Identification of MARCH2 E3 ligase-substrates using proximity-dependent biotin labeling method

MARCH2 is known to be involved in intracellular vesicular trafficking. To identify its substrates, we have recently developed a method based on proximity-dependent biotin labelling. In this protocol, the MARCH2 ubiquitin ligase of interest is expressed as a fusion to Escherichia coli biotin ligase BirA together with a biotin acceptor peptide(AP)-tagged ubiquitin. The BirA-directed biotin labeling of AP depends on the proximity of the two fusion proteins in the cell, which leads to preferential labeling of ubiquitinated E3 substrates. In this study, we applied this procedure to MARCH2 and identified its substrates.