A smaller promiscuous biotin ligase for proximity biotinylation called BioID2 enables more-selective targeting of fusion proteins, requires less biotin supplementation, exhibits enhanced labeling of proximate proteins, and demonstrates the use of a flexible linker to modulate the biotin-labeling radius.

Summary The nuclear envelope (NE) is a critical cellular structure whose constituents and roles in a myriad of cellular processes seem ever expanding. To determine the underlying mechanisms by which these NE constituents participate in various cellular events, it is necessary to understand the nature of their protein-protein associations. BioID (proximity-dependent biotin identification) is a recently established method to generate a history of protein-protein association as they occur over time in living cells. BioID is based on fusion of a bait protein to a promiscuous biotin ligase. Expression of the BioID-fusion protein in a relevant cellular environment enables biotinylation of vicinal and interacting proteins of the bait protein, permitting isolation and identification by conventional biotin-affinity capture and mass-spec analysis. In this way, BioID provides unique capabilities to identify protein-protein associations at the NE. In this chapter we provide a detailed protocol for the application of BioID to the study of NE proteins.