Phospholipid packing defects in Fld1/Ldb16 complex mutants. (A) Helical-wheel representation of the AH of Pct1 and Kes1 as predicted and drawn by Heliquest (Gautier et al., 2008). Both AHs display a well-defined face enriched in hydrophobic residues (yellow). In contrast, the polar faces are very distinct: in Pct1, it is enriched in charged residues (blue and red), whereas in Kes1, the abundance of noncharged serine and threonine residues (purple) dominates. (B) Localization of Pct1 and Kes1 in wt and ldb16Δ cells grown in SC media to early stationary phase. Proteins were expressed from their endogenous locus as C-terminal mCherry fusions. Nuclear envelope is labeled by endogenously expressed Hmg1-GFP. LDs are stained with the neutral lipid dye MDH. Yellow arrowheads indicate supersized LDs; white arrowheads indicate LD aggregates. Bar, 5 µm. (C) Electron micrographs showing immunolocalization of endogenously expressed Pct1-GFP in ldb16Δ cells. Logarithmic cultures in YPD were processed for immuno-EM as described in the Materials and methods section. Arrowheads point to 12 nm immunogold particles specifically labeling Pct1-GFP in association with nuclear LDs. NE, nuclear envelope; N, nucleus. Magnified insets are shown on the right of each micrograph. Bars, 200 nm. (D) Localization of endogenously expressed Kes1-GFP and Pct1-mCherry in wt, ldb16Δ, and fld1Δ cells. Late logarithmic cultures in SC media or SC supplemented with 75 µM inositol (SC + INO) were imaged. LDs are stained with the neutral lipid dye MDH. Bar, 5 µm.