Exocytic carrier sequestration relies on EHBP-1’s binding to RAB-10 and PI(4,5)P2. (A) The interaction between EHBP-1 and RAB-10(Q68L) predicted by AlphaFold2-Multimer. Red sticks indicate contacts between EHBP-1 and RAB-10(Q68L) within 4.0 Å. (B) Cartoon image showing domain architectures of Mito-EHBP-1^6AALAA, Mito-ΔC2, Mito-ΔCC^6AALAA, Mito-ΔCH^6AALAA, and Mito-αCH2^6AALAA. (C and C′) Confocal images showing colocalization between VIT-2-mNeonGreen-3xFlag^KI and mitochondria. The statistical analysis was calculated as Pearson’s correlation coefficient and shown as mean ± SD (n = 18 cells from six animals of each genotype; one-way ANOVA test with Dunn’s multiple comparison). (D) Cartoon image showing domain architecture of EHBP-1 truncation and domain substitution variants. (E and E′) Confocal images showing the distribution of VIT-2-mNeonGreen-3xFlag^KI in the C. elegans intestine, oocytes, and embryos in different gene knockdown backgrounds. Statistical data are shown as box-and-whisker plots with 10th–90th percentile (n = 24 cells from eight animals of each genotype; dots, outliers; boundaries, quartiles; one-way ANOVA test with Dunn’s multiple comparison).