Identifying the binding partners of a highly specific centrosome-targeting domain

Centrosomes are the main microtubule-organizing centers in mammalian cells, playing important roles in cell division, ciliogenesis, and a variety of other cellular processes. PACT (pericentrin-AKAP-450 centrosomal targeting) domain is a conserved centrosomal targeting domain present in pericentrin and AKAP450 – coiled-coil proteins of the pericentriolar material that recruit structural and regulatory proteins to the centrosome. Because the PACT domain is sufficient to target AKAP450 and pericentrin to the centrosome, it has been hypothesized that it might provide a critical link between the core of the centrosome and the components of the pericentriolar material. Thus, we can gain valuable insights into the organization and function of the centrosome by identifying the centrosomal proteins that interact with the PACT domain. However, due to the insolubility of these proteins and the difficulty in purifying centrosomes in their native state, it has proven difficult to study protein-protein interactions using conventional biochemical techniques. To overcome this problem we apply the BioID (proximity-dependent biotin identification) technique. Specifically we use it to screen for the proteins that associate with and/or are proximate to the PACT domain in the centrosome. Herein we describe our progress in narrowing down the list of candidate proteins derived from the BioID method to the proteins that have been previously identified in proteomic studies of the centrosome. We also describe our progress in characterizing these proteins’ possible function in the formation of centrosome structure.