(A) Commd1, Commd1 (R133Q, H134A, K167A), Commd7, Commd9 and Commd10 were incubated with POPC (100%), POPC/POPE (90:10, molar ratio), POPC/POPE/POPS (60:10:30, molar ratio) and POPC/POPE/PIP (80:10:10, molar ratio) liposomes doped with different phosphoinositides to perform liposome pelleting assay by ultracentrifugation and subsequent protein content analysis of the supernatant (S) and pellet (P) fractions. (B) (Left) Homology model of Commd1 COMM domain shown as a transparent electrostatic surface overlaid with the ribbon representation, highlighting the residues crucial for forming the positively charged lipid-binding pocket. Inset shows the close up of the lipid-binding pocket. (Right) The electrostatic surface representation of Commd9 COMM domain, highlighting the absence of basic patch. (C) Sequence alignment of Commd1 and Commd9 COMM domains. Red asterisks mark the positively charged amino acids constituting the basic patch on Commd1, which are absent in Commd9. (D–H) Binding of liposomes containing various phosphoinositides to Commd1 measured at different concentrations and Commd1 (R133Q, H134A, K167A) using the BLiTz system. Binding kinetics was calculated using sigma-plot (Systat Software Inc.). (I) Lentiviral constructs of GFP-tagged Commd1 and Commd1 (R133Q, H134A, K167A) were transfected into HeLa cells and colocalisation with the Fam21 WASH subunit imaged by confocal immunofluorescence microscopy. The mutant Commd1 is still recruited to endosomes, presumably due to incorporation into heteromeric COMMD complexes and the CCC/Retreiver subunits.