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Molecular and Cellular Biology, March 1999, p. 2069-2079, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

In Vivo Chaperone Activity of Heat Shock Protein 70 and Thermotolerance

Ellen A. A. Nollen,1 Jeanette F. Brunsting,1 Han Roelofsen,2 Lee A. Weber,3 and Harm H. Kampinga1,*

Department of Radiobiology, Faculty Medical Sciences, University of Groningen, 9713 BZ Groningen,1 and Division of Gastroenterology and Hepatology, Department of Internal Medicine, Groningen Institute for Drug Studies, Groningen, 9713 GZ Groningen,2 The Netherlands, and Department of Biology, University of Nevada, Reno, Nevada 895573

Received 27 April 1998/Returned for modification 18 June 1998/Accepted 30 November 1998

Heat shock protein 70 (Hsp70) is thought to play a critical role in the thermotolerance 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 means of the tetracycline-regulated gene expression system. This single-cell-line approach circumvents problems associated with clonal variation and indirect effects resulting from constitutive overexpression of Hsp70. The in vivo chaperone function of Hsp70 was quantitatively investigated by using firefly luciferase as a reporter protein. Chaperone activity was found to strictly 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 resistance of cells that expressed tetracycline-regulated Hsp70 was compared to thermotolerant cells expressing the same level of Hsp70 plus all of the other heat shock proteins. Overexpression of Hsp70 alone was sufficient to induce a similar recovery of cytoplasmic luciferase activity, as does expression of all Hsps in thermotolerant cells. However, when the luciferase reporter 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-induced thermotolerant cells containing additional Hsps showed increased resistance to thermal killing but were more sensitive than thermotolerant cells. These results suggest that the inducible form of Hsp70 contributes to the stress-tolerant state by increasing the chaperone activity in the cytoplasm. However, its expression alone is apparently insufficient for protection of other subcellular compartments to yield clonal heat resistance to the level observed in thermotolerant cells.

* Corresponding author. Mailing address: Department of Radiobiology, Faculty Medical Sciences, University of Groningen, Bloemsingel 1, 9713 BZ Groningen, The Netherlands. Phone: 31-50-3632903/2911. Fax: 31-50-3632913. E-mail: H.H.Kampinga{at}med.rug.nl.

Molecular and Cellular Biology, March 1999, p. 2069-2079, Vol. 19, No. 3
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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