Expression, localization, and activity of Rab1 MitoID constructs. (A) Confocal images of HEK293A cells expressing the indicated Rab1 MitoID constructs, in addition to the BirA control construct. Cells were stained for the HA tag in the MitoID constructs and the mitochondrial protein Tom20. Both Rab1A and Rab1B contain mutations that lock them in a either a GTP- or GDP-bound form (QL or SN, respectively). (B) Representative immunoblot showing comparable expression levels of all MitoID constructs used in the proteomics study. (C) Volcano plots comparing the abundances of biotinylated proteins obtained with MitoID of GTP-locked Rab1A or Rab1B (Rab1A Q70L; Rab1B Q67L) vs BirA alone negative control. Rab1 itself is not shown, and known effectors and regulators are indicated. (D) Structure of the Rab1A:GTP:Mg²⁺:PPP1R37 complex as predicted by AlphaFold 3, with accompanying PAE plot. Source data are available for this figure: SourceData FS1.