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

Abstract

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 Hsp150Δ–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, Hsp150Δ–β-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Δ– 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 Hsp150Δ–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.

Biology of the Heat Shock Response and Protein Chaperones: Budding Yeast (Saccharomyces cerevisiae) as a Model System

“…This is consistent with the observation that the other ER NEF, SIL1 (suppressor of the ire1⌬ lhs1⌬ double mutant) (see below) can partially compensate for LHS1 in translocation when overexpressed (471). Lhs1 was found to be required for the refolding, solubilization, and reactivation of the marker protein Hsp150⌬-␤-lactamase after heat denaturation but interestingly played no role in its translocation, folding, and secretion under normal conditions (387). Two general conclusions can be drawn from these data.…”

Cer1p Functions as a Molecular Chaperone in the Endoplasmic Reticulum of Saccharomyces cerevisiae

“…They concluded from similar CPY pulse-chase refolding experiments with DTT that no translocation or folding defects are evident in cer1⌬ strains (Fig. 9C) (35). However, a translocation defect is clearly evident in their cer1⌬ strain pulse-chase 0-min time point.…”

“…The shorter times used in our study allowed the defect to be observed. However, the long times used points in the Saris et al (35) study did indicate that CPY does not accumulate and remain in the ER of a cer1 deletion strain for extended periods. Either the protein eventually folds in the ER and is transported to the Golgi in the absence of Cer1p or it is degraded in the ER.…”

Modulation of Chaperone Gene Expression in Mutagenized Saccharomyces cerevisiae Strains Developed for Recombinant Human Albumin Production Results in Increased Production of Multiple Heterologous Proteins

“…Maintaining Kar2p in its active, cycling state may both keep luminal load down and keep Ire1p in its inactive form. To complicate this picture, Lhs1p has been shown to have chaperone activity itself, aside from its interaction with Kar2p (29).…”