LD formation in the absence of preexisting LDs. (A) Widefield microscopy of WT and 4Δ yeast cells expressing LRAT-GFP (green) and Sec63-mCherry (red), showing the differential interference contrast (DIC; white) and fluorescence channels (colors and merge). (B) Widefield microscopy of WT and Δ4 yeast cells, with or without expressing LRAT, 2 h after incubation with or without 2 mM ROH (− or +). After staining, images of UV autofluorescence (green), BODIPY (red), and brightfield were taken. (C) Widefield microscopy of WT yeast cells expressing LRAT and Erg6-mCherry (red). Cells were imaged 2 (top) or 120 (bottom) min after addition of 2 mM ROH. Images of UV autofluorescence (green), BODIPY (red), and brightfield (white) were taken. Scale bars indicate 5 µm. (D) LC-MS contour plots of retention times between 3.5 and 6.5 min (x axis) and m/z values between 350 and 900 (y axis). Detector signal is represented by color code (see key at right). Numbered boxes indicate areas with ions from (1) sterols, (2) DAGs and ceramides, (3) SEs and TAGs [M+H-2RCOOH]⁺, (4) TAG [M+H-RCOOH]⁺, and (5) TAG [M+H]⁺. IS, internal standard TAG (15:0/15:0/15:0). Representative samples of WT (left) and 4Δ+LRAT (right) yeast strains, incubated with 2 mM ROH, are shown. (For experimental details, see Fig. 2). (E) Relative quantification of summed peak area of all TAG ions except internal standard from box 5 (D) of WT (left panel; red) and Δ4+LRAT (right panel; marine blue) yeast cells in the presence or absence of 2 mM ROH. AUC, area under the curve. Bar plot indicates mean ± SD.