Purification of PA700, the 19S regulatory complex of the 26S proteasome

The 26S proteasome is a 2,400,000-Da protease complex that selectively degrades proteins modified by polyubiquitin chains. The 26S proteasome is composed of two 700,000-Da multisubunit complexes: the 20S proteasome, which serves as the proteolytic core of the complex, and PA700, an ATPase regulatory complex responsible for the binding, modification, and delivery of substrates to the proteolytic chamber. Thus, PA700 mediates multiple functions essential for ubiquitin-dependent proteolysis by the 26S proteasome. This chapter reviews briefly the structure and function of PA700, details the methodology for its large-scale purification from mammalian tissues, and describes a simple functional PA700 assay based on the stimulation of proteasome activity.