The use of BioID to identify new substrates of the E3 ubiquitin ligase MARCH6

Scott NA and Brown AJ

School of Biotechnology and Biomolecular Sciences, UNSW Sydney, NSW 2052, Australia..

BioID is a new method used to detect interacting proteins within cells. This technique works by fusing a promiscuous biotin ligase, BirA, onto the protein of interest to tag nearby proteins with biotin. Because biotinylation is a rare modification we can isolate and identify the proteins via streptavidin-based approaches. Our protein of interest is MARCH6, an E3 ubiquitin ligase that targets two key cholesterol synthesis enzymes and several others outside of this for degradation. We have used BioID to identify new candidate substrates and interacting partners for MARCH6. Insight into these interacting partners will lead to a better understanding of the function of MARCH6 and its network of interactors, including novel substrates. All E3 ubiquitin ligases interact with complex networks of proteins, however, these networks are poorly understood. As key regulators of protein stability, E3 ubiquitin ligases themselves are of interest as pharmaceutical targets in the treatment of numerous diseases.