Use of the LC3B-fusion technique for biochemical and structural studies of proteins involved in the N-degron pathway

The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the wellestablished in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein1A/1Blight chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield ofhomogeneoustarget proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes.Tovalidate our results,wedetermined the structures of select proteins in the N-degron pathway and compared them with the Protein Data Bank-deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.