Figure 4.

SMGL-1 functions as a guanine nucleotide exchange factor for RAB-8. (A and B) Confocal images of intestinal cells expressing GFP-tagged RAB-8. Compared with wild-type animals, smgl-1 knockout reduced the intensity of GFP::RAB-8-labeled puncta. Overexpression of CRISPR/Cas9-editing resistant SMGL-1 rescued the localization defects of GFP::RAB-8 in smgl-1 knockout animals. The signals from the apical membrane were avoided by manual ROI selection. Data are shown as mean ± SD (n = 18 each, six animals of each genotype sampled in three different unit regions of each intestine defined by a 100 × 100 [pixel²] box positioned at random). For multiple comparisons, statistical significance was determined using a one-way ANOVA followed by a post-hoc test (Dunn’s Multiple Comparison Test). ***, P < 0.001. Data distribution was assumed to be normal, but this was not formally tested. Scale bars: 10 μm. Colored asterisks indicate intestinal lumen. (C) Western blot showing GFP-tagged RAB-8 and RAB-5 in worm lysate, supernatant, and pellet. The membrane-to-cytosol (pellet-to-supernatant) ratio of RAB-8 decreased in smgl-1(ycxEx1659) mutants. In contrast, the ratio of GFP::RAB-5 (early endosome marker) was not affected by SMGL-1 knockout. (D) Western blot showing GST pull-down with in vitro translated HA-tagged proteins. GST-SMGL-1 interacted with HA-RAB-8(T22N) while displaying little affinity for HA-RAB-8(Q67L). (E) In a co-immunoprecipitation assay, SMGL-1 precipitated predominantly with RAB-8 (T22N) inactive form. (F) In vitro GEF assay. MANT-GDP release from RAB-8 was measured by adding GST-only, GST-SMGL-1, and GST-SMGL-1(WD40 domain). GST-SMGL-1 and GST-SMGL-1(WD40 domain) promoted the release of MANT-GDP from RAB-8. However, GST-only failed to trigger the MANT-GDP release from RAB-8. Source data are available for this figure: SourceData F4.