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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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β-Lactamase activity of the gradient fractions. Strain  H393 (sec18) was incubated at 37°C (A), then at 50°C (B), and  thereafter for 6 h at 24°C with CHX (C). The cells were lysed and  subjected to sucrose gradient centrifugation as described in Fig. 2,  A–C. The fractions were assayed for β-lactamase activity.
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Figure 3: β-Lactamase activity of the gradient fractions. Strain H393 (sec18) was incubated at 37°C (A), then at 50°C (B), and thereafter for 6 h at 24°C with CHX (C). The cells were lysed and subjected to sucrose gradient centrifugation as described in Fig. 2, A–C. The fractions were assayed for β-lactamase activity.

Mentions: Next we assayed the β-lactamase activity after fractionation. After incubation of H393 cells at 37°C, β-lactamase activity coincided with 35S-Hsp150Δ–β-lactamase in fractions 1–3 of the gradient (Fig. 3 A). After the thermal insult, no activity could be detected in the fractions (Fig. 3 B), whereas after 6 h at 24°C in the presence of CHX, ∼22% of the original β-lactamase activity reappeared in the top fractions (Fig. 3 C). We conclude that our marker protein was soluble when accumulated in the ER, aggregated by thermal insult to the living cells, and resolubilized in an ATP-dependent fashion, even in the absence of de novo protein synthesis, during recovery of the cells at 24°C.


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)

β-Lactamase activity of the gradient fractions. Strain  H393 (sec18) was incubated at 37°C (A), then at 50°C (B), and  thereafter for 6 h at 24°C with CHX (C). The cells were lysed and  subjected to sucrose gradient centrifugation as described in Fig. 2,  A–C. The fractions were assayed for β-lactamase activity.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: β-Lactamase activity of the gradient fractions. Strain H393 (sec18) was incubated at 37°C (A), then at 50°C (B), and thereafter for 6 h at 24°C with CHX (C). The cells were lysed and subjected to sucrose gradient centrifugation as described in Fig. 2, A–C. The fractions were assayed for β-lactamase activity.
Mentions: Next we assayed the β-lactamase activity after fractionation. After incubation of H393 cells at 37°C, β-lactamase activity coincided with 35S-Hsp150Δ–β-lactamase in fractions 1–3 of the gradient (Fig. 3 A). After the thermal insult, no activity could be detected in the fractions (Fig. 3 B), whereas after 6 h at 24°C in the presence of CHX, ∼22% of the original β-lactamase activity reappeared in the top fractions (Fig. 3 C). We conclude that our marker protein was soluble when accumulated in the ER, aggregated by thermal insult to the living cells, and resolubilized in an ATP-dependent fashion, even in the absence of de novo protein synthesis, during recovery of the cells at 24°C.

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