FIGURE 4.

Analysis of phosphatidic acid binding by MAM7 mce domains. Liposome association assays with individual mce domains and liposomes prepared from PC and PA and containing increasing concentrations of PA as indicated (0–80 mol %). Supernatant and pellet fractions were analyzed by SDS-PAGE and visualized by Coomassie staining (supplemental Fig. S1). % Bound protein were determined by densitometry of gels and used to compare the affinities of mce1–7 constructs (A). Shown is the densitometry of mce6 (weakest binding to PA) compared with mce2 (tightest binding) and three mce6 point mutants (B). A pulldown assay of liposomes containing 50 mol % PA and 50 mol % PC on immobilized GST-mce domains is shown (C). A sequence alignment of mce2 and mce6 is shown (D). Positions of point mutations in mce6 are shown in blue (S646K), dark purple (Q664K), and pink (H703R), respectively. aa., amino acids. *, identical aa; :, well conserved aa; ., conserved aa.