Proteasomes begin ornithine decarboxylase digestion at the C terminus.
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.
Zhang
Mingsheng
M
Department of Microbiology and Immunology, University of California-San Francisco, 513 Parnassus Avenue, San Francisco, CA 94143, USA.
MacDonald
Alasdair I
AI
Hoyt
Martin A
MA
Coffino
Philip
P
D000818:Animals; D003513:Cycloheximide / Q000494:pharmacology; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005822:Genetic Vectors; D008727:Methotrexate / Q000494:pharmacology; D051379:Mice; D009097:Multienzyme Complexes / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009955:Ornithine Decarboxylase / Q000737:chemistry* / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010957:Plasmids; D016133:Polymerase Chain Reaction; D046988:Proteasome Endopeptidase Complex; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D013379:Substrate Specificity
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D003513:Cycloheximide; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D009955:Ornithine Decarboxylase; D008727:Methotrexate