Figure 8. Mutation of the putative phosphoinositide binding site impairs COMMD1 localization to VPS35-positive endosomes.
(A) Ribbon and space filling models of the COMMD domain of COMMD1 modeled on COMMD9 (PDB: 6BP6) (Healy et al., 2018). Positively charged residues within the predicted phosphorylated phosphatidylinositol (PPI) binding site are indicated. (B–C) COMMD1-/- HeLa cells were transiently transfected with either wild-type COMMD1 or COMMD1 mutants (EQE and QAA), then fixed, permeabilized, and co-stained with antibodies against COMMD1 and VPS35. EQE: R133E/H134Q/K167E, and QAA: R133Q/H134A/ K167A. The percent of the total COMMD1 residing on VPS35-positive endosomes was quantified. Data presented as mean ± SE. Statistical significance from three independent experiments were analyzed using one-way ANOVA and Tukey’s post hoc test. *p < 0.05, and ns, not significant. Bar: 10 µm.
Figure 8—figure supplement 1. Validation of COMMD1-/- HeLa cells.
