Reconstitution of proteasome activator PA28 from isolated subunits: optimal activity is associated with an α,β‐heteromultimer

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.