Probing mammalian centrosome structure using BioID proximity-dependent biotinylation

Abstract

Understanding the structure and function of the centrosome will require identification of its constituent components and a detailed characterization of the interactions among these components. Here, we describe the application of proximity-dependent biotin identification (BioID) to identify spatial and temporal relationships among centrosome proteins. The BioID method relies on protein fusions to a promiscuous mutant of the Escherichia coli biotin ligase BirA, which biotinylates proteins that are in a similar to 10 nm labeling radius of the enzyme. The biotinylated proteins are captured by affinity and are identified by mass spectrometry. Proteins identified in this way are referred to as "proximity interactors." Application of BioID to a set of centrosome proteins demonstrated the utility of this approach in overcoming inherent limitations in probing centrosome structure. These studies also demonstrated the potential of BioID for building large-scale proximity interaction maps among centrosome proteins. In this chapter, we describe the work flow for identification of proximity interactions of centrosome proteins, including materials and methods for the generation and characterization of a BirA*-fusion protein expression plasmid, expression of BirA*-fusion proteins in cells, and purification and identification of proximity partners by mass spectrometry.