of heat inactivation o f firefly luciferase transiently expressed in hamster O23 fibroblasts was analyzed in cells co-transfected with the gene encoding the human Hsp40 (Ohtsuka, K. (1993) Biochem. Biophys. Res. Commun. 197, 235-240) , the human inducible Hsp70 (Hunt, C., and Morimoto, R. I. (1985) Proc . Natl. Acad. Sci. U.S.A. 82, 6455-6459), or a combination of both, Wh ereas the expression of human Hsp70 alone in hamster cells was suffici ent for the protection of firefly luciferase during heat shock, expres sion of the human Hsp40 alone was not, Rather, this led to a small but significant increase in the heat sensitivity of luciferase. The expre ssion of the human Hsp40 only led to heat protection when the human Hs p70 was expressed as well. Under such conditions the rate of luciferas e reactivation from the heat-inactivated state was increased, but the rate of inactivation during heat shock was not affected. Using constru cts that direct firefly luciferase either to the cytoplasm or to the n ucleus (Michels, A. A., Nguyen, V. -T., Konings, A. W. T., Kampinga, H . H., and Bensaude, O. (1995) Eur. J. Biochem. 234, 382-389), it was d emonstrated that these chaperone functions are found in both compartme nts. Our data provide the first evidence on how the Hsp40/Hsp70 chaper one complex acts as heat protector in mammalian cells in vivo.