ABSTRACT
Loss of functional RAB18 causes the autosomal recessive condition Warburg Micro syndrome. To better understand this disease, we used proximity biotinylation in HEK293 and HeLa cells to generate an inventory of potential RAB18 effectors. In HeLa cells, we expressed BirA*-RAB18 in cells in which RAB18-guanine nucleotide exchange factor (GEF) activity was disrupted with CRISPR. We found that most RAB18-interactions are regulated independently by its different GEFs; the binary RAB3GAP1-RAB3GAP2 complex and the TRAPPC9-containing TRAPPII complex. RAB3GAP-dependent RAB18 interactions included a group of microtubule-interacting/membrane shaping proteins, a group of proteins involved in membrane tethering and docking, and a group of lipid-modifying/lipid transport proteins. We demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation. Further, we provide confirmatory evidence for several of the interactors (SPG20/SPART, SEC22A and TMCO4) as well as functional evidence supporting a role for RAB18 in modulating the close apposition of membranes and in cholesterol mobilization.
SUMMARY STATEMENT We used proximity biotinylation together with guanine nucleotide exchange factor (GEF)-null cell lines to discriminate functional RAB18-interactions. We anticipate that this approach will be broadly applicable in small GTPase research.