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 subunits, alpha and beta. In reconstitution experiments, alpha-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 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, heteromultimers formed, concomitant with a marked increase in stimulatory activity when compared with that of alpha-homooligomers. The reconstituted PA28alpha,beta protein is indistinguishable from purified PA28 by several criteria: it displays the same molecular mass, shows the same abundance of alpha- and beta-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 alpha- and beta-subunits in fixed stoichiometry, most likely as an alpha3beta3-heterohexamer.
Kuehn
L
L
Biochemische Abteilung, Diabetes-Forschungsinstitut, Düsseldorf, Germany.
Dahlmann
B
B
D015153:Blotting, Western; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D005260:Female; D006801:Humans; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism*; D009124:Muscle Proteins; D010920:Placenta / Q000737:chemistry; D011247:Pregnancy; D046988:Proteasome Endopeptidase Complex; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology*
D009097:Multienzyme Complexes; D009124:Muscle Proteins; C085047:PSME1 protein, human; D011506:Proteins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex