The N terminus of antizyme promotes degradation of heterologous proteins.
Regulated degradation of ornithine decarboxylase (ODC) is mediated by its association with the inducible protein antizyme. The N terminus of antizyme (NAZ), although unneeded for the interaction with ODC, must be present to induce degradation. We report here that covalently grafting NAZ to ODC confers lability that normally results from the non-covalent association of native antizyme and ODC. To determine whether NAZ could act similarly as a modular functional domain when grafted to other proteins, we fused it to a region of cyclin B (amino acids 13-90) capable of undergoing degradation or to cyclin B (amino acids 13-59), which is not subject to degradation. The association with NAZ made both NAZ-cyclin B13-90 and NAZ-cyclin B13-59 unstable. Furthermore, NAZ and cyclin B 13-59 were together able to induce in vitro degradation of Trypanosoma brucei ODC, a stable protein. The ODC-antizyme complex bound to the 26 S protease but not the 20 S proteasome, consistent with the observation that ODC degradation is mediated by the 26 S protease. The association was shown to be independent of NAZ, suggesting that NAZ does not act as a recognition signal.
Li
X
X
Department of Microbiology and Immunology, University of California, San Francisco, 94143, USA.
Stebbins
B
B
Hoffman
L
L
Pratt
G
G
Rechsteiner
M
M
Coffino
P
P
D000818:Animals; D019926:Cyclin B; D016213:Cyclins / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005982:Glutathione Transferase / Q000378:metabolism; D007700:Kinetics; D009097:Multienzyme Complexes / Q000378:metabolism*; D009955:Ornithine Decarboxylase / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014346:Trypanosoma brucei brucei / Q000201:enzymology
D019926:Cyclin B; D016213:Cyclins; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011506:Proteins; D011993:Recombinant Fusion Proteins; C098520:cyclin B (13-59); C031017:ornithine decarboxylase antizyme; D005982:Glutathione Transferase; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase