RABI-8 interacts with SMGL-1 and reduces its GEF activity toward RAB-8. (A) RABI-8 precipitated GFP::SMGL-1 in a representative Co-IP assay. (B) A representative immunoblot showing the GST-tagged SMGL-1 was pulled down by in vitro translated HA-RABI-8. (C) Confocal images and quantification showing colocalization between RABI-8::GFP and MC::SMGL-1 in the intestinal cells. Broad-spectrum intestinal autofluorescent lysosome-like organelles can be seen in blue. Pearson’s correlation coefficients for GFP and mCherry signals were calculated (n = 12 animals). The signals from the apical membrane were avoided by manual ROI selection. Scale bars: 10 μm. White asterisk indicates the intestinal lumen. (D) In vitro GEF assay. MANT-GDP release from RAB-8 was measured by adding GST only, GST-SMGL-1, and GST-SMGL-1 and GST-RABI-8. GST-SMGL-1 promoted the release of MANT-GDP from RAB-8, while the presence of RABI-8 inhibited the promotion. Data are shown as mean ± SD from three independent replicates. (E) In vitro GEF assay. MANT-GDP release from RAB-8 was facilitated by adding GST only, GST-SMGL-1, GST-SMGL-1 (crosslinked) and GST-SMGL-1 (crosslinked), and GST-RABI-8. Crosslinking assay was performed using DSG. Data are shown as mean ± SD from three independent replicates. Source data are available for this figure: SourceData F4.