Rab GTPases act in defined locations throughout the endolysosomal system by recruiting trafficking effectors to guide cargo transport. In turn, accurate sorting of cargo to the plasma membrane dictates how a cell interacts with its environment through behaviours such as migration. Understanding the crosstalk between trafficking and cell behaviour requires probing the complex network of Rabs and their effectors. In this Tools and Resources paper (Wilson et al., 2023), Patrick Caswell and colleagues use BioID proximity labelling to define the proximal interactomes for Rab4, Rab11 and Rab25, which have known roles in invasive migration in cancer cells. Although proximity labelling does not necessarily indicate a physical interaction, the authors here use a sensitive analysis of mass spectrometry data to infer direct interactions with Rab4, Rab11 or Rab25 by quantifying the frequency of biotin tagging in specific peptides, identifying potentially novel interacting domains. To validate the functional significance of these interactions, they employ a knock-sideways system and demonstrate that Rab4, Rab11 or Rab25 artificially targeted to mitochondria can specifically recruit endogenous candidate effectors such as sorting nexins. Finally, they show that knockdown of several candidates impacts migration behaviour in ovarian and breast cancer cells that require Rab4, Rab11 or Rab25 for motility, confirming the robust utility of proximity proteomic data in extracting mechanistically relevant interactions from within the intricate network of trafficking machinery.
