Figure 2.
Chm7 binds directly to PA-rich lipid membranes. (A) GST-Chm7 preferentially binds to PA and cardiolipin. At left is a schematic showing the location of lipids immobilized on membranes (lipid strip). These lipid strips were probed with either GST (middle) or GST-Chm7 (right). Specific binding was detected with anti-GST primary antibodies followed by HRP-conjugated secondary antibodies and ECL. Position of PA and cardiolipin is indicated with arrowheads. (B) GST-Chm7 specifically binds PA-rich liposomes. At left is a schematic of liposome flotation assay in which proteins and liposomes of defined lipid compositions are mixed, overlaid with a Nycodenz gradient, and then subjected to ultracentrifugation. Fractions that float (bound) are separated by SDS-PAGE, and proteins are detected with Coomassie staining; all liposomes are composed of 75% phosphatidylcholine (PC) with the indicated percentages of PA, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Numbers indicate the position of molecular weight (MW) standards in kD. (C) Plot of the percentage (relative to input) of GST-Chm7 bound to liposomes with increasing PA concentration as indicated. Error bars are SD from three independent experiments. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns (not significant) is P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. (D) Mutations in the hydrophobic stretch of Chm7 diminish binding to PA-rich membranes. Coomassie-stained gel of liposome flotation as in B but incorporating GST-chm7-N and GST-chm7-(W3AV1A). (E) Plot of the percentage of GST-Chm7, GST-chm7-(W3AV1A), and GST-chm7-N (relative to input) bound to liposomes with (+) or without (-) PA as in D. Average from two independent replicates (denoted as circles) is plotted. (F) Chm7 prefers high curvature. Coomassie-stained gel of liposome flotation and analysis as in B but changing liposome diameter as indicated above the liposome diagrams (in nm). All liposomes in this experiment are composed of 5% PA, 20% PE, and 75% PC. (G) Plot of the percentage (relative to input) of GST-Chm7 from experiments as in F. Error bars are SD from three independent experiments. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where *, P ≤ 0.05; **, P ≤ 0.01.

Chm7 binds directly to PA-rich lipid membranes. (A) GST-Chm7 preferentially binds to PA and cardiolipin. At left is a schematic showing the location of lipids immobilized on membranes (lipid strip). These lipid strips were probed with either GST (middle) or GST-Chm7 (right). Specific binding was detected with anti-GST primary antibodies followed by HRP-conjugated secondary antibodies and ECL. Position of PA and cardiolipin is indicated with arrowheads. (B) GST-Chm7 specifically binds PA-rich liposomes. At left is a schematic of liposome flotation assay in which proteins and liposomes of defined lipid compositions are mixed, overlaid with a Nycodenz gradient, and then subjected to ultracentrifugation. Fractions that float (bound) are separated by SDS-PAGE, and proteins are detected with Coomassie staining; all liposomes are composed of 75% phosphatidylcholine (PC) with the indicated percentages of PA, phosphatidylethanolamine (PE), and phosphatidylinositol (PI). Numbers indicate the position of molecular weight (MW) standards in kD. (C) Plot of the percentage (relative to input) of GST-Chm7 bound to liposomes with increasing PA concentration as indicated. Error bars are SD from three independent experiments. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where ns (not significant) is P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01. (D) Mutations in the hydrophobic stretch of Chm7 diminish binding to PA-rich membranes. Coomassie-stained gel of liposome flotation as in B but incorporating GST-chm7-N and GST-chm7-(W3AV1A). (E) Plot of the percentage of GST-Chm7, GST-chm7-(W3AV1A), and GST-chm7-N (relative to input) bound to liposomes with (+) or without (-) PA as in D. Average from two independent replicates (denoted as circles) is plotted. (F) Chm7 prefers high curvature. Coomassie-stained gel of liposome flotation and analysis as in B but changing liposome diameter as indicated above the liposome diagrams (in nm). All liposomes in this experiment are composed of 5% PA, 20% PE, and 75% PC. (G) Plot of the percentage (relative to input) of GST-Chm7 from experiments as in F. Error bars are SD from three independent experiments. P values were calculated from a one-way ANOVA with Tukey’s post hoc test where *, P ≤ 0.05; **, P ≤ 0.01.

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