Fig. 5

Plin4 AH is not adapted to classical phospholipid bilayers. a Principle of the assay. Plin4 fragments labeled with NBD were mixed with liposomes and their membrane association was determined by measuring the increase in NBD fluorescence. b Typical measurements. NBD-Plin4-4mer or the 2T→V mutant were mixed with liposomes containing PC, phosphatidylethanolamine (PE), and cholesterol (50/17/33 mol%). The acyl chains of PC and PE varied from C16:0-C18:1 to C18:1-C18:1 as indicated. The spectra in solution or with diphytanoyl-PS liposomes are also shown. These spectra were used to calculate % binding for each construct (Methods). c–f Effect of phospholipid unsaturation, PS, membrane curvature, and DOG on liposome binding of the indicated constructs as determined from experiments similar to b. The liposome composition (mol%) was: c PC (50), PE (17) and cholesterol (33) with an increasing fraction of C18:1-C18:1 at the expense of C16:0-C18:1 phospholipids; d C16:0-C18:1-PE (17), cholesterol (33) and an increasing fraction of C16:0-C18:1-PS (0 to 50) at the expense of C16:0-C18:1-PC (50–0); e C16:0-C18:1-PC (30), C16:0-C18:1-PS (20) C16:0-C18:1-PE (17) and cholesterol (33). The liposomes were extruded with polycarbonate filters of decreasing pore radius and the liposome radius was determined by dynamic light scattering; f C16:0-C18:1-PE (17), cholesterol (33) and an increasing fraction of DOG (0 to 15) at the expense of C16:0-C18:1-PC (50 to 35). g Influence of AH length on binding to diphytanoyl lipids. Liposomes contained PC (50), PE (17), and cholesterol (33) with an increasing fraction of diphytanoyl at the expense of C16:0-C18:1 phospholipids. h CD spectra of Plin4-20mer in solution or with increasing amounts of diphytanoyl-PS liposomes. Inset: titration of the CD signal at at 222 nm as a function of liposome concentration. Data shown in c, d, and g are means ± s.e.m. from three independent experiments; e–f show the results of two independent experiments