CRL2ZER1/ZYG11B recognizes small N-terminal residues for degradation

N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.N-degron pathway plays an important role in the protein quality control and maintenance of cellular protein homeostasis. ZER1 and ZYG11B, the substrate receptors of the Cullin 2-RING E3 ubiquitin ligase (CRL2), recognize N-terminal (Nt) glycine degrons and participate in the Nt-myristoylation quality control through the Gly/N-degron pathway. Here we show that ZER1 and ZYG11B can also recognize small Nt-residues other than glycine. Specifically, ZER1 preferentially binds to Nt-serine, -alanine, -threonine and -cysteine over -glycine, while ZYG11B prefers Nt-glycine but also has the capacity to recognize Nt-serine, -alanine and -cysteine in vitro. Nt-serine, -alanine and -cysteine undergo Nt-acetylation by NatA, thereby shielding them from recognition by ZER1/ZYG11B in cells. We found that ZER1/ZYG11B is able to target the non-acetylation of small Nt-residue degrons for degradation in NatA-deficient cells, implicating its role in the Nt-acetylation quality control. Furthermore, we present the crystal structures of ZER1 and ZYG11B bound to various small Nt-residues and uncover the molecular mechanism of non-acetylated substrate recognition by ZER1 and ZYG11B.