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

Aggregation and degradation of Hsp150Δ–β-lactamase  in the absence of Lhs1p. The same experiments as described in  the legend of Fig. 2, A–C, were performed using strain H621  (lhs1− sec18). B and C were exposed five times longer than A.  The sedimentation of BSA (4.6 S) and thyroglobulin (19.3 S) is  indicated at the bottom.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139846&req=5

Figure 4: Aggregation and degradation of Hsp150Δ–β-lactamase in the absence of Lhs1p. The same experiments as described in the legend of Fig. 2, A–C, were performed using strain H621 (lhs1− sec18). B and C were exposed five times longer than A. The sedimentation of BSA (4.6 S) and thyroglobulin (19.3 S) is indicated at the bottom.

Mentions: In the absence of the LHS1 gene (strain H621, lhs1− sec18), 35S-Hsp150Δ–β-lactamase labeled at 37°C was soluble and migrated in SDS-PAGE again as a doublet of 95 and 110 kD (Fig. 4 A). After the 50°C treatment, it was found in aggregates (Fig. 4 B), and after the recovery period it was barely detectable (Fig. 4 C). After the thermal insult and the recovery period, very little of the marker protein could be immunoprecipitated. Exposure time for B and C was five times longer than for A. Fractions 1–3 contained β-lactamase activity when the cells had been incubated at 37°C, whereas after thermal insult and recovery, no activity could be detected in the gradients (not shown). In the rescue strain H645 (lhs1− LHS1+ sec18), ATP-dependent solubilization of the Hsp150Δ–β-lactamase– containing aggregates occurred similarly as in strain H393 (see Fig. 2; not shown). Thus, heat-denatured Hsp150Δ–βlactamase remained in aggregates and was susceptible for proteolysis in the absence of Lhs1p. Degradation in this experiment occurred mainly by cellular proteases after cell lysis since less degradation occurred in vivo, as shown below. We conclude that thermal insult caused aggregation of ERlocated Hsp150Δ–β-lactamase, whether Lhs1p was present or not. In the absence of Lhs1p, the Hsp150Δ–β-lactamase–containing aggregates failed to be solubilized and were susceptible to proteolytic degradation in vitro.


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)

Aggregation and degradation of Hsp150Δ–β-lactamase  in the absence of Lhs1p. The same experiments as described in  the legend of Fig. 2, A–C, were performed using strain H621  (lhs1− sec18). B and C were exposed five times longer than A.  The sedimentation of BSA (4.6 S) and thyroglobulin (19.3 S) is  indicated at the bottom.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: Aggregation and degradation of Hsp150Δ–β-lactamase in the absence of Lhs1p. The same experiments as described in the legend of Fig. 2, A–C, were performed using strain H621 (lhs1− sec18). B and C were exposed five times longer than A. The sedimentation of BSA (4.6 S) and thyroglobulin (19.3 S) is indicated at the bottom.
Mentions: In the absence of the LHS1 gene (strain H621, lhs1− sec18), 35S-Hsp150Δ–β-lactamase labeled at 37°C was soluble and migrated in SDS-PAGE again as a doublet of 95 and 110 kD (Fig. 4 A). After the 50°C treatment, it was found in aggregates (Fig. 4 B), and after the recovery period it was barely detectable (Fig. 4 C). After the thermal insult and the recovery period, very little of the marker protein could be immunoprecipitated. Exposure time for B and C was five times longer than for A. Fractions 1–3 contained β-lactamase activity when the cells had been incubated at 37°C, whereas after thermal insult and recovery, no activity could be detected in the gradients (not shown). In the rescue strain H645 (lhs1− LHS1+ sec18), ATP-dependent solubilization of the Hsp150Δ–β-lactamase– containing aggregates occurred similarly as in strain H393 (see Fig. 2; not shown). Thus, heat-denatured Hsp150Δ–βlactamase remained in aggregates and was susceptible for proteolysis in the absence of Lhs1p. Degradation in this experiment occurred mainly by cellular proteases after cell lysis since less degradation occurred in vivo, as shown below. We conclude that thermal insult caused aggregation of ERlocated Hsp150Δ–β-lactamase, whether Lhs1p was present or not. In the absence of Lhs1p, the Hsp150Δ–β-lactamase–containing aggregates failed to be solubilized and were susceptible to proteolytic degradation in vitro.

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