Proteasome activator PA28 and its interaction with 20 S proteasomes

An activator of the 20 S proteasome has been purified to apparent homogeneity from rabbit erythrocytes, liver, and skeletal muscle. The activator displays an M(r) of about 200,000 upon sizing chromatography and, as judged by gel electrophoresis under denaturing conditions, is composed of two species of subunit of about equal abundance and with M(r) of 31 and 29 kDa. Upon isoelectric focusing, the activator is resolved into two major bands with pI values in the range of pH 5.1 and 5.5, corresponding to the two subunits. Limited proteolytic cleavage with trypsin results, for each subunit, in a distinct fragmentation pattern, indicating that in the rabbit, the native activator molecule occurs either as two homomultimers or as heteromultimers. The activator shows no hydrolytic activity by itself. However, when combined with proteasomes, it enhances, in a dose-related manner, the distinct peptidase activities of the proteinase. The activation process requires binding of the activator protein to the proteinase. This association, however, is reversible with recovery of active proteinase and activator protein. In vitro experiments suggest that, in vivo, the activator is bound to 20 S proteasomes rather than occurring as the free molecule.