The Autophagic Adaptor p62 Binds to Destabilizing N-terminal Residues of the N-end Rule Pathway.

Abstract

The N-end rule pathway is a proteolytic system in which a destabilizing N-terminal residue {type 1 (Arg, Lys, His) and type 2 (Phe, Trp, Tyr, Lue, Ile) in mammals} acts as a degradation signal (N-degron). Known functions of the pathway include the regulated proteolysis of short-lived proteins through the ubiquitin-proteasome system. So far, known N-recognins include UBR1, UBR2, UBR4 and UBR5, which possess an evolutionally conserved 72-residue UBR box that is required for recognizing N-degrons. We used synthetic peptides carrying N-terminal destabilizing residues to purify recognition components (N-recognins) of the pathway. Our proteomic screen for N-degron binding proteins identified p62/SQSTM1 known to mediate autophagic degradation of aberrant proteins as a new recognition component of the N-end rule pathway. In this study, we characterized the binding specificity and other biochemical properties of p62 as a potential N-recognin of the N-end rule pathway. Pull-down assays with synthetic N-end rule peptides show that its ZZ domain whose function had remained unclear is responsible for the binding to N-end rule N-termini. p62 binds N-terminal Arg and other type-1 peptides (Lys and His) as well as a subset of type-2 peptides (Phe, Trp and Tyr) but not Leu and Ile. Site-directed mutagenesis revealed specific residues critical for the interaction with N-end rule peptides and conserved in p62 and known N-recognins. And the surface plasmon resonance biacore assay again demonstrated that the interaction between p62 and destabilizing N-end rule N-termini. Our results suggest that p62 may be a new recognition component of the N-end rule pathway and hints that to be an effective autophagy inducer, a ligand should have high affinity to the ZZ domain.