Drs2 flippase activity is required for CUPS biogenesis. Cells genomically expressing pGalS-Drs2-3xGFP and Grh1-2xmCherry were transformed with an empty vector, WT Drs2, or mutant Drs2 (D560N) plasmids in a galactose-containing medium. Cells were switched to glucose medium, in order to repress the expression of Drs2-3xGFP, 72 h prior to starvation in 2% potassium acetate. Cells were visualized by epifluorescence microscopy at the indicated times (early, 30- to 35-min incubation; late, 2-h incubation). (A) Representative images of Grh1-positive foci. Scale bar = 2 μm. (B) Quantification of the percentage of cells showing one to three foci per cell.