Degradation signals in the lysine-asparagine sequence space.
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.
Suzuki
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T
Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA.
Varshavsky
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A
D000595:Amino Acid Sequence; D001216:Asparagine / Q000235:genetics*; D005656:Fungal Proteins / Q000378:metabolism; D006388:Hemagglutinins / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008025:Ligases; D008239:Lysine / Q000235:genetics*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D044767:Ubiquitin-Protein Ligases; D001616:beta-Galactosidase / Q000378:metabolism
D005656:Fungal Proteins; D006388:Hemagglutinins; D011506:Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D001216:Asparagine; D013762:Tetrahydrofolate Dehydrogenase; C066066:UBR1 protein, S cerevisiae; D044767:Ubiquitin-Protein Ligases; D001616:beta-Galactosidase; D008025:Ligases; D008239:Lysine