Figure 5. VARP is required in vitro to reconstitute the proposed SNX27/Retromer/ESCPE-1 endosomal ‘supercomplex’.

(A) Purified recombinant human SNX27, Retromer, SNX2/SNX6 (ESCPE-1), and VARP were incubated with PDZbm cargo motif from 5HT4(a)R, either alone or together on PI(3)P-enriched liposomes. DOPC/DOPE was used as a negative control. Samples were subjected to ultracentrifugation followed by SDS-PAGE and Coomassie staining of unbound supernatant (S) and bound pellet (P) fractions. In the presence of VARP, all endosomal coat complexes (SNX2/SNX6, SNX27, and Retromer) are recruited to PI3P-enriched membranes. (B) Binding of proteins to phosphoinositide-enriched membranes visualized by SDS-PAGE was quantified by measuring relative protein band intensities (ImageJ). Relative gel band intensities corresponding to Supernatant (S) and Pellet (P) fractions were calculated for each protein sample and plotted as a heat map. (C) Representative negative stain EM image visualizing PI(3)P liposomes incubated with the SNX27/Retromer/ ESCPE-1/VARP ‘supercomplex’ in presence of the PDZbm cargo (scale bar = 500 nm). The supercomplex can both assemble (A) and tubulate membranes in the presence of VARP (C).