Limits...
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.

Show MeSH

Related in: MedlinePlus

Effect of Lhs1p  on acquisition of thermotolerance. (A) Strains H541  (WT, open circles), H540  (lhs1−, squares), H656 (lhs1−  c-myc–LHS1+, closed circles), and H454 (hsp104−, triangles) were incubated for  different times at 37°C and  then for 20 min at 50°C, and  plated at 24°C. (B) Strains  H656 (closed circles) and  H540 (squares) were incubated for 45 min at 37°C and  then for different times at  50°C, and plated at 24°C. The  colonies were counted to determine the percentage of  survival, which was plotted  against incubation times.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139846&req=5

Figure 10: Effect of Lhs1p on acquisition of thermotolerance. (A) Strains H541 (WT, open circles), H540 (lhs1−, squares), H656 (lhs1− c-myc–LHS1+, closed circles), and H454 (hsp104−, triangles) were incubated for different times at 37°C and then for 20 min at 50°C, and plated at 24°C. (B) Strains H656 (closed circles) and H540 (squares) were incubated for 45 min at 37°C and then for different times at 50°C, and plated at 24°C. The colonies were counted to determine the percentage of survival, which was plotted against incubation times.

Mentions: Next we studied whether Lhs1p affected the ability of yeast cells to acquire thermotolerance. Strain H540 (lhs1−), the parental strain H541 (WT), and strain H656 (lhs1− c-myc–LHS1+) were incubated for different times at 37°C and thereafter for 20 min at 50°C and plated at 24°C (Fig. 10 A). More than 50% of H541 (Fig. 10 A, open circles) and H656 cells (closed circles) formed colonies when preconditioned for 45 min at 37°C before the 50°C treatment, whereas only 4% of H540 cells (Fig. 10 A, squares) survived. Strain H454 lacking a functional HSP104 gene (Fig. 10 A, triangles) served as a negative control: only 0.1% of them were able to form colonies after the thermal treatments. When the assay was performed by preincubating strains H540 (Fig. 10 B, lhs1−; squares) and H656 (lhs1− LHS1+; circles) for 45 min at 37°C followed by different times at 50°C, lack of LHS1 again caused a 10-fold decrease in survival.


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)

Effect of Lhs1p  on acquisition of thermotolerance. (A) Strains H541  (WT, open circles), H540  (lhs1−, squares), H656 (lhs1−  c-myc–LHS1+, closed circles), and H454 (hsp104−, triangles) were incubated for  different times at 37°C and  then for 20 min at 50°C, and  plated at 24°C. (B) Strains  H656 (closed circles) and  H540 (squares) were incubated for 45 min at 37°C and  then for different times at  50°C, and plated at 24°C. The  colonies were counted to determine the percentage of  survival, which was plotted  against incubation times.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 10: Effect of Lhs1p on acquisition of thermotolerance. (A) Strains H541 (WT, open circles), H540 (lhs1−, squares), H656 (lhs1− c-myc–LHS1+, closed circles), and H454 (hsp104−, triangles) were incubated for different times at 37°C and then for 20 min at 50°C, and plated at 24°C. (B) Strains H656 (closed circles) and H540 (squares) were incubated for 45 min at 37°C and then for different times at 50°C, and plated at 24°C. The colonies were counted to determine the percentage of survival, which was plotted against incubation times.
Mentions: Next we studied whether Lhs1p affected the ability of yeast cells to acquire thermotolerance. Strain H540 (lhs1−), the parental strain H541 (WT), and strain H656 (lhs1− c-myc–LHS1+) were incubated for different times at 37°C and thereafter for 20 min at 50°C and plated at 24°C (Fig. 10 A). More than 50% of H541 (Fig. 10 A, open circles) and H656 cells (closed circles) formed colonies when preconditioned for 45 min at 37°C before the 50°C treatment, whereas only 4% of H540 cells (Fig. 10 A, squares) survived. Strain H454 lacking a functional HSP104 gene (Fig. 10 A, triangles) served as a negative control: only 0.1% of them were able to form colonies after the thermal treatments. When the assay was performed by preincubating strains H540 (Fig. 10 B, lhs1−; squares) and H656 (lhs1− LHS1+; circles) for 45 min at 37°C followed by different times at 50°C, lack of LHS1 again caused a 10-fold decrease in survival.

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