GOP-1 interacts with UNC-108 and disrupts the UNC-108–GDI-1 complex. (A and B) The interaction between GOP-1 and UNC-108 was examined by yeast two-hybrid analyses (Y2H; A) and GST pull-down assays (B). GOP-1 specifically interacts with the GDP-bound and nucleotide-free forms of UNC-108 in Y2H (A), whereas significantly more GOP-1-HIS is pulled down by GST-UNC-108(S20N) (GDP-bound) and GST-UNC-108(N119I) (nucleotide-free) than by GST-UNC108(WT) and GST-UNC-108(Q65L) (GTP-bound; B). At least four independent experiments were performed, and data are shown as mean ± SD. One-way ANOVA with Tukey’s posttest was performed to compare mutant datasets with wild type. *, P < 0.05; N.S., no significance. (C) Cherry::UNC-108 is coprecipitated with GFP::GOP-1 by GBP pull-down in lysates prepared from adult worms coexpressing both fusion proteins. (D–D″′) DIC and fluorescent images of the wild-type germ line coexpressing GFP::GOP-1 and Cherry::UNC-108. GOP-1 and UNC-108 colocalized on the phagosome (arrowheads). n, number of worms quantified. Bars, 5 µm. (E) GOP-1 disrupts the UNC-108–GDI-1 complex. Schematic illustration of the GDI displacement assay is shown at left. Addition of GST-GOP-1 but not GST led to association of UNC-108 but not GDI-1 with GST-beads. At least three independent experiments were performed, and a representative result is shown.