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© The Rockefeller University Press, 0021-9525/1997//813 $5.00
The Journal of Cell Biology, Volume 137, Number 4, , 1997 813-824

The Hsp70 Homologue Lhs1p Is Involved in a Novel Function of the Yeast Endoplasmic Reticulum, Refolding and Stabilization of Heat-denatured Protein Aggregates

Nina Saris^*, Heidi Holkeri^*, Rachel A. Craven, Colin J. Stirling, and Marja Makarow^*

^* Institute of Biotechnology, University of Helsinki, Helsinki, Finland; and School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom

Heat stress is an obvious hazard, and mechanisms to recover from thermal damage, largely unknown as of yet, have evolved in all organisms. We have recently shown that a marker protein in the ER of Saccharomyces cerevisiae, denatured by exposure of cells to 50°C after preconditioning at 37°C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24°C. Here we show that refolding of the marker enzyme Hsp150–β-lactamase, inactivated and aggregated by the 50°C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150–β-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150– β-lactamase, whereas no association with native marker protein molecules could be detected. Similar findings were obtained for a natural glycoprotein of S. cerevisiae, pro-carboxypeptidase Y (pro-CPY). Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150–β-lactamase or pro-CPY, suggesting that the machinery repairing heat-damaged proteins may have specific features as compared to chaperones assisting de novo folding. After preconditioning and 50°C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24°C, but only 10% were able to form colonies, as compared to wild-type cells. We suggest that Lhs1p is involved in a novel function operating in the yeast ER, refolding and stabilization against proteolysis of heatdenatured protein. Lhs1p may be part of a fundamental heat-resistant survival machinery needed for recovery of yeast cells from severe heat stress.

Support from the Academy of Finland and the Foundation of the University of Helsinki is gratefully acknowledged. M. Makarow is a Biocentrum Helsinki fellow. C.J. Stirling is a Lister Institute Jenner Research Fellow.

Address all correspondence to Marja Makarow, Institute of Biotechnology, P.O. Box 56, 00014 University of Helsinki, Finland. Tel.: 358-970859419. Fax: 358-9-70859366. E-mail: Makarow{at}operoni.helsinki.fi

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