Figure 4.
Analysis of CPY transport and invertase secretion in sly1(L140K) and sed5(F10A) mutants. (A) Cells of wild-type (MSUC3D), sly1(L140K) (RPY238), sed5(F10A) (RPY215) and sec23-1 (RH227-3A) strains were grown at 25°C before incubation at 35°C for 10 min. The cells were labeled with Tran35S-label at 35°C for 10 min and chased for 10 or 30 min. CPY was immunoprecipitated and analyzed by SDS–PAGE, followed by autoradiography. ER core-glycosylated (p1), Golgi-modified (p2) and mature form (m) of CPY are indicated. (B) Cells of wild-type, sly1(L140K), sed5(F10A), sly1(L140K)/sed5(F10A) double-mutant (RPY249) and sec23-1 strains grown at 25°C were transferred to 0.1% glucose medium to induce invertase synthesis for 60 min at either 25 or 35°C (nonpermissive temperatures). Spheroplasts were prepared and pelleted to obtain the periplasmic (e) and intracellular (i) fractions. Proteins of both fractions were separated on nondenaturing polyacrylamide gel, followed by activity staining of invertase. S, secreted invertase; ER, ER core-glycosylated invertase.