Fig. 8. The defects of different vps1 mutants in vacuolar protein sorting. (A) Abnormal secretion of CPY in the vps1 and the GTPase mutants. Strains as indicated (YMC457-461) were cultured in YEPD media to saturation at 24°C. The cell densities from these cultures were normalized and 5 µl of them was spotted onto YEPD plates. After 2 days at 24°C, cells were replica-plated onto YEPD plates, overlaid with a nitrocellulose membrane, and incubated overnight at 30°C. The membranes were then probed with a monoclonal antibody against CPY. (B) Over-expression of the vps1 GTPase mutants caused the sorting defects of CPY in wild-type cells. Strains of YMC462-466 were first cultured in raffinose media at 24°C for 2 days. Cell numbers were normalized and 5 µl of them was spotted onto galactose-containing plates. After an overnight incubation at 24°C, cells were replica-plated onto refresh galactose plates, overlaid with a nitrocellulose membrane and incubated overnight at 30°C. The membranes were probed with a monoclonal antibody against CPY. (C) Over-expression of the C-terminal region of Vps1p resulted in the secretion of CPY in wild-type cells. Strains as indicated were assayed for CPY sorting as described in (B).