Identification of an activation region in the proteasome activator REGα

Proteasomes can be markedly activated by associating with 19S regulatory complexes to form the 26S protease or by binding 11S protein complexes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coli, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in vitro activity assay, we have isolated and characterized 36 inactive, single-site mutants of recombinant REG alpha. Most are monomers that produce functional proteasome activators when mixed with REG beta subunits. Five REG alpha mutants that remain inactive in the mixing assay contain amino acid substitutions clustered between Arg-141 and Gly-149. The crystal structure of the REG alpha heptamer shows that this region forms a loop at the base of each REG alpha subunit. One mutation in this loop (N146Y) yields a REG alpha heptamer that binds the proteasome as tightly as wild-type REG alpha but does not activate peptide hydrolysis. Corresponding amino acid substitutions in REG beta (N135Y) and REG gamma (N151Y) produce inactive proteins that also bind the proteasome and inhibit proteasome activation by their normal counterparts. Our studies clearly demonstrate that REG binding to the proteasome can be separated from activation of the enzyme. Moreover, the dominant negative REGs identified here should prove valuable for elucidating the role(s) of these proteins in antigen presentation.