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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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Secretion of β-lactamase activity. Strains (A) H602  (lhs1−) and (B) H604 (WT) were incubated at 37°C. The β-lactamase activity of the culture medium (closed circles, EX) and lysed  cells (open circles, IN) was determined and plotted against incubation time.
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Figure 8: Secretion of β-lactamase activity. Strains (A) H602 (lhs1−) and (B) H604 (WT) were incubated at 37°C. The β-lactamase activity of the culture medium (closed circles, EX) and lysed cells (open circles, IN) was determined and plotted against incubation time.

Mentions: Lhs1p is required for translocation into the ER of a subset of proteins (2, 6). Thus, we studied the role of Lhs1p in translocation, folding, and secretion of de novo–synthesized Hsp150Δ–β-lactamase in more detail. Strain H602 (lhs1−) (Fig. 8 A) and its parental strain H604 (WT) (Fig. 8 B), both wild-type for secretion, were incubated at 37°C, and the β-lactamase activities of the culture medium samples (Fig. 8, closed circles, EX) and lysed cell samples (open circles, IN) were determined. In both strains, β-lactamase activity externalized to the culture medium increased linearly, a little faster in the parental strain than in the lhs1− mutant. In the parental strain, little intracellular activity accumulated (0.12 U/ml in 90 min), indicating efficient secretion of the marker enzyme. In the lhs1− mutant, twice as much intracellular activity accumulated during the incubation, and its level in the beginning of the experiment was high. This suggests that a fraction of the newly synthesized marker protein remained cell associated in active form and gradually built up an intracellular pool in the absence of a functional LHS1 gene.


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)

Secretion of β-lactamase activity. Strains (A) H602  (lhs1−) and (B) H604 (WT) were incubated at 37°C. The β-lactamase activity of the culture medium (closed circles, EX) and lysed  cells (open circles, IN) was determined and plotted against incubation time.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139846&req=5

Figure 8: Secretion of β-lactamase activity. Strains (A) H602 (lhs1−) and (B) H604 (WT) were incubated at 37°C. The β-lactamase activity of the culture medium (closed circles, EX) and lysed cells (open circles, IN) was determined and plotted against incubation time.
Mentions: Lhs1p is required for translocation into the ER of a subset of proteins (2, 6). Thus, we studied the role of Lhs1p in translocation, folding, and secretion of de novo–synthesized Hsp150Δ–β-lactamase in more detail. Strain H602 (lhs1−) (Fig. 8 A) and its parental strain H604 (WT) (Fig. 8 B), both wild-type for secretion, were incubated at 37°C, and the β-lactamase activities of the culture medium samples (Fig. 8, closed circles, EX) and lysed cell samples (open circles, IN) were determined. In both strains, β-lactamase activity externalized to the culture medium increased linearly, a little faster in the parental strain than in the lhs1− mutant. In the parental strain, little intracellular activity accumulated (0.12 U/ml in 90 min), indicating efficient secretion of the marker enzyme. In the lhs1− mutant, twice as much intracellular activity accumulated during the incubation, and its level in the beginning of the experiment was high. This suggests that a fraction of the newly synthesized marker protein remained cell associated in active form and gradually built up an intracellular pool in the absence of a functional LHS1 gene.

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