The N-terminal cysteine is a dual sensor of oxygen and oxidative stress.

Cellular homeostasis requires the sensing of and adaptation to intracellular oxygen (O 2 ) and reactive oxygen species (ROS). The Arg/N-degron pathway targets proteins that bear destabilizing N-terminal residues for degradation by the proteasome or via autophagy. Under normoxic conditions, the N-terminal Cys (Nt-Cys) residues of specific substrates can be oxidized by dioxygenases such as plant cysteine oxidases and cysteamine (2-aminoethanethiol) dioxygenases and arginylated by ATE1 R-transferases to generate Arg-CysO 2 (H) (R-C O2 ). Proteins bearing the R-C O2 N-degron are targeted via Lys48 (K48)-linked ubiquitylation by UBR1/UBR2 N-recognins for proteasomal degradation. During acute hypoxia, such proteins are partially stabilized, owing to decreased Nt-Cys oxidation. Here, we show that if hypoxia is prolonged, the Nt-Cys of regulatory proteins can be chemically oxidized by ROS to generate Arg-CysO 3 (H) (R-C O3 ), a lysosomal N-degron. The resulting R-C O3 is bound by KCMF1, a N-recognin that induces K63-linked ubiquitylation, followed by K27-linked ubiquitylation by the noncanonical N-recognin UBR4. Autophagic targeting of Cys/N-degron substrates is mediated by the autophagic N-recognin p62/SQTSM-1/Sequestosome-1 through recognition of K27/K63-linked ubiquitin (Ub) chains. This Cys/N-degron-dependent reprogramming in the proteolytic flux is important for cellular homeostasis under both chronic hypoxia and oxidative stress. A small-compound ligand of p62 is cytoprotective under oxidative stress through its ability to accelerate proteolytic flux of K27/K63-ubiquitylated Cys/N-degron substrates. Our results suggest that the Nt-Cys of conditional Cys/N-degron substrates acts as an acceptor of O 2 to maintain both O 2 and ROS homeostasis and modulates half-lives of substrates through either the proteasome or lysosome by reprogramming of their Ub codes.