Figure 8.
Endocytic transport in the cdc50Δ mutant. (A) Internalization of the fluorescent lipophilic dye FM4–64 in wild-type and cdc50Δ strains. Wild-type (YKT38) and cdc50Δ mutant (YKT249) cells were labeled with FM4–64 dye for 15 min at 30 or 18°C, and then chased in fresh medium for 1 h or 3 h, respectively. (B) Fluid phase endocytosis in wild-type strain and cdc50Δ mutant. Wild-type (YKT38) and the cdc50Δ mutant (YKT249) cells were incubated for 12 h at 18°C, stained with Lucifer yellow for 2, 4, and 8 h at 18°C. Bar, 5 μm (C) Endocytic delivery and degradation of the Ste3p-myc protein. Wild-type (YKM42) and cdc50Δ mutant (YKM43) cells, both harboring plasmids pSL2099 encoding the STE3-myc gene under the control of the galactose-inducible GAL1 promoter, were grown at 30°C in minimal media containing galactose. The cultures were then shifted to 18°C, and glucose was added to shut off expression of STE3-myc. Aliquots of cells were removed at the indicated time points, and protein extracts were prepared immediately from the cells. Samples were separated by SDS-PAGE and analyzed by Western blotting using a mouse anti-myc antibody.