PROTEASOME ACTIVATION BY REG MOLECULES LACKING HOMOLOG-SPECIFIC INSERTS

The peptidase activities of eukaryotic proteasomes are markedly activa ted by the 11 S REG or PA28, The three identified REG subunits, design ated alpha, beta, and gamma, differ significantly in sequence over a s hort span of 15-30 amino acids that we call homolog-specific inserts. These inserts were deleted from each REG to produce the mutant protein s REG alpha Delta i, REG beta Delta i, and REG gamma Delta i. The puri fied recombinant proteins were then tested for their ability to oligom erize and activate the proteasome, Both REG alpha Delta i and REG gamm a Delta i formed apparent heptamers and activated human red cell prote asomes to the same extent as their full-length counterparts. By contra st, REG beta Delta i exhibited, at low protein concentrations, reduced proteasome activation when compared with the wild-type REG beta prote in. REG beta Delta i was able to form hetero-oligomers with a single s ite, monomeric REG alpha mutant and with REG alpha Delta i. At low con centrations, the REG alpha Delta i/REG beta Delta i hetero-oligomers s timulated the proteasome less than REG alpha/REG beta oligomers formed from wild-type subunits, and the reduced activation by REG alpha Delt a i/REG beta Delta i was due to removal of the REG beta insert, not th e REG alpha insert. These studies demonstrate that the REG alpha and R EG gamma inserts play virtually no role in oligomerization or in prote asome activation. By contrast, removal of REG beta insert reduces bind ing of this subunit and REG alpha/REG beta oligomers to proteasomes. O n the whole, however, our findings show that BEG inserts are not requi red for binding and activating the proteasome, We speculate that they serve to localize REG-proteasome complexes within cells, possibly by b inding components in endoplasmic reticulum membranes.