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

Coimmunoprecipitation of Lhs1p with Hsp150Δ–β-lactamase. (A and B) H647 (lhs1− c-myc–LHS1+ sec18) cells were  incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2  and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes  4 and 9). The samples were lysed under mild detergent conditions  and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti– β-lactamase antiserum (α-bla; A and B, lanes 1–4) and the other  with preimmune serum (pim; A and B, lanes 6–9). The precipitates were separated by SDS-PAGE, blotted, and immunostained  (IS) using anti–c-myc antibody (α-c-myc; A, lanes 1–10) or anti– β-lactamase antiserum (B, lanes 1–10). Lanes 5 and 10 in both  panels contained total lysate sample. c-myc–Lhs1p (lhs1p) and  Hsp150Δ–β-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in  lane M. (C) A cell sample incubated at 37° and 50°C, like above  in lane 2, was lysed like above and centrifuged in a 10–40% sucrose gradient as described in Fig. 2. The fractions (lanes 1–10)  and pellet material (lane P) were immunoprecipitated with anti– β-lactamase antiserum and subjected to blotting and immunostaining with anti–c-myc antibody, as in A. T, total cell lysate  sample stained with anti–c-myc antibody. Arrowhead indicates  c-myc–Lhs1p.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139846&req=5

Figure 5: Coimmunoprecipitation of Lhs1p with Hsp150Δ–β-lactamase. (A and B) H647 (lhs1− c-myc–LHS1+ sec18) cells were incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2 and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes 4 and 9). The samples were lysed under mild detergent conditions and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti– β-lactamase antiserum (α-bla; A and B, lanes 1–4) and the other with preimmune serum (pim; A and B, lanes 6–9). The precipitates were separated by SDS-PAGE, blotted, and immunostained (IS) using anti–c-myc antibody (α-c-myc; A, lanes 1–10) or anti– β-lactamase antiserum (B, lanes 1–10). Lanes 5 and 10 in both panels contained total lysate sample. c-myc–Lhs1p (lhs1p) and Hsp150Δ–β-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in lane M. (C) A cell sample incubated at 37° and 50°C, like above in lane 2, was lysed like above and centrifuged in a 10–40% sucrose gradient as described in Fig. 2. The fractions (lanes 1–10) and pellet material (lane P) were immunoprecipitated with anti– β-lactamase antiserum and subjected to blotting and immunostaining with anti–c-myc antibody, as in A. T, total cell lysate sample stained with anti–c-myc antibody. Arrowhead indicates c-myc–Lhs1p.

Mentions: Coimmunoprecipitation experiments were performed to study whether Lhs1p was associated with Hsp150Δ–β-lactamase. H647 cells (lhs1− c-myc–LHS1+ sec18) were incubated successively for 1 h at 37°C (Fig. 5, A and B, lanes 1), 20 min at 50°C (lanes 2), and with CHX at 24°C for 3 h (lanes 3) or 6 h (lanes 4). The cells were lysed under nondenaturing conditions and divided in two. One lysate set was immunoprecipitated with anti–β-lactamase antiserum (Fig. 5, A and B, lanes 1–4) and the other with preimmune serum (lanes 6–9). Both sets were divided in two and subjected to SDS-PAGE and Western analysis using anti– c-myc antibody (Fig. 5 A) or anti–β-lactamase antiserum (Fig. 5 B). After incubation of the cells at 37°C, no precipitation of c-myc–Lhs1p with anti–β-lactamase antiserum could be detected (Fig. 5 A, lane 1). However, after thermal insult at 50°C and recovery at 24°C, c-myc–Lhs1p was coimmunoprecipitated with anti–β-lactamase antiserum (lanes 2–4). The total cell lysate samples (Fig. 5 A, lanes 5 and 10) show that a minor portion of Lhs1p was coprecipitated with Hsp150Δ–β-lactamase. Anti–β-lactamase antiserum precipitated Hsp150Δ–β-lactamase of ∼110 kD from all samples (Fig. 5 B, lanes 1–4). Neither Lhs1p (Fig. 5 A, lanes 6–9) nor the marker protein (Fig. 5 B, lanes 6–9) were precipitated with preimmune serum. Thus, Lhs1p appeared to occur in association predominantly with heatdenatured marker protein molecules. Whether the association was direct or mediated by other components is not known.


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)

Coimmunoprecipitation of Lhs1p with Hsp150Δ–β-lactamase. (A and B) H647 (lhs1− c-myc–LHS1+ sec18) cells were  incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2  and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes  4 and 9). The samples were lysed under mild detergent conditions  and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti– β-lactamase antiserum (α-bla; A and B, lanes 1–4) and the other  with preimmune serum (pim; A and B, lanes 6–9). The precipitates were separated by SDS-PAGE, blotted, and immunostained  (IS) using anti–c-myc antibody (α-c-myc; A, lanes 1–10) or anti– β-lactamase antiserum (B, lanes 1–10). Lanes 5 and 10 in both  panels contained total lysate sample. c-myc–Lhs1p (lhs1p) and  Hsp150Δ–β-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in  lane M. (C) A cell sample incubated at 37° and 50°C, like above  in lane 2, was lysed like above and centrifuged in a 10–40% sucrose gradient as described in Fig. 2. The fractions (lanes 1–10)  and pellet material (lane P) were immunoprecipitated with anti– β-lactamase antiserum and subjected to blotting and immunostaining with anti–c-myc antibody, as in A. T, total cell lysate  sample stained with anti–c-myc antibody. Arrowhead indicates  c-myc–Lhs1p.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Coimmunoprecipitation of Lhs1p with Hsp150Δ–β-lactamase. (A and B) H647 (lhs1− c-myc–LHS1+ sec18) cells were incubated for 1 h at 37°C (lanes 1 and 6), 20 min at 50°C (lanes 2 and 7), and at 24°C with CHX for 3 h (lanes 3 and 8) or 6 h (lanes 4 and 9). The samples were lysed under mild detergent conditions and divided in two. One set of samples was subjected under nondenaturing conditions to immunoprecipitation (IP) with anti– β-lactamase antiserum (α-bla; A and B, lanes 1–4) and the other with preimmune serum (pim; A and B, lanes 6–9). The precipitates were separated by SDS-PAGE, blotted, and immunostained (IS) using anti–c-myc antibody (α-c-myc; A, lanes 1–10) or anti– β-lactamase antiserum (B, lanes 1–10). Lanes 5 and 10 in both panels contained total lysate sample. c-myc–Lhs1p (lhs1p) and Hsp150Δ–β-lactamase (bla) are indicated by arrowheads. Molecular mass markers of 212, 170, 116, and 76 kD are indicated in lane M. (C) A cell sample incubated at 37° and 50°C, like above in lane 2, was lysed like above and centrifuged in a 10–40% sucrose gradient as described in Fig. 2. The fractions (lanes 1–10) and pellet material (lane P) were immunoprecipitated with anti– β-lactamase antiserum and subjected to blotting and immunostaining with anti–c-myc antibody, as in A. T, total cell lysate sample stained with anti–c-myc antibody. Arrowhead indicates c-myc–Lhs1p.
Mentions: Coimmunoprecipitation experiments were performed to study whether Lhs1p was associated with Hsp150Δ–β-lactamase. H647 cells (lhs1− c-myc–LHS1+ sec18) were incubated successively for 1 h at 37°C (Fig. 5, A and B, lanes 1), 20 min at 50°C (lanes 2), and with CHX at 24°C for 3 h (lanes 3) or 6 h (lanes 4). The cells were lysed under nondenaturing conditions and divided in two. One lysate set was immunoprecipitated with anti–β-lactamase antiserum (Fig. 5, A and B, lanes 1–4) and the other with preimmune serum (lanes 6–9). Both sets were divided in two and subjected to SDS-PAGE and Western analysis using anti– c-myc antibody (Fig. 5 A) or anti–β-lactamase antiserum (Fig. 5 B). After incubation of the cells at 37°C, no precipitation of c-myc–Lhs1p with anti–β-lactamase antiserum could be detected (Fig. 5 A, lane 1). However, after thermal insult at 50°C and recovery at 24°C, c-myc–Lhs1p was coimmunoprecipitated with anti–β-lactamase antiserum (lanes 2–4). The total cell lysate samples (Fig. 5 A, lanes 5 and 10) show that a minor portion of Lhs1p was coprecipitated with Hsp150Δ–β-lactamase. Anti–β-lactamase antiserum precipitated Hsp150Δ–β-lactamase of ∼110 kD from all samples (Fig. 5 B, lanes 1–4). Neither Lhs1p (Fig. 5 A, lanes 6–9) nor the marker protein (Fig. 5 B, lanes 6–9) were precipitated with preimmune serum. Thus, Lhs1p appeared to occur in association predominantly with heatdenatured marker protein molecules. Whether the association was direct or mediated by other components is not known.

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