A proximity ligation screen identifies SNAT2 as a novel target of the MARCH1 E3 ubiquitin ligase.

E3 ubiquitin ligases include hundreds of members that can regulate the half-life of other proteins but also modulate their cellular localization and functions. While MARCH1 appears to target principally immune cell components, such as MHC class II molecules and the co-stimulatory molecule CD86, the repertory of its targets remains to be fully documented. Here, we adapted a proximity-dependent biotin identification (BioID)-based screening approach in live HEK293 cells. We transfected a fusion protein consisting of mouse MARCH1 linked to YFP at its N-terminus and to the biotin ligase of Aquifex aeolicus at its C-terminus. Upon transient overexpression in the presence of biotin, we could recover biotinylated proteins that are presumably found within 10 nm of MARCH1. CD98 and CD71, two previously described targets of MARCH1, were identified. Of 16 other biotinylated proteins identified by semi-quantitative mass spectrometry, ten were tested directly by flow cytometry to monitor their expression in the presence or absence of transfected MARCH1. SNAT2 was particularly sensitive to the presence of MARCH1 and was found to be ubiquitinated on western blots. Thus, BioID2 is an effective mean of characterizing the interactome of MARCH1, and the identification of SNAT2 suggests a role of this ubiquitin ligase in cellular metabolism.