Probing the ubiquitin/proteasome system with ornithine decarboxylase, a ubiquitin-independent substrate

Ornithine decarboxylase (ODC) is an unusual proteasome substrate-ubiquitin conjugation plays no part in its turnover. It can therefore be used as a probe to distinguish proteasome-mediated actions that do or do not depend on the activity of the ubiquitin system. A 37 residue region of ODC suffices for proteasome interactions, and within this sequence functionally critical residues have been identified. Because no posttranslational modifications are required for substrate preparation, ODC and derived constructs can be readily generated as substrates for either in vitro or in vivo studies. This chapter describes methodologies that allow the use of ODC as a reporter to examine the ubiquitin-proteasome system, both in reconstituted in vitro systems and in living cells.