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

Conformational maturation of Hsp150Δ–β-lactamase and pro-CPY. (A) Strains  H602 (lhs1−) and (B) H604  (WT) were preincubated for 10  min in the absence or presence  of DTT and labeled with  [35S]methionine/cysteine for 1 h  in the absence (lanes 1 and 2) or  presence of the reducing agent  (lanes 3 and 4). CHX was  added and the incubations continued for 20 min. In lanes 5  and 6, the labeling was performed in the presence of DTT  as above, after which DTT was  removed and the cells incubated further in fresh medium  with CHX for 1 h. All incubations were at 37°C. The culture  medium (m; lanes 1, 3, and 5)  and cell lysate samples (c; lanes  2, 4, and 6) were immunoprecipitated with anti–β-lactamase  antiserum before SDS-PAGE  analysis. Apparent molecular  masses of Hsp150Δ–β-lactamase–related proteins (kD) are  indicated on the right, and the  molecular mass markers (kD)  on the left. (C) Strains H602  (lhs1−) and (D) H604 (WT)  were 35S-labeled for 1 h after a  preincubation of 10 min in the  presence of DTT (lanes 1–3),  washed, and incubated with  CHX for 45 min (lanes 2) or 90  min (lanes 3). In lanes 4, the labeling was as in lanes 1, but in  the absence of DTT. All incubations were at 24°C. The cell  lysates were precipitated with  anti-CPY antiserum, followed  by SDS-PAGE analysis.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139846&req=5

Figure 9: Conformational maturation of Hsp150Δ–β-lactamase and pro-CPY. (A) Strains H602 (lhs1−) and (B) H604 (WT) were preincubated for 10 min in the absence or presence of DTT and labeled with [35S]methionine/cysteine for 1 h in the absence (lanes 1 and 2) or presence of the reducing agent (lanes 3 and 4). CHX was added and the incubations continued for 20 min. In lanes 5 and 6, the labeling was performed in the presence of DTT as above, after which DTT was removed and the cells incubated further in fresh medium with CHX for 1 h. All incubations were at 37°C. The culture medium (m; lanes 1, 3, and 5) and cell lysate samples (c; lanes 2, 4, and 6) were immunoprecipitated with anti–β-lactamase antiserum before SDS-PAGE analysis. Apparent molecular masses of Hsp150Δ–β-lactamase–related proteins (kD) are indicated on the right, and the molecular mass markers (kD) on the left. (C) Strains H602 (lhs1−) and (D) H604 (WT) were 35S-labeled for 1 h after a preincubation of 10 min in the presence of DTT (lanes 1–3), washed, and incubated with CHX for 45 min (lanes 2) or 90 min (lanes 3). In lanes 4, the labeling was as in lanes 1, but in the absence of DTT. All incubations were at 24°C. The cell lysates were precipitated with anti-CPY antiserum, followed by SDS-PAGE analysis.

Mentions: Proteins that normally acquire disulfide bonds are retained in the yeast ER if cotranslocational disulfide formation is prevented by incubating cells with a reducing agent like DTT, though the secretory machinery remains functional. When oxidizing conditions are reestablished by removing DTT, the sulfhydryls are oxidized, and the proteins undergo conformational maturation resulting in resumption of secretion (19, 44). To study whether Lhs1p was involved in conformational maturation of newly synthesized proteins, H602 (lhs1−) cells were 35S-labeled in the presence of DTT to allow translocation, while folding of Hsp150Δ–β-lactamase was inhibited. Immunoprecipitation with anti–β-lactamase antiserum of the culture medium sample showed that very little of the marker protein was secreted (Fig. 9 A, lane 3). In the absence of DTT, mature Hsp150Δ–β-lactamase of 145 kD was immunoprecipitated from the culture medium (Fig. 9 A, lane 1), and some 66- and 145-kD protein from the cell lysate (lane 2). The intracellular reduced molecules were difficult to detect (Fig. 9 A, lane 4), as noted before (43). When cells labeled under reducing conditions were washed and chased in the absence of DTT and presence of CHX, secretion of the marker protein was resumed, since it could be detected in the culture medium (Fig. 9 A, lane 5). Some 66-, 110-, and 145-kD proteins remained cell associated (Fig. 9 A, lane 6). Similar results were obtained for the parental strain H604: DTT prevented reversibly the secretion of the marker protein (Fig. 9 B), but less intracellular proteins were detected in the cell lysates as compared to H602 (Fig. 9 B, lanes 2, 4, and 6).


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)

Conformational maturation of Hsp150Δ–β-lactamase and pro-CPY. (A) Strains  H602 (lhs1−) and (B) H604  (WT) were preincubated for 10  min in the absence or presence  of DTT and labeled with  [35S]methionine/cysteine for 1 h  in the absence (lanes 1 and 2) or  presence of the reducing agent  (lanes 3 and 4). CHX was  added and the incubations continued for 20 min. In lanes 5  and 6, the labeling was performed in the presence of DTT  as above, after which DTT was  removed and the cells incubated further in fresh medium  with CHX for 1 h. All incubations were at 37°C. The culture  medium (m; lanes 1, 3, and 5)  and cell lysate samples (c; lanes  2, 4, and 6) were immunoprecipitated with anti–β-lactamase  antiserum before SDS-PAGE  analysis. Apparent molecular  masses of Hsp150Δ–β-lactamase–related proteins (kD) are  indicated on the right, and the  molecular mass markers (kD)  on the left. (C) Strains H602  (lhs1−) and (D) H604 (WT)  were 35S-labeled for 1 h after a  preincubation of 10 min in the  presence of DTT (lanes 1–3),  washed, and incubated with  CHX for 45 min (lanes 2) or 90  min (lanes 3). In lanes 4, the labeling was as in lanes 1, but in  the absence of DTT. All incubations were at 24°C. The cell  lysates were precipitated with  anti-CPY antiserum, followed  by SDS-PAGE analysis.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 9: Conformational maturation of Hsp150Δ–β-lactamase and pro-CPY. (A) Strains H602 (lhs1−) and (B) H604 (WT) were preincubated for 10 min in the absence or presence of DTT and labeled with [35S]methionine/cysteine for 1 h in the absence (lanes 1 and 2) or presence of the reducing agent (lanes 3 and 4). CHX was added and the incubations continued for 20 min. In lanes 5 and 6, the labeling was performed in the presence of DTT as above, after which DTT was removed and the cells incubated further in fresh medium with CHX for 1 h. All incubations were at 37°C. The culture medium (m; lanes 1, 3, and 5) and cell lysate samples (c; lanes 2, 4, and 6) were immunoprecipitated with anti–β-lactamase antiserum before SDS-PAGE analysis. Apparent molecular masses of Hsp150Δ–β-lactamase–related proteins (kD) are indicated on the right, and the molecular mass markers (kD) on the left. (C) Strains H602 (lhs1−) and (D) H604 (WT) were 35S-labeled for 1 h after a preincubation of 10 min in the presence of DTT (lanes 1–3), washed, and incubated with CHX for 45 min (lanes 2) or 90 min (lanes 3). In lanes 4, the labeling was as in lanes 1, but in the absence of DTT. All incubations were at 24°C. The cell lysates were precipitated with anti-CPY antiserum, followed by SDS-PAGE analysis.
Mentions: Proteins that normally acquire disulfide bonds are retained in the yeast ER if cotranslocational disulfide formation is prevented by incubating cells with a reducing agent like DTT, though the secretory machinery remains functional. When oxidizing conditions are reestablished by removing DTT, the sulfhydryls are oxidized, and the proteins undergo conformational maturation resulting in resumption of secretion (19, 44). To study whether Lhs1p was involved in conformational maturation of newly synthesized proteins, H602 (lhs1−) cells were 35S-labeled in the presence of DTT to allow translocation, while folding of Hsp150Δ–β-lactamase was inhibited. Immunoprecipitation with anti–β-lactamase antiserum of the culture medium sample showed that very little of the marker protein was secreted (Fig. 9 A, lane 3). In the absence of DTT, mature Hsp150Δ–β-lactamase of 145 kD was immunoprecipitated from the culture medium (Fig. 9 A, lane 1), and some 66- and 145-kD protein from the cell lysate (lane 2). The intracellular reduced molecules were difficult to detect (Fig. 9 A, lane 4), as noted before (43). When cells labeled under reducing conditions were washed and chased in the absence of DTT and presence of CHX, secretion of the marker protein was resumed, since it could be detected in the culture medium (Fig. 9 A, lane 5). Some 66-, 110-, and 145-kD proteins remained cell associated (Fig. 9 A, lane 6). Similar results were obtained for the parental strain H604: DTT prevented reversibly the secretion of the marker protein (Fig. 9 B), but less intracellular proteins were detected in the cell lysates as compared to H602 (Fig. 9 B, lanes 2, 4, and 6).

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