Cell-free assay for ubiquitin-independent proteasomal protein degradation.

The ATP-dependent degradation of ornithine decarboxylase is an exceptional case whereby a protein is targeted to the 26S proteasome independently of ubiquitin conjugation. Rather, prior association with the polyamine-induced regulatory protein, antizyme, confers susceptibility of ornithine decarboxylase to proteasomal degradation. In this chapter we describe ornithine decarboxylase/antizyme-based in vivo and in vitro systems for the measurement of ATP-dependent, ubiquitin-independent proteasomal degradation, as well as the application of ornithine decarboxylase as a reporter for the targeting of proteins to the 26S proteasome.