Reconstitution of PA700, the 19S regulatory particle, from purified precursor complexes

Here, we describe methodology for the in vitro reconstitution of PA700, the 19S regulatory particle of the 26S proteasome, from three purified subcomplexes that closely represent cellular assembly intermediates. These PA700 subcomplexes (denoted PS-1, PS-2, and PS-3) account for all subunits present in purified PA700 but have no overlapping or non-PA700 components. The reconstituted PA700 displays functional features indistinguishable from independently purified PA700, including ATPase activity, deubiquitylating activity, and ATP-dependent binding and activation of the 20S proteasome. This reconstitution assay -provides a platform for exploration of critical biochemical and molecular features of PA700 assembly and for insights to 26S proteasome assembly in intact cells.