Dept Biochemistry & Molecular Biology, University of Melbourne, Bio21 Institute, Melbourne, 3010, Victoria, Australia
Recently, techniques have been developed to biotinylate interacting and proximal proteins within a living cell. These techniques include a promiscuous biotin ligase gene BirA* (BioID) and an engineered ascorbate peroxidase (APEX2) to provide genetic tags for the protein of interest. Due to the shorter half-lives of the intermediate reactive species, APEX2 is likely to have a smaller labelling radius than BioID. The application of both labelling techniques may provide information on the molecular neighbourhood of a given membrane protein. Our lab is interested in identifying the organisation of the molecular machinery that regulates membrane trafficking to and from the trans-Golgi network (TGN). Small G proteins of the Arl/Arf family are important regulators of membrane trafficking. Effectors of small G proteins interact only transiently with the active G protein and are therefore difficult to identify using standard technology. One Arl family member, Arl5b, regulates transport between the TGN and endosomes. To identify the effectors of Arl5b in vivo both BioID and APEX2 tagging systems were used. BirA*-Arl5b fusion protein and APEX2-Arl5b were expressed in HeLa cells and localisation was analysed by confocal imaging. Both fusion proteins were located to the TGN. Addition of biotin to BirA*-Arl5b transfected cells or APEX2-Arl5b transfected cells resulted in extensive biotinylation of proteins within the region of the TGN as detected by fluorescent Streptavidin staining. Immunoblotting detected a number of biotinylated proteins from transfected cell lysates. Purified biotinylated proteins from transfected HeLa cell lysates were analysed by mass spectrometry and the identified proteins examined by Ingenuity Pathway Analysis (IPA) to reveal potentially novel interacting partners of Arl5b.