Proteasomes Begin Ornithine Decarboxylase Digestion at the C Terminus

• Published in: The Journal of biological chemistry Vol. 279; no. 20; pp. 20959 - 20965
• Main Authors: Zhang, Mingsheng, MacDonald, Alasdair I, Hoyt, Martin A, Coffino, Philip
• Format: Journal Article
• Language: English
• Published: United States American Society for Biochemistry and Molecular Biology 14-05-2004
• Subjects: Animals; Cycloheximide - pharmacology; Cysteine Endopeptidases - metabolism; Genetic Vectors; Methotrexate - pharmacology; Mice; Multienzyme Complexes - metabolism; Mutagenesis, Insertional; Ornithine Decarboxylase - chemistry; Ornithine Decarboxylase - drug effects; Ornithine Decarboxylase - genetics; Ornithine Decarboxylase - metabolism; Peptide Fragments - chemistry; Peptide Fragments - metabolism; Plasmids; Polymerase Chain Reaction; Proteasome Endopeptidase Complex; Protein Denaturation; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - metabolism; Saccharomyces cerevisiae - metabolism; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M314043200

Proteasomes denature folded protein substrates and thread them through a narrow pore that leads to the sequestered sites of
proteolysis. Whether a protein substrate initiates insertion from its N or C terminus or in a random orientation has not been
determined for any natural substrate. We used the labile enzyme ornithine decarboxylase (ODC), which is recognized by the
proteasome via a 37-residue C-terminal tag, to answer this question. Three independent approaches were used to assess orientation
as follows. 1) The 461-residue ODC protein chain was interrupted at position 305. The C-terminal fragment was degraded by
purified proteasomes, but because processivity requires continuity of the polypeptide chain, the N-terminal fragment was spared.
2) A proteasome-inhibitory viral sequence prevented degradation when introduced near the C terminus but not when inserted
elsewhere in ODC. 3) A bulky tightly folded protein obstructed in vivo degradation most effectively when positioned near the C terminus. These data demonstrate that the proteasome initiates degradation
of this native substrate at the C terminus. The co-localization of entry site and degradation tag to the ODC C terminus suggests
that recognition tags determine the site for initiating entry. Flexibility of a polypeptide terminus may promote the initiation
of degradation.