Regulation and recovery of functions of Saccharomyces cerevisiae chaperone BiP/Kar2p after thermal insult

Abstract

We described earlier a novel mode of regulation of Hsp104, a cytosolic chaperone directly involved in the refolding of heat-denatured proteins, and designated it delayed upregulation, or DUR. When Saccharomyces cerevisiae cells grown at the physiological temperature of 24 degrees C, preconditioned at 37 degrees C, and treated briefly at 50 degrees C were shifted back to 24 degrees C, Hsp104 expression was strongly induced after 2.5 h of recovery and returned back to normal after 5 h. Here we show that the endoplasmic reticulum (ER) chaperones BiP/Kar2p and Lhs1p and the mitochondrial chaperone Hsp78 were also upregulated at the physiological temperature during recovery from thermal insult. The heat shock element (HSE) in the KAR2 promoter was found to be sufficient to drive DUR. The unfolded protein element could also evoke DUR, albeit weakly, in the absence of a functional HSE. BiP/Kar2p functions in ER translocation and assists protein folding. Here we found that the synthesis of new BiP/Kar2p molecules was negligible for more than an hour after the shift of the cells from 50 degrees C to 24 degrees C. Concomitantly, ER translocation was blocked, suggesting that preexisting BiP/Kar2p molecules or other necessary proteins were not functioning. Translocation resumed concomitantly with enhanced synthesis of BiP/Kar2p after 3 h of recovery, after which ER exit and protein secretion also resumed. For a unicellular organism like S. cerevisiae, conformational repair of denatured proteins is the sole survival strategy. Chaperones that refold proteins in the cytosol, ER, and mitochondria of S. cerevisiae appear to be subject to DUR to ensure survival after thermal insults.

FIG. 1.

Upregulation of Kar2p expression during recovery from thermal insult at 24°C. (A) Schematic presentation of thermal treatments. (B) Metabolic labeling of Kar2p. Cells (strain H245) grown at 24°C were divided into 10 parallel samples and labeled with [³⁵S]methionine-cysteine for successive 45-min periods, as indicated by the bars in panel A (a to j). After the termination of labeling, the cells were lysed for immunoprecipitation with Kar2p antiserum, and the immunoprecipitates were resolved by SDS-PAGE, followed by autoradiography. (C) Phosphorimager quantification of [³⁵S]Kar2p levels in panel B. Normalization was done against the level of [³⁵S]Kar2p in untreated cells (sample a). (D) The experiment in panel C was repeated, but with the 50°C treatment omitted.

FIG. 2.

Real-time quantitative PCR analysis of mRNA levels. (A) Cells (H245) were exposed to heat treatments and harvested at the indicated time points. Total RNA was extracted, and 2-μg samples were subjected to reverse transcription-PCR analysis with KAR2 (B), LHS1 (C), or HSP78 primers (D) (see Materials and Methods). The value for untreated cells at 24°C was set to 1.

FIG. 3.

Contribution of different promoter elements of KAR2 to DUR. (A) Metabolic labeling of β-galactosidase. The figure shows a schematic presentation of the thermal treatments. Cells (strain H1806) harboring a β-galactosidase reporter construct regulated by the KAR2 promoter were grown at 24°C, divided into 10 parallel samples, and labeled with [³⁵S]methionine-cysteine for successive 45-min periods, as indicated by the bars (a to j). After the termination of labeling, the cells were lysed for immunoprecipitation with β-galactosidase antiserum, and the immunoprecipitates were resolved by SDS-PAGE, followed by autoradiography. (B) Phosphorimager quantification of [³⁵S]β-galactosidase. Normalization was done against the level of [³⁵S]β-galactosidase in untreated cells (sample a). Data are means of six experiments. (C to L) Summary of a series of metabolic labeling experiments and β-galactosidase assays. Metabolic labeling and immunoprecipitation of β-galactosidase were performed with strains harboring different KAR2 promoter-lacZ variants, as shown in panel A. The basal expression of β-galactosidase in the strains was quantified by enzyme activity measurements. The magnitude of β-galactosidase expression in strain H1806, harboring a KAR2 promoter (−280 to −109) directly upstream of the lacZ reporter, was used as a reference for β-galactosidase expression at 24°C (basal), after 1 h at 37°C (HS), and at 24°C 3 h after thermal insult (DUR). 0, no expression; =, same expression as that in H1806; <, expression weaker than that in H1806.

FIG. 4.

Translocation and secretion of a reporter glycoprotein during recovery from thermal insult. (A) Schematic presentation of the experiment. Cells (strain H335) were divided into six parallel samples, subjected to thermal treatments, labeled with [³⁵S]methionine-cysteine for 5 min, as indicated by the bars, and chased for different periods, as indicated in panel B. After the termination of labeling, the growth media were separated from the cells, which were then lysed. The cell lysates and growth media were subjected to immunoprecipitation with β-lactamase antiserum, and the immunoprecipitates were resolved by SDS-PAGE, followed by autoradiography. (B) Cytoplasmic (66 kDa; triangles), ER-specific (110 kDa; squares), and mature (145 kDa; circles) forms of β-lactamase were quantified by phosphorimaging and plotted against the chase time.