Drs2 flippase activity is acutely required for Gcs1 endosomal localization. (A) Gcs1(186–352)-GFP localization was monitored in drs2Δ cells carrying wild-type DRS2 or a drs2 temperature-sensitive allele (drs2^ts) grown at 27°C or shifted to 38°C for 1 h. Bars, 5 µm. (B) The percentage of mCherry-Tlg1 punctae showing colocalization with Gcs1(186–352)-GFP from 15 cells of each strain was quantified. ***, P < 0.001. (C) Time-lapse images of Gcs1-GFP expressed on 2μ plasmids, which exist in multiple copies per cell, in wild-type and drs2^ts as cells warmed from room temperature to ∼37°C on a heated stage. Arrows indicate the positions of TGN/EE structures marked with Gcs1-GFP. Bar, 5 µm.