Characterization of the E3 ligase KCMF1 as a ZZ/N-recognin of the autophagic Arg/N-degron pathway.

In the Arg/N-degron pathway, Arg/N-degrons share the N-terminal (Nt) arginine (Nt-Arg) residue and hydrophobic amino acid residues that can be generated through Nt-arginylation by ATE1-encoded R-transferases (EC 2.3.2). In the ubiquitin-proteasome system (UPS), N-degrons are recognized by the UBR box of N-recognins that facilitate ubiquitination and proteasomal degradation. Arg/N-degrons also modulate the lysosomal degradation of proteins and other biomaterials via the autophagy-lysosome system (ALS). In this autophagic process, N-degrons function through their recognition by the ZZ-type zinc finger domain of the N-recognin p62/SQSTM1-1/Sequestosome-1. Recently, we identified the E3 ligase KCMF1 (potassium channel modulatory factor 1) as an autophagic N-recognin at the crossroads of the UPS and ALS. KCMF1 binds Nt-Arg and structurally related Nt-motifs through its ZZ domain, a structural equivalent to the ZZ domain of p62 as well as the UBR box of N-recognins. Under oxidative stress such as prolonged hypoxia where protein aggregates accumulate, the cysteine (Cys) residue at position 2 is Nt-exposed through the Nt-methionine (Nt-Met) excision and undergoes chemical oxidation into Cys sulfonic acid (CysO₃) followed by Nt-arginylation. The resulting Arg-CysO₃ N-degron binds KCMF1 to induce the assembly of lysine 63 (Lys63)-linked Ub chains, to which p62-type autophagic receptors bind via their Ub-associated (UBA) domain for autophagic degradation. Through this collaboration between the UPS and ALS, Arg-CysO₃ N-degrons contribute the degradation of harmful protein species generated under cellular stresses. Here, we describe biochemical assays to characterize KCMF1 as an emerging N-recognin, including its interaction with synthetic N-degrons and its activity to undergo self-polymerization stimulated by N-degrons.