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The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates.

Saris N, Holkeri H, Craven RA, Stirling CJ, Makarow M - J. Cell Biol. (1997)

Bottom Line: In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded.Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-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 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

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 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-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 Hsp150Delta-beta-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 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees 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.

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Dependence of reactivation of heat-denatured Hsp150Δ– β-lactamase on Lhs1p. (Top) Scheme of the renaturation assay.  After growth at 24°C, cells were incubated for 60–70 min at 37°C  (preconditioning), thereafter for 20 min at 50°C (thermal insult),  and then for 6 h at 24°C (recovery). Strains (A) H621 (lhs1−  sec18), (B) H393 (sec18), and (C) H645 (lhs1− LHS1+ sec18)  were preincubated for 10 min at 37°C, pelleted, resuspended in  prewarmed medium, and incubated at 37°C for 1 h and then at  50°C for 20 min. CHX (closed circles) or NaN3 (open circles) was  added, and the cells were incubated for the indicated times at  24°C. The β-lactamase activity of lysed cells was determined and  plotted against time. The absence (lhs1−) and presence (WT) of  the LHS1 gene is indicated. The designation wild-type (WT) refers only to the LHS1 gene.
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Figure 1: Dependence of reactivation of heat-denatured Hsp150Δ– β-lactamase on Lhs1p. (Top) Scheme of the renaturation assay. After growth at 24°C, cells were incubated for 60–70 min at 37°C (preconditioning), thereafter for 20 min at 50°C (thermal insult), and then for 6 h at 24°C (recovery). Strains (A) H621 (lhs1− sec18), (B) H393 (sec18), and (C) H645 (lhs1− LHS1+ sec18) were preincubated for 10 min at 37°C, pelleted, resuspended in prewarmed medium, and incubated at 37°C for 1 h and then at 50°C for 20 min. CHX (closed circles) or NaN3 (open circles) was added, and the cells were incubated for the indicated times at 24°C. The β-lactamase activity of lysed cells was determined and plotted against time. The absence (lhs1−) and presence (WT) of the LHS1 gene is indicated. The designation wild-type (WT) refers only to the LHS1 gene.

Mentions: Our assay to study heat-denaturation and refolding of ERlocated proteins in living S. cerevisiae cells is presented at the top of Fig. 1. Cells grown at 24°C are incubated in liquid medium for an h at 37°C. The marker protein Hsp150Δ–β-lactamase is expressed under the heat-activated HSP150 promoter in a temperature-sensitive mutant (sec18), where ER-derived transport vesicles are unable to fuse with Golgi membranes at 37°C. The 37°C incubation thus preconditions the cells to survive a subsequent thermal insult at 50°C, upregulates the synthesis of the marker protein, and causes its retention in the preGolgi compartment. The cells are then incubated for 20 min at 50°C, resulting in inactivation of the β-lactamase fusion protein, and shifted to 24°C to monitor reactivation of the marker enzyme. In tens of experiments, 20–70% of the original β-lactamase activity was resumed in 3–6 h at 24°C in the presence of CHX (21). The variation in the reactivation yields evidently reflects the vulnerability of the cells to extreme temperature. Hsp150Δ–β-lactamase consists of Escherichia coli β-lactamase fused to the COOH terminus of the Hsp150Δ-carrier, an NH2-terminal 321–amino acid fragment of the yeast secretory glycoprotein Hsp150 (20). The carrier is needed for translocation and to promote proper folding of foreign proteins that by themselves are not secretion-competent in yeast (25, 43).


The Hsp70 homologue Lhs1p is involved in a novel function of the yeast endoplasmic reticulum, refolding and stabilization of heat-denatured protein aggregates.

Saris N, Holkeri H, Craven RA, Stirling CJ, Makarow M - J. Cell Biol. (1997)

Dependence of reactivation of heat-denatured Hsp150Δ– β-lactamase on Lhs1p. (Top) Scheme of the renaturation assay.  After growth at 24°C, cells were incubated for 60–70 min at 37°C  (preconditioning), thereafter for 20 min at 50°C (thermal insult),  and then for 6 h at 24°C (recovery). Strains (A) H621 (lhs1−  sec18), (B) H393 (sec18), and (C) H645 (lhs1− LHS1+ sec18)  were preincubated for 10 min at 37°C, pelleted, resuspended in  prewarmed medium, and incubated at 37°C for 1 h and then at  50°C for 20 min. CHX (closed circles) or NaN3 (open circles) was  added, and the cells were incubated for the indicated times at  24°C. The β-lactamase activity of lysed cells was determined and  plotted against time. The absence (lhs1−) and presence (WT) of  the LHS1 gene is indicated. The designation wild-type (WT) refers only to the LHS1 gene.
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Figure 1: Dependence of reactivation of heat-denatured Hsp150Δ– β-lactamase on Lhs1p. (Top) Scheme of the renaturation assay. After growth at 24°C, cells were incubated for 60–70 min at 37°C (preconditioning), thereafter for 20 min at 50°C (thermal insult), and then for 6 h at 24°C (recovery). Strains (A) H621 (lhs1− sec18), (B) H393 (sec18), and (C) H645 (lhs1− LHS1+ sec18) were preincubated for 10 min at 37°C, pelleted, resuspended in prewarmed medium, and incubated at 37°C for 1 h and then at 50°C for 20 min. CHX (closed circles) or NaN3 (open circles) was added, and the cells were incubated for the indicated times at 24°C. The β-lactamase activity of lysed cells was determined and plotted against time. The absence (lhs1−) and presence (WT) of the LHS1 gene is indicated. The designation wild-type (WT) refers only to the LHS1 gene.
Mentions: Our assay to study heat-denaturation and refolding of ERlocated proteins in living S. cerevisiae cells is presented at the top of Fig. 1. Cells grown at 24°C are incubated in liquid medium for an h at 37°C. The marker protein Hsp150Δ–β-lactamase is expressed under the heat-activated HSP150 promoter in a temperature-sensitive mutant (sec18), where ER-derived transport vesicles are unable to fuse with Golgi membranes at 37°C. The 37°C incubation thus preconditions the cells to survive a subsequent thermal insult at 50°C, upregulates the synthesis of the marker protein, and causes its retention in the preGolgi compartment. The cells are then incubated for 20 min at 50°C, resulting in inactivation of the β-lactamase fusion protein, and shifted to 24°C to monitor reactivation of the marker enzyme. In tens of experiments, 20–70% of the original β-lactamase activity was resumed in 3–6 h at 24°C in the presence of CHX (21). The variation in the reactivation yields evidently reflects the vulnerability of the cells to extreme temperature. Hsp150Δ–β-lactamase consists of Escherichia coli β-lactamase fused to the COOH terminus of the Hsp150Δ-carrier, an NH2-terminal 321–amino acid fragment of the yeast secretory glycoprotein Hsp150 (20). The carrier is needed for translocation and to promote proper folding of foreign proteins that by themselves are not secretion-competent in yeast (25, 43).

Bottom Line: In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded.Lhs1p had no significant role in folding or secretion of newly synthesized Hsp150Delta-beta-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 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees C, but only 10% were able to form colonies, as compared to wild-type cells.

View Article: PubMed Central - PubMed

Affiliation: Institute of Biotechnology, University of Helsinki, Helsinki, Finland.

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 degrees C after preconditioning at 37 degrees C, was reactivated by an ATP-dependent machinery, when the cells were returned to physiological temperature 24 degrees C. Here we show that refolding of the marker enzyme Hsp150Delta-beta-lactamase, inactivated and aggregated by the 50 degrees C treatment, required a novel ER-located homologue of the Hsp70 family, Lhs1p. In the absence of Lhs1p, Hsp150Delta-beta-lactamase failed to be solubilized and reactivated and was slowly degraded. Coimmunoprecipitation experiments suggested that Lhs1p was somehow associated with heat-denatured Hsp150Delta- beta-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 Hsp150Delta-beta-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 degrees C treatment, cells lacking Lhs1p remained capable of protein synthesis and secretion for several hours at 24 degrees 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.

Show MeSH
Related in: MedlinePlus