RECONSTITUTION OF PROTEASOME ACTIVATOR PA28 FROM ISOLATED SUBUNITS - OPTIMAL ACTIVITY IS ASSOCIATED WITH AN ALPHA,BETA-HETEROMULTIMER

PA28, a 200 kDa activator of 20S proteasomes, was purified from human placenta and was gel electrophoretically resolved into two different s ubunits, alpha and beta. In reconstitution experiments, alpha-subunits alone mere found to re-associate forming homooligomers with an M(r) o f about 200 kDa, which elicit a stimulatory effect on proteasomal pept ide-hydrolyzing activity, albeit at a moderate level, Under the same c onditions, isolated beta-subunits were neither found to associate nor did they display stimulatory activity. Significantly, when both alpha- and beta-subunits were present in the reconstitution assay, heteromul timers formed, concomitant with a marked increase in stimulatory activ ity when compared with that of alpha-homooligomers. The reconstituted PA28 alpha,beta protein is indistinguishable from purified PA28 by sev eral criteria: it displays the same molecular mass, shows the same abu ndance of alpha- and beta-subunits and has a similar stimulatory activ ity toward 20S proteasomes, These results indicate that optimal PA28 a ctivity is associated with a heteromultimeric structure which contains the alpha- and beta-subunits in fixed stoichiometry, most likely as a n alpha(3) beta(3)-heterohexamer.