Rab1A and Rab1B MitoID ident ifies novel Rab1 effectors. (A) Schematic of the Rab1-MitoID approach in which Rab1 is fused to the promiscuous biotin ligase BirA* and a mitochondrial localization signal from monoamine oxidase (depicted in red), resulting in the relocalization of Rab1 to the mitochondrial membrane. Rab1 interactors will be efficiently biotinylated, while other Golgi proteins and Rab interactors are not. (B) Two-dimensional plot comparing the enrichment of proteins biotinylated by Rab1A-MitoID vs control (BirA alone), plotted against the enrichment of proteins biotinylated with GTP-locked Rab1A (QL) vs GDP-locked Rab1A (SN). For the x axis, the value plotted is that for the nucleotide form of Rab1 that gave the greatest fold change over background. For clarity, Rab1A itself is not shown, as it is part of the BirA* construct. Known effectors and regulators are indicated, with known effectors being enriched in the upper right quadrant. Zoomed regions to the right show a region demarcated by well-enriched known effectors with novel proteins of note identified. For all protein identities and enrichment values see Table S1. (C) as for (B) except with Rab1B rather than Rab1A. (D) Overrepresented GO terms for biological process of the proteins within the regions demarcated by known effectors shown in B and C with the known effectors removed. Ranking is by the summed FDR and enrichment rank. (E) Immunoblots of binding to GST-Rab1A–coated beads of the indicated proteins from HEK293A cell lysates. Rab1A was either Q70L (GTP) or S25N (GDP). The known Rab1 effector RABEP1 (rabaptin-5) is included as positive control (Valsdottir et al., 2001). Source data are available for this figure: SourceData F1.