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Fig. 2. Intracellular transport of proteins in sec24-1 mutant. (A) Hsp150 secretion. sec24-1 (H1101; lanes 1-10) and sec18-1 (H4, lane 11) cells were pulse-labeled for 5 minutes with ³⁵S-methionine/cysteine and chased in the presence of CHX at 37°C as indicated. Preincubation before labeling at 37°C was for 15 minutes, except in the case of lanes 9 and 10, where it was 30 minutes. Immunoprecipitation of cell lysate (c) and medium (m) samples before SDS-PAGE analysis was with Hsp150 antiserum. Migration of ER (97 kDa) and mature (150 kDa) forms of Hsp150 are indicated on the right. (B) Transport to the vacuole of pro-CPY. sec24-1 cells (H1101) were preincubated for 15 minutes and ³⁵S-labeled for 5 minutes and chased with CHX as indicated, at 37°C (lanes 1-4) or at 24°C (lanes 5-8). Cell lysates were immunoprecipitated with CPY antiserum. Migration of cytosolic (c, 59 kDa), ER (p1, 67 kDa), Golgi (p2, 69 kDa) and vacuolar (m, 62 kDa) forms of CPY are indicated. (C) Invertase secretion. Control (H23, lane 1), sec18-1 (H4, lane 2) and sec24-1 (H1101, lanes 3 and 4) cells were preincubated at 38°C (lanes 1-3) or 24°C (lane 4) for 15 minutes, shifted to low glucose (0.1%) medium to derepress invertase synthesis and incubated for 1 hour at the same temperatures. After nondenaturing gel electrophoresis the gel was stained for invertase activity. Migration of mature invertase is indicated. T, temperature.

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