Identification of the Ac/N-degron recognition domain in the MARCHF6 E3 ubiquitin ligase

Nα-terminal (Nt-) acetylation is a prevalent post-translational modification that regulates protein stability and turnover. The Ac/N-degron pathway, a branch of the N-degron pathways, recognizes Nt-acetyl groups as degradation signals (Ac/N-degrons), mediating proteolysis. MARCHF6, an endoplasmic reticulum (ER)-transmembrane E3 ubiquitin ligase, acts as a principal Ac/N-recognin, targeting Ac/N-degron-bearing substrates for polyubiquitylation and subsequent proteasomal degradation. However, the molecular mechanisms underlying Ac/N-degron recognition by MARCHF6 remain elusive. Here, we utilized a comprehensive alanine-stretch mutational screen combined with split-ubiquitin (Split-Ub) assays to define the Ac/N-degron recognition domain (Ac/N-domain) within MARCHF6. Sequence alignment with its yeast ortholog, Doa10, revealed conserved cytosolic residues essential for substrate recognition. Biochemical approaches, including chemical crosslinking and co-immunoprecipitation, identified key residues critical for Ac/N-degron recognition, while truncation and Split-Ub assays delineated the specific Ac/N-domain necessary for binding. These findings establish a mechanistic framework for Ac/N-degron recognition by MARCHF6, deepening our understanding of Nt-acetylation-mediated proteostasis and its therapeutical implications for diseases linked to dysregulation of Nt-acetylation or MARCHF6, including cancer, birth defects, and metabolic and neurological disorders.