Loss of sorf-1 and sorf-2 leads to PtdIns3P enrichment and defective endosomal transport. (A) Representative projection images of 2xFYVE::GFP or GFP::EEA-1 on early endosomes in N2, sorf-1(tm3855), and sorf-2(tm5210) coelomocytes. Images were obtained with the same exposure time. Projections were made for the whole cell. (B and C) Intensity of 2xFYVE::GFP or GFP::EEA-1 (arbitrary units [AU]) on endosomes. ≥300 endosomes were measured per genotype. N2 and mutants were compared with t tests. Error bars represent SEM. ***, P < 0.001. (D) Time course of endosomal TR-BSA trafficking in coelomocytes in animals of the indicated genotypes. TR-BSA was injected into the body cavity and examined at the indicated time points after injection (left). Bars, 5 µm. Early endosomes (EEs) were quantified for retention of TR-BSA (right). 60 or more endosomes were scored at each time point for each genotype. Error bars represent SEM, and data are from three independent experiments. (E–G) Interaction of SORF-1 and SORF-2. Myc-tagged SORF-2 (E) or its different subregions (G) were coexpressed with GFP-SORF-1 in HEK293 cells. IPs were performed with Myc antibody, and precipitated proteins were detected with Myc and GFP antibodies. ³⁵S-labeled SORF-2 was incubated with MBP-SORF-1 and precipitated with maltose-binding beads (F).