Fig. 2. sar1-1, sec31-1 and pmm1-1 accumulate ER forms of acid phosphatase. Equal quantities of cell extracts were resolved by SDS-PAGE, transferred to a nylon membrane and probed with the anti-acid phosphatase monoclonal antibody 7B4 (Schweingruber et al., 1986) unless otherwise stated. (A) Wild-type (lanes 1-4) and sar1-1 (lanes 5-8) cells were harvested after 0, 1, 2 or 4 hours at 36°C. (B) Wild-type (lanes 1,2) and sec31-1 (lanes 3,4) cells were harvested after incubation for 0 or 4 hours at 36°C. (C) Wild-type (lanes 1,2) and pmm1-1 (lanes 3,4) cells were treated with cycloheximide for 1 hour at the permissive temperature, washed, and released into pre-warmed media without cycloheximide at 36°C. Untreated cells and cells incubated for 4 hours at 36°C were harvested. (D) Wild-type (lanes 1,2), ypt1-VN (lanes 3,4) and ypt2-VN (lanes 5,6) cells were harvested after 0 and 4 hours at 36°C. Three times as much protein was loaded for the ypt mutants at 25°C to allow better visualization of the acid phosphatase. The arrow indicates the 72 kDa core glycosylated ER form (A,B), the 45-75 kDa incompletely glycosylated ER forms (C) or the high molecular weight Golgi modified forms of acid phosphatase (D).
