This is SPrTAC: A New Method to Distinguish Multisubunit Complexes with Highly Similar Compositions In Vivo

Large multisubunit complexes conduct the vast majority of essential cellular processes in eukaryotes. These complexes are often constructed of dozens or even hundreds of highly similar but non‐equivalent subunits. Further, the exact subunit composition of these complexes is frequently altered to fine‐tune their biological activities. For example, changes to the subunit composition of the proteasome promote antigen processing, suppress self‐recognition by immune cells, and lead to chemotherapy resistance in some cancers. Thus, a difficult and pervasive challenge in deciphering the biology of multisubunit machines is distinguishing complexes with non‐identical but extremely similar compositions from one another in vivo . To address this important challenge, we have developed a new approach, called S plit Pr otein‐based T racking of A ssembly or C omposition (SPrTAC). This method exploits split protein complementation to produce growth‐based, or potentially, fluorescence‐based readouts for particular subunit configurations, subcomplexes, or the presence of accessory factors within a given multisubunit complex in vivo. We demonstrate the utility of SPrTAC to detect a rare proteasome isoform containing two α4 subunits and whose production is driven by several known oncogenes in humans. Further, we use this approach to show that disruption of subunit expression stoichiometry compromises normal proteasome biogenesis in vivo . This methodology can be applied to any multisubunit complex for which a basic subunit architecture is known, and can be used to monitor the composition and abundance of such complexes with minimal perturbation to cell function.