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. 2011 Nov 28;195(5):889–902. doi: 10.1083/jcb.201104040

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© 2011 Karotki et al.

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Figure 5. — Pil1 and Lsp1 membrane binding requires an N-terminal segment and a patch of positively charged amino acids on their BAR domain surface. (A) Computational rigid body fitting of the Lsp1 BAR domain dimer x-ray structure to cryo-EM density maps of Lsp1 tubules and Lsp1 bound to PC liposomes containing 1.5% PI(4,5)P₂. The top view of tubules (top) shows Lsp1 BAR domain monomer chains colored blue to red from N terminus to C terminus. The side view of tubules (bottom) shows the Lsp1 helix colored blue to red from the bottom to the top. (B) A close-up of the side view and intersection of the tubules. Lsp1 BAR domain monomer chains are colored blue to red from the N terminus to the C terminus. Density that might be occupied by the flexible tips of the x-ray structure, adopting a slightly different conformation in the tubules than in the crystal, is indicated by green arrows. The density that could be filled by the C termini, which are missing in the x-ray structure, is indicated by red arrows. (C) Negative staining and EM of recombinant Pil1 or Lsp1 proteins incubated with PC liposomes containing either 1.5 or 3.5% PI(4,5)P₂. Mutants with an N-terminal truncation (lsp1ΔN) or changes in the positively charged amino acid patch of the concave BAR domain surface of Pil1 or Lsp1 (lsp1KRE) retain the ability to bind and tubulate PI(4,5)P₂-containing liposomes. Combination of both types of mutation (lsp1ΔNKRE) abolishes membrane binding. Protein-covered membrane tubules are marked with yellow arrowheads. Bar, 100 nm. (D) Spin-down experiments of Lsp1, lsp1KRE, lsp1ΔN, or lsp1ΔNKRE incubated with or without PC liposomes containing 0.1, 1, 1.5, or 3.5% PI(4,5)P₂ as indicated. Panels showing different experimental conditions are separated by dotted lines for better visibility. P, pellet; S, supernatant. (E) Quantification of protein amounts in pellet fractions of experiments analogous to D. n = 3. Error bars represent SDs of three independent experiments.