FIG. 2.

(A) Underglycosylation phenotype of rer2 mutants. Wild-type (WT) RER2 (SNY9) and rer2-2 (SNH23-7D) cells were grown in YPD medium to 10⁷ cells/ml at 23°C and then shifted to 37°C for the indicated times. Total cell extracts were prepared, and proteins (70 μg) were analyzed by immunoblotting with the anti-Sec12p antibody. (B) Biosynthesis of CPY in rer2 mutants. The rer2-2 mutant (SNH23-7A) and wild-type (SNY9) cells were preincubated for 1 h at 23 or 37°C and then labeled for 4 min with Tran³⁵S-label and chased for the indicated times at the same temperature. CPY was immunoprecipitated from cell extracts and treated with or without endo H. Samples were analyzed by SDS-PAGE and fluorography. p1, ER form; p2, Golgi form; m, mature vacuolar form; ∗, unglycosylated pro-CPY. (C) Biosynthesis of a GPI anchor protein, Gas1p, in rer2 mutants. After preincubation at 37°C for 1 h, rer2-2 (SNH23-7A) and wild-type (SNY9) cells were labeled for 4 min with Tran³⁵S-label and chased at 37°C. Gas1p was immunoprecipitated and analyzed by SDS-PAGE and fluorography.