Figure 10.

Grd20p is required for rapid endocytic delivery of Ste3p to the vacuole. The following strain/plasmid combinations were grown for several generations at 22°C in minimal media containing galactose: SNY121-12A/pSB18 + pSL2099 (GRD20 END4), SBY4-10A/pSB1-125 + pSL2099 (grd20-1 END4), and SNY44-13B/pSL2099 (GRD20 end4-1). The cultures were then shifted to nonpermissive temperature (36°C), and 15 min later glucose was added to shut off expression of Ste3-myc. Equivalent volumes of culture were withdrawn at 0, 30, 60, and 90 min after addition of glucose. Protein extracts were immediately prepared from the cells, separated by SDS-PAGE, and analyzed by Western blotting using a mouse antic-myc antibody. The blots were developed as described in MATERIALS AND METHODS.