PA28, a 200 kDa activator of 20S proteasomes, was purified from human placenta and was gel electrophoretically resolved into two different subunits, α and β. In reconstitution experiments, α-subunits alone were found to re-associate forming homooligomers with an M r of about 200 kDa, which elicit a stimulatory effect on proteasomal peptide-hydrolyzing activity, albeit at a moderate level. Under the same conditions, isolated β-subunits were neither found to associate nor did they display stimulatory activity. Significantly, when both α- and β-subunits were present in the reconstitution assay, heteromultimers formed, concomitant with a marked increase in stimulatory activity when compared with that of α-homooligomers. The reconstituted PA28α,β protein is indistinguishable from purified PA28 by several criteria: it displays the same molecular mass, shows the same abundance of α- and β-subunits and has a similar stimulatory activity toward 20S proteasomes. These results indicate that optimal PA28 activity is associated with a heteromultimeric structure which contains the α- and β-subunits in fixed stoichiometry, most likely as an α 3 β 3-heterohexamer.
