Figure S5.

CZW-1 interacts with SMGL-1 and promotes the subcellular localization of SMGL-1. (A) CZW-1 colocalized with a subset of RAB-8-positive endosomal structures. 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. (B) GST-SMGL-1 pulled down HA-CZW-1 in an in vitro pull-down assay. (C) In vitro GEF assay. MANT-GDP release from RAB-8 was measured by adding GST-only, GST-SMGL-1, and GST-SMGL-1&GST-CZW-1. (D) The intracellular distribution of GFP::CZW-1 was not affected in smgl-1(ycxEx1659) mutants. (E and F) Overexpression of CZW-1 failed to rescue the localization defects of RAB-8 and PGP-1 in smgl-1(ycxEx1659) mutants. (G) GFP::RER-1 was diffusive and labeled dispersed small puncta in CZW-1 knockdown animals. Overexpression of SMGL-1 or RAB-8 failed to rescue the localization defects of GFP::RER-1. 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). Statistical significance was determined using a two-tailed, unpaired Student’s t test. 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. A dotted line indicates the outline of the intestine. Source data are available for this figure: SourceData FS5.