Figure 3.

CrPHOT promotes NBD-phospholipid flipping by flippases in a BL-dependent manner. (a) NBD-PE internalization by CrPHOT under BL. Wild type (WT) harbouring vector plasmid, fpk1Δ fpk2Δ mutant harbouring vector, CrPHOT or KDm plasmids, and KKT274 (fpk1Δ fpk2Δ lem3Δ) harbouring vector or CrPHOT plasmids were grown in SC medium in darkness or under 10 μmol m⁻² s⁻¹ BL at 30 °C and treated with NBD-PE. A representative cell image obtained by microscopic observation is shown. Scale bar = 5 μm. (b) Quantification of internalized NBD-labelled phospholipids. Cells grown in SDA-U medium in the dark at 30 °C were labelled with NBD-PE or -PC for 60 min in the dark or under 10 μmol m⁻² s⁻¹ BL, and then washed with SD containing 2.5% BSA before flow cytometry. (c) Time course of NBD-labelled PE internalization by BL. Cells grown in the dark were incubated for a total of 3 h in the indicated light condition (dark, BL 0.5 h, BL 1.0 h, or BL 3.0 h), in which the last 1 h was incubated with the NBD-PE. The internalized NBD-phospholipids were quantitated by flow cytometry. Data are presented as the average percentage ± SD relative to wild-type measurements of three independent experiments (10,000 cells per sample) in each light condition (*p < 0.05, **p < 0.01; Tukey’s test).