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. 2022 May 23;221(7):e202111125. doi: 10.1083/jcb.202111125

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© 2022 Wang et al.

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Figure S2. — SMGL-1 interacts with RAB-8 and acts as a guanine nucleotide exchange factor for RAB-8. (A and B) Confocal images of intestinal cells expressing GFP-tagged RAB-8. SMGL-1 knockdown reduced the number of GFP::RAB-8-labeled puncta. (C) Expression level of GFP::RAB-8 in wild-type and smgl-1(ycxEx1659) animals. (D) Levels of rab-8 mRNA in wild-type and smgl-1(ycxEx1659) animals. Each is the average of three replicates. (E and F) The intracellular distribution of GFP::RAB-5 did not change in smgl-1(ycxEx1659) mutants. (G) Western blot showing GFP-tagged RAB-7 in worm lysate, supernatant and pellet. The membrane-to-cytosol (pellet-to-supernatant) ratio of GFP::RAB-7 (late endosome marker) was not affected by SMGL-1 knockout. (H) In a co-IP assay, GFP::SMGL-1 precipitated with MC::RAB-8. (I and J) In vitro GEF assay. MANT-GDP release from RAB-5 or RAB-8(T22N) was not facilitated by adding GST-SMGL-1. (K) In a co-IP assay, EHBP-1::GFP preferentially precipitated with MC::RAB-8(Q67L). (L) EHBP-1::GFP co-immunoprecipitated by MC::RAB-8 were reduced in SMGL-1 knockdown animals. Levels of EHBP-1::GFP were normalized to the corresponding actin (set to one in wild-type animals). (M and N) The intensity of GFP::RAB-8 was increased in rabi-8(tm2518) and reduced in smgl-1(RNAi); rabi-8(tm2518) 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). 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. (O) The expressional level of GFP::RAB-8 remained intact in rabi-8(tm2518) mutants. (P) The membrane-to-cytosol ratio of GFP::RAB-8 increased in rabi-8(tm2518) mutants. Actin served as the loading control. Levels of RAB-8 were normalized to the corresponding controls and set to one in the supernatant fraction. (Q) The membrane-to-cytosol ratio of GFP::RAB-8 decreased in smgl-1(RNAi); rabi-8(tm2518) animals. Actin served as the loading control. Levels of RAB-8 were normalized to the corresponding controls and set to one in the supernatant fraction. Source data are available for this figure: SourceData FS2.