Figure 8. Details in the interaction of AtCP with the membrane containing phosphatidic acid.

A Sequence comparison of C-terminal parts of CPα (CPα-Cterm) from different species. The mafft algorith [40] was used to construct multiple alignments and the final figure was produced using the Jalview alignment editor [56]. Abbreviations used: At – Arabidopsis thaliana, Gg – Gallus gallus, Hs – Homo sapiens, Mb - Monosiga brevicollis, Os – Oryza sativa, Pp – Physcomitrella patens, Sc - Saccharomyces cerevisiae, Sm - Selaginella moellendorffii, Sp - Schizosaccharomyces pombe. Red asterisk marks conserved Lys in plants. B A detailed view of AtCP interaction with membrane containing 20% POPA (charge −2)/POPC. This figure was prepared using VMD [54]. C Protein-lipid overlay assay for detecting interacting lipids. CPα-Cterm shows a preference for PA and PPIs. GST-CPα-Cterm bound to the lipids was detected by immunoblotting with an antibody against GST. Figure shows a representative result from 3 different experiments. D Liposome-binding assay of CPα-Cterm. PA binding was determined using 200 nm-sized vesicles containing 20% PA/PC or PC alone. After incubation of GST-AtCPα-Cterm with the vesicles, they were recovered by ultracentrifugation and protein bound was analysed by SDS-PAGE. As negative control, GST alone was used. Figure shows representative result from 4 different experiments.