Fig. 7. Trafficking phenotype of the gea2-V698G mutant. Strains CJY092 gea1gea2/pSKP1(pCEN GEA2) and CJY093 gea1gea2/pCEN-gea2-V698G were grown to exponential phase and incubated for 1 hour at 38°C, pulse-labeled for 10 minutes with [³⁵S]methionine/cysteine, then chased with non-radioactive amino acids for 30 minutes, all at 38°C. (A) Proteins secreted into the medium were recovered and analyzed as described in Materials and Methods. Strains CJY062-10-3 gea1-4 gea2 and APY022 gea1-6 gea2 (Peyroche et al., 2001) were treated similarly and included as controls. (B) Strains listed above were grown at 30°C and shifted to the indicated temperature for 1 hour before labeling for 5 minutes. Cell lysates were prepared at the chase times indicated and immunoprecipitated with anti-CPY antibodies. The ER-early-Golgi localized p1 precursor, late-Golgi p2 precursor, and mature (m) forms of CPY are indicated. Note that at 20°C, transport through the secretory pathway is significantly slowed, and no mature CPY accumulates during the short time course of this experiment.