Comparative proximity biotinylation implicates RAB18 in cholesterol mobilization and biosynthesis

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. A restricted set of 25 RAB18-interactions were regulated by the binary RAB3GAP1-RAB3GAP2 RAB18-guanine nucleotide exchange factor (GEF) complex. These included three groups of functionally interrelated proteins: 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 provide confirmatory evidence for several of the interactors (SPG20/SPART, SEC22A, TMCO4). Further we provide functional evidence that RAB18 links the Δ8-Δ7 sterol isomerase emopamil binding protein (EBP) to a molecular machinery mobilizing the products of EBP-catalysis. The cholesterol precursor lathosterol accumulates in RAB18-null cells, and de novo cholesterol biosynthesis is reduced. Our data demonstrate that GEF-dependent Rab-interactions are highly amenable to interrogation by proximity biotinylation and suggest that Micro syndrome is a cholesterol biosynthesis disorder. SUMMARY STATEMENT We used proximity biotinylation together with guanine nucleotide exchange factor (GEF)-null cell lines to discriminate functional RAB18-interactions. This approach revealed that RAB18 mediates lathosterol mobilization and cholesterol biosynthesis.