hSNX16 oligomerizes into higher-order assemblies on membranes via its CC domain. (A and B) hSNX16^ΔCC reduces and hSNX16^3A promotes self-association compared with hSNX16^WT on PI(3)P liposomes. 5 µM purified hSNX16, hSNX16^3A, or hSNX16^ΔCC were incubated with 1% (5 µM) PI(3)P liposomes followed by BS³ cross-linking, and then tested for liposome cosedimentation. (A) Coomassie-stained gels of cross-linked high-molecular-weight assemblies in protein–liposome pellets with increasing BS³ concentration (125 nM, 2.5 µM, 5 µM, 12.5 µM, and 25 µM). (B) Coomassie-stained gels of unbound proteins in the lipid cosedimentation assay from same experiments as in A. Black, white, and red triangles point to monomers, dimers, and higher-order assemblies, respectively. (C) Schematic of the GUV assay. (D–F) hSNX16^3A fails to recover after photobleaching at lower protein concentrations compared with wild-type hSNX16, while hSNX16^ΔCC retains high mobility at all concentrations measured. (D) Representative time-lapse images of 100 nM hSNX16 and hSNX16^3A before and after photobleaching. (E and F) Quantification of protein and lipid fluorescence of GUVs bound by hSNX16 variants at 100 nM and 500 nM. Protein and lipid fluorescence were normalized to a nonbleached region on the same GUV to correct for photobleaching. Protein fluorescence was then further normalized by subtracting from all time points the intensity at t = 0 and normalizing prebleach intensity to 1. Quantification is from five GUVs incubated with 100 nM hSNX16; six GUVs incubated with 100 nM hSNX16^3A, 500 nM hSNX16, or 500 nM hSNX16^ΔCC; and eight GUVs incubated with 500 nM hSNX16^3A. Data are presented as mean ± SEM. Scale bars, 10 µm.