Degradation signals in the lysine-asparagine sequence space

EMBO J. 1999 Nov 1;18(21):6017-26. doi: 10.1093/emboj/18.21.6017.


The N-degrons, a set of degradation signals recognized by the N-end rule pathway, comprise a protein's destabilizing N-terminal residue and an internal lysine residue. We show that the strength of an N-degron can be markedly increased, without loss of specificity, through the addition of lysine residues. A nearly exhaustive screen was carried out for N-degrons in the lysine (K)-asparagine (N) sequence space of the 14-residue peptides containing either K or N (16 384 different sequences). Of these sequences, 68 were found to function as N-degrons, and three of them were at least as active and specific as any of the previously known N-degrons. All 68 K/N-based N-degrons lacked the lysine at position 2, and all three of the strongest N-degrons contained lysines at positions 3 and 15. The results support a model of the targeting mechanism in which the binding of the E3-E2 complex to the substrate's destabilizing N-terminal residue is followed by a stochastic search for a sterically suitable lysine residue. Our strategy of screening a small library that encompasses the entire sequence space of two amino acids should be of use in many settings, including studies of protein targeting and folding.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Amino Acid Sequence
  • Asparagine / genetics*
  • Fungal Proteins / metabolism
  • Hemagglutinins / genetics
  • Hemagglutinins / metabolism
  • Kinetics
  • Ligases*
  • Lysine / genetics*
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Proteins / metabolism*
  • Recombinant Fusion Proteins / metabolism
  • Saccharomyces cerevisiae
  • Saccharomyces cerevisiae Proteins*
  • Tetrahydrofolate Dehydrogenase / genetics
  • Tetrahydrofolate Dehydrogenase / metabolism
  • Ubiquitin-Protein Ligases*
  • beta-Galactosidase / metabolism


  • Fungal Proteins
  • Hemagglutinins
  • Proteins
  • Recombinant Fusion Proteins
  • Saccharomyces cerevisiae Proteins
  • Asparagine
  • Tetrahydrofolate Dehydrogenase
  • UBR1 protein, S cerevisiae
  • Ubiquitin-Protein Ligases
  • beta-Galactosidase
  • Ligases
  • Lysine