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Fig. 7. Trafficking phenotype of the gea2-V698G mutant. Strains CJY092 gea1{Delta}gea2{Delta}/pSKP1(pCEN GEA2) and CJY093 gea1{Delta}gea2{Delta}/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{Delta} and APY022 gea1-6 gea2{Delta} (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.





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