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.