Figure 8.

Analysis of the secretory pathway in the desaturase mutant strain. (A) Maturation of Gas1p was analyzed in wild-type (W303), and ole1^ts (YRS1327) by pulse-chase analysis and immunoprecipitation. Cells were preshifted to 37°C for 15 min before labeling with [³⁵S]methionine for 5 min and chasing for the time points indicated. Gas1p was recovered by immunoprecipitation, subjected to SDS-PAGE, and visualized by autoradiography. The position of the precursor 105-kDa (p, ER) and mature 125-kDa (m, Golgi) form of Gas1p is indicated to the right. (B) Maturation of CPY. Wild-type (W303) and ole1^ts (YRS1327) mutant cells were incubated at 30°C or preshifted to 37°C for 15 min before labeling with [³⁵S]methionine for 5 min and chasing for the time points indicated. CPY was recovered by immunoprecipitation, subjected to SDS-PAGE, and visualized by autoradiography. The positions of precursors (p1, ER; p2, Golgi) and that of the mature form of CPY (m/vacuolar) are indicated. (C) Secretion of invertase was analyzed in wild-type (W303) and ole1^ts (YRS1327) mutant cells. After invertase induction by low glucose, cells were preshifted to 37°C for 15 min. Samples were removed at the time points indicated and the activity of internal (i) and secreted (e) invertase was determined. (D) Secretion of Kar2p was analyzed in wild-type (wt, W303), emp24Δ (RH1961), and ole1^ts (YRS1327) mutant cells. Exponentially growing cells were washed, resuspended in fresh media, and incubated at either 30 or 37°C for 1 h. Proteins secreted into the media were analyzed by Western blot with an anti-Kar2p antibody.