PXA domain of Mdm1 binds FAs. (A) TLC on lipids extracted from proteins purified from E. coli: PXA-SUMO-hisx6 and PXA-hisx6. SUMO-hisx6 was used as a negative control. Red box: FFA bands. (B) TLC on lipids extracted from recombinant Mdm1^PXA-hisx6 before and after denaturation by using guanidinium hydrochloride. (C) Native PAGE of the PXA domain with or without fluorescent palmitic acid (BODIPY-C16) or BODIPY. Coomassie staining (top) and fluorescence (UV excitation; bottom). (D) Native PAGE of the GST with or without fluorescent palmitic acid (BODIPY-C16) or BODIPY used as a negative control for B. Coomassie staining (top) and fluorescence (bottom). (E) Native PAGE of BODIPY-loaded PXA after flotation assay in the presence and absence of liposomes. Coomassie staining of the PXA protein (top; arrows) and fluorescence corresponding to bound lipids (bottom, arrow and box). (F) Left: Light microscopy of yeast expressing PXA-Helix1 or PXA-Helix2 N-terminally tagged with GFP. LDs (gray) visualized by MDH staining. Scale bar, 2 µm. Right: Helix1 and Helix2 helical wheel representations generated by using HeliQuest. (G) TLC quantification of FFAs (in micrograms) extracted from WT yeast overexpressing Mdm1^FL or Mdm1^IMD+PXA (with or without oleate; 4 h). Data represent mean ± SD normalized to cell pellet weight from TLC plate; n = 2. *, P < 0.01; **, P < 0.005; two-way ANOVA.