Fig. 2. Secretion into the medium in gea mutants. (A) WT (CJY049-2-3), gea1-4 (CJY054), sec18-1 (RSY271) and sec21-3 (EGY1213) strains were preincubated at 38°C for 45 minutes, pulse-labeled for 10 minutes with [35S]methionine/cysteine, then chased with non-radioactive amino acids for 30 minutes at 38°C. Proteins secreted into the medium were recovered as described in Materials and Methods, and analyzed by SDS-PAGE and fluorography. Filled circles, proteins that continue to be secreted in sec21-3 and gea mutants; arrowheads, proteins whose secretion is blocked or severely retarded. (B) Wild-type strain CJY049-11-1 GEA1 and the mutant APY026 gea1-19 were preincubated for 20 minutes at 37°C, then pulse-labeled for 10 minutes and chased for the indicated times at 37°C. Proteins secreted into the medium were recovered and analyzed. (C) Strains CJY049-2-3 GEA1 and CJY054 gea1-4 were preincubated at 38°C for the times indicated, then pulse-labeled for 10 minutes and chased for 5 minutes before analyzing proteins secreted into the medium. (D,E) Strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS6-gea1-6 (D) and strains CJY52-10-2/pCLJ90-GEA1 and CJY052-10-2/pNTS19-gea1-19 (E) were preincubated at 37°C for the times indicated, then pulse-labeled for 10 minutes and chased for 20 minutes (D) or 30 minutes (E) prior to recovery of medium-secreted proteins.
