In vivo chaperone activity of heat shock protein 70 and thermotolerance

Heat shock protein 70 (Hsp70) is thought to play a critical role in the the rmotolerance of mammalian cells, presumably due to its chaperone activity. We examined the chaperone activity and cellular heat resistance of a clonal cell line in which overexpression of Hsp70 was transiently induced by mean s of the tetracycline-regulated gene expression system. This single-cell-li ne approach circumvents problems associated with clonal variation and indir ect effects resulting from constitutive overexpression of Hsp70. The in viv o chaperone function of Hsp70 was quantitatively investigated by using fire fly luciferase as a reporter protein. Chaperone activity was found to stric tly correlate to the level of Hsp70 expression. In addition, we observed an Hsp70 concentration dependent increase in the cellular heat resistance. in order to study the contribution of the Hsp70 chaperone activity, heat resi stance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the ot her heat shock proteins. Overexpression of Hsp70 alone was sufficient to in duce a similar recovery of cytoplasmic luciferase activity, as does express ion of all asps in thermotolerant cells. However, when the luciferase repor ter protein was directed to the nucleus, expression of Hsp70 alone was not sufficient to yield the level of recovery observed in thermotolerant cells. In addition, cells expressing the same level of Hsp70 found in heat-induce d thermotolerant cells containing additional Hsps showed increased resistan ce to thermal killing but were more sensitive than thermotolerant cells. Th ese results suggest that the inducible form of Hsp70 contributes to the str ess-tolerant state by increasing the chaperone activity in the cytoplasm. H owever, its expression alone is apparently insufficient for protection of o ther subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.