The cytoplasmic chaperone hsp104 is required for conformational repair of heat-denatured proteins in the yeast endoplasmic reticulum

Abstract

Severe heat stress causes protein denaturation in various cellular compartments. If Saccharomyces cerevisiae cells grown at 24 degrees C are preconditioned at 37 degrees C, proteins denatured by subsequent exposure to 48-50 degrees C can be renatured when the cells are allowed to recover at 24 degrees C. Conformational repair of vital proteins is essential for survival, because gene expression is transiently blocked after the thermal insult. Refolding of cytoplasmic proteins requires the Hsp104 chaperone, and refolding of lumenal endoplasmic reticulum (ER) proteins requires the Hsp70 homologue Lhs1p. We show here that conformational repair of heat-damaged glycoproteins in the ER of living yeast cells required functional Hsp104. A heterologous enzyme and a number of natural yeast proteins, previously translocated and folded in the ER and thereafter denatured by severe heat stress, failed to be refolded to active and secretion-competent structures in the absence of Hsp104 or when an ATP-binding site of Hsp104 was mutated. During recovery at 24 degrees C, the misfolded proteins persisted in the ER, although the secretory apparatus was fully functional. Hsp104 appears to control conformational repair of heat-damaged proteins even beyond the ER membrane.

Figure 1

Dependence of reactivation of heat-inactivated Hsp150Δ-β-lactamase on Hsp104. (Top) Scheme of thermal treatments. After growth at 24°C, cells were incubated at 37°C (preconditioning), thereafter at 50°C (thermal insult), and then at 24°C (recovery). Strains H393 (A), H534 (B, ●), H850 (B, ○), and H941 (C) were preincubated for 10 min at 37°C, pelleted, resuspended in prewarmed medium, and incubated at 37°C for 1 h and then at 50°C for 20 min, after which CHX was added and the cells remained at 24°C. The β-lactamase activity of lysed cell samples was determined and plotted against incubation time.

Figure 2

Glucose consumption of cells after thermal insult in the absence and presence of Hsp104. H393 (A) and H534 (B) cells were resuspended in YPD medium (25 × 10⁷ cells/ml) and incubated for 1 h at 37°C and then for 20 min at 48°C (○) or 50°C (●). The cells were pelleted and resuspended in fresh YPD medium and shifted to 24°C. Duplicate samples were withdrawn at the indicated times, and the glucose concentration of the medium was determined and plotted against incubation time at 24°C.

Figure 3

The role of Hsp104 in the resumption of the secretion competence of Hsp150Δ-β-lactamase. sec18-1 (A; H393) and Δhsp104 sec18-1 (B; H534) cells were preincubated for 10 min at 37°C, labeled with [³⁵S]methionine/cysteine for 60 min at the same temperature, and incubated for 15 min at 48°C. The labeling medium was replaced by SC medium with excess unlabeled methionine and cysteine, and the cells were shifted to 24°C for the indicated times (lanes 1, 2, and 5–12). In lanes 3 and 4, the 48°C treatment was omitted. In lanes 13 and 14, sodium azide was included in the recovery mixture. The culture medium samples (lanes with uneven numbers) and lysed cell samples (lanes with even numbers) were immunoprecipitated with β-lactamase antiserum and analyzed by SDS-PAGE. Mature (145 kDa) and ER-specific (110 kDa) Hsp150Δ-β-lactamase are indicated.

Figure 4

Resumption of the secretion competence of β-lactamase activity. sec18-1 cells (H393) were treated as described in the scheme at the top of Figure 1, except that the thermal insult was for 20 min at 48°C. The β-lactamase activity of cell lysates (●) and medium samples (○) was determined and plotted against time.

Figure 5

Recovery of synthesis and modification and secretion of Hsp150Δ-β-lactamase in the absence of Hsp104. The experimental design is shown in the scheme at the top of the figure. Parallel samples of Δhsp104 sec18-1 cells (H534) were labeled with ³⁵S during successive 1-h periods at 24°C (samples a–e), lysed, immunoprecipitated with β-lactamase antiserum, and analyzed by SDS-PAGE. The cytoplasmic (66 kDa), ER-specific (110 kDa), and mature (145 kDa) forms of Hsp150Δ-β-lactamase and the molecular mass markers are indicated. m, medium; c, cell lysate.

Figure 6

Secretion kinetics of newly synthesized Hsp150Δ-β-lactamase. H393 (A) and H534 (B) cells were treated as indicated in the scheme at the top of the figure. After 4 h of recovery at 24°C, the cells were labeled with ³⁵S for 5 min and then chased with CHX at 24°C for the indicated times. The medium (m) and cell lysate (c) samples were subjected to precipitation with β-lactamase antiserum and analyzed by SDS-PAGE. Mature (145 kDa) and ER-specific (110 kDa) Hsp150Δ-β-lactamase are indicated.

Figure 7

The role of Hsp104 in the resumption of the secretion competence of proCPY. H1 (A), H453 (B), H826 (C), and H924 (D) cells were preincubated for 1 h and then pulse labeled for 5 min with [³⁵S]methionine/cysteine at 37°C (lanes 1). Parallel cells remained at 48°C for 20 min, after which the labeling medium was changed to SC medium with excess unlabeled methionine and cysteine and the cells were shifted to 24°C for 2 h (lanes 2), 4 h (lanes 3), or 6 h (lanes 4). The cells were lysed and subjected to immunoprecipitation with CPY antiserum followed by SDS-PAGE analysis. ProCPY (67 kDa) and CPY(61 kDa) are indicated.

Figure 8

The role of Hsp104 in the fate of heat-affected cell wall mannoproteins. Strains H4 (A and B) and H534 (C) were preincubated in YPD medium containing 0.1% glucose for 15 min at 37°C and labeled with [³H]mannose for 30 min. (A) The cells were pelleted, washed, and resuspended in fresh YPD medium containing 4% glucose and returned to 24°C (see scheme at top). (B and C) The ³H-labeled cell samples were incubated for 20 min at 50°C before being shifted to 24°C (see scheme at top). Duplicate samples were withdrawn, washed, and subjected to cell wall removal. The spheroplast-associated (●) and cell wall–associated (○) ³H radioactivity was counted and plotted against incubation time at 24°C.