Figure 7.

gyp1Δ missorts CPY to the cell surface. (A) Cells were labeled with ³⁵S for 10 min at 30°C in minimal medium and chased with unlabeled methionine and cysteine for 30 min. CPY was immunoprecipitated from total lysates, separated on SDS-PAGE, and quantified by phosphorimaging. P1, ER form; P2, Golgi form; M, mature form. (B) Overexpression of Ypt1p enhances the CPY missorting phenotype of gyp1Δ. The same number of cells was spotted on YPD and YP-raffinose-galactose plates. Wet nitrocellulose membranes were overlaid on top of the plates. The plates were incubated at 30°C overnight. The amount of CPY absorbed on the membrane was determined by Western blot. (C) ypt1-2 suppresses the CPY missorting phenotype of gyp1Δ. The same number of cells was spotted on YPD plate at 30°C. CPY secretion was determined by the overlay assay.