Panel A shows a protein schematic comparing mp2.1 construct with ALPS deleted mutant and highlighted ALPS sequence region. Panel B shows protein gels and western blot confirming expression of mp2.1 and ALPS deleted streptagged proteins. Panel C shows a line graph comparing liposome diameter distributions after filtration through different pore size filters. Panel D shows density gradient centrifugation images before and after separation of synaptic vesicle like liposomes. Panel E shows fluorescence and immunoblot dot images comparing mp2.1 association with liposomes of different diameters. Panel E prime shows a bar graph comparing top and bottom protein fractions for mp2.1 across liposome diameters. Panel F shows fluorescence and immunoblot dot images comparing ALPS deleted mp2.1 association with liposomes of different diameters. Panel F prime shows a bar graph comparing top and bottom protein fractions for ALPS deleted mp2.1 across liposome diameters.
An N-terminal Piccolino ALPS motif binds to artificial, SV-like liposomes. (A) Schematic of Piccolino (black) including the location of the ALPS motif (yellow/purple) within the region used for protein–lipid binding studies (mp 2.1; gray). WT mp 2.1 without the ALPS motif (mp2.1ΔALPS) was used as a negative control. (B) SDS-PAGE with purified Pclo fragments labeled with the total protein stain TCE (left). Western blots of purified Pclo fragments mp2.1::strep and mp2.1ΔALPS::strep fragments stained with anti-strep (right). (C) Diameter of SV-like liposomes measured by DLS after extrusion through polycarbonate filters. (D) Detection of NBD-labeled lipids in centrifuge tubes with sucrose step gradients (0–50%) before (left) and after (right) ultracentrifugation. (E–F′) Dot blots of collected fractions (T, top; B, bottom) after liposome floatation assays and quantification of bound protein for WT mp2.1::strep (E and E′) and mutant mp2.1ΔALPS::strep (F and F′). The graphs in C, E′, and F′ show the mean values of n = 3 individual experiments. mp, mouse Piccolino; NBD, nitrobenzoxadiazole; Zf, zinc finger. Source data are available for this figure: SourceData F5.