Opt2 plays an important role in adaptation to altered lipid asymmetry mediated by the Rim101 pathway. (A) SEY6210 (WT, wild type), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK2027 (rim21Δ), YYS52 (rim21Δ lem3Δ), YOK3260 (rim21Δ lem3Δ P_ADH-HA-OPT2) and YOK3259 (opt2Δ lem3Δ P_ADH-HA-OPT2) cells were grown to stationary phase, serially diluted at 1∶10, spotted onto YPD plates and grown at 30°C for 28 h. (B) DNA microarray analysis was performed on SEY6210 (WT, wild type), YOK2030 (lem3Δ), YOK2368 (pdr5Δ) and YOK2209 (rim21Δ lem3Δ) cells. Expression levels of OPT2 (closed circles) and, to demonstrate uniform use of poly (A)⁺ RNAs, ACT1 (open circles) are shown as a relative value to that in wild-type cells. (C) Total lysates were prepared from YYS351 (OPT2-myc), YYS352 (OPT2-myc lem3Δ), YYS354 (OPT2-myc pdr5Δ), YYS355 (OPT2-myc rim21Δ) and YYS356 (OPT2-myc rim21Δ lem3Δ) cells in log phase and subjected to immunoblotting with an anti-Myc antibody. Ponceau S staining was used to demonstrate uniform protein loading. (D) SEY6210 (WT, wild type), YOK2027 (rim21Δ), YOK2030 (lem3Δ), YYS12 (opt2Δ), and YYS13 (opt2Δ lem3Δ) cells harboring pFI1 (HA-RIM101) were grown to log phase and exposed to an alkaline medium (pH 8.0) for 20 min. Total lysates were then prepared and imunoblotted with an anti-HA antibody. FL and ΔC denote the full-length and processed Rim101, respectively. (E) SEY6210 (WT, wild type), YYS5 (lem3Δ), YOK2053 (rim21Δ), YYS4 (rim21Δ lem3Δ), YYS12 (opt2Δ) and YYS13 (opt2Δ lem3Δ) cells in stationary phase were serially diluted at 1∶10, spotted onto YPD plates, and grown at pH 8.0 and 30°C for 91 h.

Opt2 cycles between the plasma membrane and the late Golgi. (A) YYS250 (GFP-OPT2 SEC7-mCherry) cells were grown to log phase and subjected to fluorescence microscopy. Arrowheads indicate GFP–Opt2 (green) colocalized with Snf7–mCherry (red). (B) YOK3206 (GFP-OPT2 LAS17-HA-AID) cells were grown to log phase, treated with 500 µM 3-indoleacetic acid (IAA) or ethanol (mock) for 30 min, and subjected to fluorescence microscopy. Scale bars: 2 µm.

Opt2 is involved in the maintenance of vacuole morphology in cells with altered lipid asymmetry. (A) SEY6210 (WT, wild type), YYS195 (lem3Δ), YOK2027 (rim21Δ), YYS52 (rim21Δ lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK3260 (rim21Δ lem3Δ P_ADH-HA-OPT2) and YOK3259 (opt2Δ lem3Δ P_ADH-HA-OPT2) cells in log phase were stained with FM4-64 and their vacuolar morphology was examined under a fluorescence microscope. Inset, magnified image of the region surrounded by the dashed line. (B) Total lysates were prepared from SEY6210 (WT, wild type), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ) and YOK3156 (vps21Δ) cells in log phase and were subjected to immunoblotting with anti-CPY, anti-Pho8 or, to demonstrate uniform protein loading, anti-Pgk1 antibody. Immunoblotting of each extracellular fraction (medium) was also performed with anti-CPY antibody. p, preform; p2, p2 Golgi form; m, mature form; s, soluble form. (C) SEY6210 (WT, wild type), YYS5 (lem3Δ), YYS12 (opt2Δ) and YYS13 (opt2Δ lem3Δ) cells in log phase were pulse-labeled with FM4-64 and examined under a fluorescence microscope. Arrowheads indicate endosomes labeled with FM4-64. Scale bars: 2 µm.

Opt2 is involved in the exposure of PtdEtn and PtdSer to the outer leaflet of the plasma membrane. (A) SEY6210 (WT, wild type), YYS5 (lem3Δ), YYS12 (opt2Δ), YYS13 (opt2Δ lem3Δ), YOK2027 (rim21Δ), YYS52 (rim21Δ lem3Δ), YOK3260 (rim21Δ lem3Δ P_ADH-HA-OPT2) and YOK3259 (opt2Δ lem3Δ P_ADH-HA-OPT2) cells were grown to stationary phase, serially diluted at 1∶10, spotted onto YPD plates with or without 5 µM duramycin or 0.25 µg/ml papuamide B, and grown at 30°C for 50 h. (B) SEY6210 (WT, wild type), YYS5 (lem3Δ) and YYS13 (opt2Δ lem3Δ) cells in log phase were collected and the surface-exposed PtdEtn was visualized using Bio-Ro and FITC-labeled streptavidin. Arrowheads indicate FITC signal. Scale bar: 2 µm. (C) The percentage of cells with FITC signal is presented as the mean±s.d. (three independent experiments); *P<0.001 (Student's t-test). (D) SEY6210 (WT, wild type) and YYS307 (P_ADH-HA-OPT2) cells in log phase were harvested and subjected to the NBD-phospholipid transfer assay. PE, phosphatidylethanolamine; PC, phosphatidylcholine; PS, phosphatidylserine. Values represent the mean±s.d. (three independent experiments); *P<0.05 (Student's t-test).