Fig. 3. CPY maturation and sorting was unaffected by loss of all OSH function. (A) Autoradiograph of CPY immunoprecipitations from wild-type (Osh⁺; SEY6210), osh osh4-1 (CBY886) and osh OSH4 (JRY6320) cultures. p1, ER/early Golgi form of CPY; p2, late Golgi CPY; m, mature vacuolar form of CPY. Cells were pulse-labeled with Tran[³⁵S]-label for 5 minutes and chased for the periods indicated at 37°C. CPY was immunoprecipitated from cell lysates and analyzed by SDS-PAGE. (B) CPY immunoblots of vps4 (CBY824), wild-type (W.T.; SEY6210), and osh P^MET3-OSH2 (JRY6326) strains. Strains were spotted on solid media with increasing concentrations of methionine (up to 100 mM), which repressed expression of OSH2 in the osh P^MET3-OSH2 strain and arrested growth. Filters were placed directly on the solid medium and CPY secreted from cells was detected with anti-CPY antibodies (Roberts et al., 1991). Filters were also probed with anti-Act1p to detect the discharge of actin from non-specific cell lysis. Equivalent amounts of cells were spotted on the solid medium as shown by the example in the bottom panel.