IDENTIFICATION OF AN ACTIVATION REGION IN THE PROTEASOME ACTIVATOR REG-ALPHA

Proteasomes can be markedly activated by associating with 19S regulato ry complexes to form the 26S protease or by binding 11S protein comple xes known as REG or PA28. Three REG subunits, alpha, beta, and gamma, have been expressed in Escherichia coil, and each recombinant protein can activate human proteasomes. Combining PCR mutagenesis with an in v itro activity assay, we have isolated and characterized 36 inactive, s ingle-site mutants of recombinant REG alpha. Most are monomers that pr oduce functional proteasome activators when mixed with REG beta subuni ts. Five REG alpha mutants that remain inactive in the mixing assay co ntain 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 t his loop (N146Y) yields a REG alpha heptamer that binds the proteasome as tightly as wild-type REG alpha but does not activate peptide hydro lysis. Corresponding amino acid substitutions in REG beta (N135Y) and REG gamma (N151Y) produce inactive proteins that also bind the proteas ome and inhibit proteasome activation by their normal counterparts. Ou r studies clearly demonstrate that REG binding to the proteasome can b e separated from activation of the enzyme. Moreover, the dominant nega tive REGs identified here should prove valuable for elucidating the ro le(s) of these proteins in antigen presentation.