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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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In vivo degradation of heat-denatured Hsp150Δ–β-lactamase. (A) H621 (lhs1− sec18), (B) H647 (lhs1− c-myc–LHS1+  sec18), and (C) H534 (Hsp104− sec18) cells were preincubated  for 15 min at 37°C, labeled in SC medium lacking methionine and  cysteine with [35S]methionine/cysteine for 20 min, diluted with excess SC medium containing unlabeled methionine and cysteine,  and incubated further at 37°C for 40 min (lanes 1). Parallel cells  were incubated thereafter for 20 min at 50°C (lanes 2), or after  this at 24°C for 2 h (lanes 3), 4 h (lanes 4), 6 h (lanes 5), or 8 h  (lanes 6). The cells were lysed and immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis.
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Figure 7: In vivo degradation of heat-denatured Hsp150Δ–β-lactamase. (A) H621 (lhs1− sec18), (B) H647 (lhs1− c-myc–LHS1+ sec18), and (C) H534 (Hsp104− sec18) cells were preincubated for 15 min at 37°C, labeled in SC medium lacking methionine and cysteine with [35S]methionine/cysteine for 20 min, diluted with excess SC medium containing unlabeled methionine and cysteine, and incubated further at 37°C for 40 min (lanes 1). Parallel cells were incubated thereafter for 20 min at 50°C (lanes 2), or after this at 24°C for 2 h (lanes 3), 4 h (lanes 4), 6 h (lanes 5), or 8 h (lanes 6). The cells were lysed and immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis.

Mentions: To study the stability of heat-aggregated Hsp150Δ–β-lactamase in the absence of Lhs1p in vivo, strain H621 (lhs1− sec18) was labeled with [35S]methionine/cysteine at 37°C (Fig. 7 A, lane 1). Parallel cells were then incubated at 50°C (Fig. 7, lane 2), or thereafter at 24°C for 2–8 h (lanes 3–6). The cell lysates were immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis. Hsp150Δ–β-lactamase persisted for about 4 h at 24°C, growing in size apparently because of some increase in O-glycosylation. During the next 4 h, it was largely degraded. In the rescue strain H647 (lhs1− c-myc–LHS1+ sec18), the amount of the marker protein remained virtually unchanged (Fig. 7 B), as shown before for strain H393 harboring the genomic LHS1 gene (21). Degradation of the marker in the absence of Lhs1p was not due to abrupt cell death, since heat-treated H621 cells were capable of protein synthesis for hours after shift to 24°C (see below). To confirm this, we studied the stability of Hsp150Δ–β-lactamase in a strain lacking a functional HSP104 gene (H534), which does not acquire thermotolerance at 50°C in spite of preconditioning at 37°C (35) (see below). In this strain, the thermal treatments inhibited protein synthesis irreversibly (see below). Hsp150Δ–β-lactamase 35S-labeled at 37°C remained after the 50°C treatment apparently undegraded at 24°C at least for 8 h (Fig. 7 C). These data show that Lhs1p suppressed in vivo degradation of the heat-denatured marker protein.


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)

In vivo degradation of heat-denatured Hsp150Δ–β-lactamase. (A) H621 (lhs1− sec18), (B) H647 (lhs1− c-myc–LHS1+  sec18), and (C) H534 (Hsp104− sec18) cells were preincubated  for 15 min at 37°C, labeled in SC medium lacking methionine and  cysteine with [35S]methionine/cysteine for 20 min, diluted with excess SC medium containing unlabeled methionine and cysteine,  and incubated further at 37°C for 40 min (lanes 1). Parallel cells  were incubated thereafter for 20 min at 50°C (lanes 2), or after  this at 24°C for 2 h (lanes 3), 4 h (lanes 4), 6 h (lanes 5), or 8 h  (lanes 6). The cells were lysed and immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis.
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Figure 7: In vivo degradation of heat-denatured Hsp150Δ–β-lactamase. (A) H621 (lhs1− sec18), (B) H647 (lhs1− c-myc–LHS1+ sec18), and (C) H534 (Hsp104− sec18) cells were preincubated for 15 min at 37°C, labeled in SC medium lacking methionine and cysteine with [35S]methionine/cysteine for 20 min, diluted with excess SC medium containing unlabeled methionine and cysteine, and incubated further at 37°C for 40 min (lanes 1). Parallel cells were incubated thereafter for 20 min at 50°C (lanes 2), or after this at 24°C for 2 h (lanes 3), 4 h (lanes 4), 6 h (lanes 5), or 8 h (lanes 6). The cells were lysed and immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis.
Mentions: To study the stability of heat-aggregated Hsp150Δ–β-lactamase in the absence of Lhs1p in vivo, strain H621 (lhs1− sec18) was labeled with [35S]methionine/cysteine at 37°C (Fig. 7 A, lane 1). Parallel cells were then incubated at 50°C (Fig. 7, lane 2), or thereafter at 24°C for 2–8 h (lanes 3–6). The cell lysates were immunoprecipitated with anti– β-lactamase antiserum, followed by SDS-PAGE analysis. Hsp150Δ–β-lactamase persisted for about 4 h at 24°C, growing in size apparently because of some increase in O-glycosylation. During the next 4 h, it was largely degraded. In the rescue strain H647 (lhs1− c-myc–LHS1+ sec18), the amount of the marker protein remained virtually unchanged (Fig. 7 B), as shown before for strain H393 harboring the genomic LHS1 gene (21). Degradation of the marker in the absence of Lhs1p was not due to abrupt cell death, since heat-treated H621 cells were capable of protein synthesis for hours after shift to 24°C (see below). To confirm this, we studied the stability of Hsp150Δ–β-lactamase in a strain lacking a functional HSP104 gene (H534), which does not acquire thermotolerance at 50°C in spite of preconditioning at 37°C (35) (see below). In this strain, the thermal treatments inhibited protein synthesis irreversibly (see below). Hsp150Δ–β-lactamase 35S-labeled at 37°C remained after the 50°C treatment apparently undegraded at 24°C at least for 8 h (Fig. 7 C). These data show that Lhs1p suppressed in vivo degradation of the heat-denatured marker protein.

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