Generation and characterization of engineered N-degrons of the N-degron pathway using the ubiquitin-reference...

In the N-degron pathway, the degradation of proteins and other cellular constituents is governed by destabilizing N-terminal (Nt) amino acids of proteins. These degradation determinants, called N-degrons, modulate cellular degradative processes by binding to N-recognins that link their clients to the ubiquitin (Ub)-proteasome system (UPS) or the autophagy-lysosome system (ALS). Physiological N-degrons can be generated when the initiator methionine (Met, M) cotranslationally cleaved by Met amino peptidases (MetAPs), exposing the second residues to the Nt-position. Alternatively, endoproteolytic cleavages of folded proteins generate C-terminal (Ct) fragments exposing N-degrons. In either case, expressing physiological or engineered N-degron substrates is technically challenging, in part because the Nt-Met excision depends on specific second-position residues. To generate engineered N-degrons, the Ub fusion technique (UFT) employs a chimeric construct in which a Ub moiety is followed by X-substrate. Ub is cotranslationally cleaved off at Ct-Gly76 by deubiquitylases (DUBs). The resulting X-substrate can expose any of the principal amino acids listed in the genetic code possibly except for proline. Moreover, the half-lives of X-substrates can be precisely measured using the Ub reference technique (URT), in which M-reference-Ub-X-substrate is cleaved into M-reference-Ub and X-substrate. By comparing the ratio of two cleavage products, the decay of X-substrate can be quantitatively measured for various N-degrons. The URT critically contributed to identification and biochemical characterization of most N-recognins known in the Arg/N-degron pathway. Here, we describe detailed protocols for designing and using URT constructs to characterize substrate degradation, validate putative N-degrons, and assess their interactions with N-recognins in the Arg/N-degron pathway.