Identification of TbUnc119-interacting proteins by proximity-based biotinylation

Both intraflagellar transport (IFT) and lipidated intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei. TbArl13 catalyses two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 was identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119 using an improved version of biotin ligase BioID2. The BioID2 tag was fused to either the N-terminus (3HA-BioID2-TbUnc119) or the C-terminus (TbUnc119-BioID2-HA) of TbUnc119 and expressed using a cumate-inducible expression system in the procyclic cells of T. brucei. We performed LC-MS/MS on the affinity-purified biotinylated samples followed by evaluation of emPAI score.