Synthetic signals for ubiquitin-dependent proteolysis.
Short-lived proteins are targeted for turnover by sequence elements known as degradation signals. Because of the large size and heterogeneity of these signals, the structural features important for their function are not well defined. In this study, we have isolated three classes of degradation signals by screening short artificial sequences for the ability to destabilize a reporter protein. Class I and class II signals were derived by inserting random nonapeptide sequences after the second residue of beta-galactosidase. Class III signals contained five-residue homopolymers at the same position. Class I beta-galactosidase turnover was inhibited in mutants lacking either the ubiquitin-conjugating enzyme Ubc2 or the ubiquitin protein ligase Ubr1. Class I random inserts functioned to promote N-terminal proteolytic processing and define a novel pathway for exposure of residues that are destabilizing according to the N-end rule. Efficient degradation of proteins containing class II signals required at least three Ubc enzymes: Ubc6, Ubc7, and either one of the related enzymes Ubc4 and Ubc5. Analysis of 56 amino acid substitutions in the class II signal suggested that it is recognized in the form of an amphipathic alpha helix. Class III signals consisted of short tracts of hydrophobic residues such as Leu and Ile. Degradation of class III proteins involved the Ubc4 and Ubc5 enzymes but not Ubc2, Ubc6, or Ubc7. Clusters of hydrophobic residues appear to be critical for the recognition of both class II and class III signals.
Sadis
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S
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Atienza
C
C
Finley
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D015723:Gene Library; D017930:Genes, Reporter; D006868:Hydrolysis; D008025:Ligases / Q000145:classification / Q000378:metabolism*; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000145:classification / Q000235:genetics / Q000378:metabolism*; D015202:Protein Engineering; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013329:Structure-Activity Relationship; D014452:Ubiquitins / Q000378:metabolism*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D001616:beta-Galactosidase; D008025:Ligases