RhoG facilitates CG docking through interaction with Munc13-4 and cooperative binding to plasma membrane lipids. (A) Affinity purification of STREP-HA-tagged RhoG protein complexes identified novel interacting protein partners of RhoG, including Munc13-4. (B) Coimmunoprecipitation of endogenous RhoG with Munc13-4 from a lysate of unstimulated or phorbol 12-myristate 13-acetate + ionomycin (PMA/I)-stimulated Jurkat cells. (C) Quantification of colocalization of RhoG and Munc13-4 in Jurkat cells (representative microscopy images are shown in supplemental Figure 4F). (D) A proposed model of RhoG-mediated exocytosis specific for human lymphocytes. (E) RHOG and UNC13D constructs used in reconstitution experiments and lipid-binding assay. (F) Graph demonstrating reconstitution of degranulation in RHOG KO NK-92 cells with constructs encoding WT and truncated (Δ182) RhoG, WT, and truncated (ΔC1) Munc13-1. (G) Scheme of PIP-strip membrane depicting spots of lipid species. (H) PIP-strip assay with recombinant WT RhoG protein alone or in a mixture with purified Munc13-4, showing binding pattern to the lipids indicated in panel G. (I) Quantification of PIP-strip assay results, showing a lipid-binding pattern of recombinant proteins.