Abstract

Significance Proximity-dependent biotinylation (BioID) is a readily accessible method for identifying protein associations that occur in living cells. Fusion of a promiscuous biotin ligase to a bait protein for expression in live cells enables covalent biotin labeling, and thus identification, of proteins proximate to the bait. Here we used BioID to probe the organization of the nuclear pore complex, a large structure that regulates molecular transport between the nucleus and cytoplasm. These studies enhance our understanding of major subcomplexes within the nuclear pore complex and demonstrate the utility of BioID for studying the organization of large protein assemblies. Additionally, we have measured the labeling radius of BioID, thus enabling the rational application of this method and more meaningful data interpretation.