Colocalization of GFP-Syb1 with internalized FM4-64, normal D4H distribution in osh2Δ osh3Δ double mutants and relative expression levels of Ltc1-sfGFP under native, p^pom1 and p^act1 promoters. (A) GFP-Syb1 labels an endosomal compartment. Cells were incubated with FM-64 and dye distribution was evaluated after 5, 30, and 60 min after labeling. FM4-64 signal initially labels PM and endosomes, which strongly colocalize with GFP-Syb1. Endosomal and weak vacuolar staining can be observed 30 min after staining, and the dye reaches vacuoles within 60 min of incubation. Scale bar, 2 µm. (B) mCherry-D4H in WT and osh2Δ osh3Δ double mutant treated or not with CK-666 for 1 h. Scale bar, 2 µm. (C) Ltc1-sfGFP was expressed either under ltc1 promoter at the native genomic locus, or under p^pom1 or p^act1 promoters. Untagged and Ltc1-sfGFP–expressing strains imaged in identical conditions are shown at the top. The images are contrasted optimally for either p^act1-driven expression (top) or native expression (bottom). Arrowheads indicate sites of Ltc1 peripheral localization. Scale bar, 2 µm. (D) Quantification of total fluorescence in cells as in C. (E) Quantification of fluorescence in cortical punctae in cells as in C. A.U., arbitrary units.