Purification and assay of proteasome activator PA200

PA200, the most recently discovered activator of the 20S proteasome, is a nuclear protein thought to play a role in DNA repair. Homologs of PA200 have been found in rat, frog, birds, worms, and budding yeast, where it is called Blm3p (now known as Blm10p), but not in Drosophila or fission yeast. Western blots of SDS-PAGE transfers reveal 160 and 200K forms of mammalian PA200, and organ surveys demonstrate that the 200K species is highest in testis. PA200 purified from bovine testis binds the ends of the cylindrical 20S proteasome, forming volcano-shaped structures in negatively stained EM images. In vitro assays demonstrate that binding of PA200 activates peptide hydrolysis by the 20S proteasome. This chapter describes the purification and assay of bovine testis PA200.