Identification of a Ubiquitin-Independent Degron by a Reporter Assay.

Ubiquitin-independent degradation (UID) by the 20S proteasome represents a key pathway for mainly regulating intrinsically disordered proteins (IDPs), yet the degron motifs that govern this process remain poorly defined. In this chapter, we describe experimental strategies to identify UID degrons that mediate degradation by the catalytic 20S proteasome particle. Combining high-throughput 20S substrate assays in vitro with analyses of PSMA3 C-terminal binding proteins, we identified numerous IDPs, including p21, as potential substrates. Using bimolecular fluorescence complementation (BiFC) and split-luciferase reporter assays, we mapped the degron within p21. Systematic mutagenesis and peptide- and fragment-based assays delineated the minimal degron sequence, while CRISPR-Cas9-mediated editing of the endogenous protein confirmed its physiological role in proteasomal turnover. This integrated workflow-spanning degron mapping to functional validation-provides a generalizable framework for uncovering ubiquitin-independent degrons.