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.