The proteasome activator 11 S regulator or PA28: Contribution by both α and β subunits to proteasome activation

The proteasome 11 S regulator (REG) consists of two homologous subunits, REGα and REGβ. Each subunit is capable of activating the proteasome, and when combined, they form superactive REGα/REGβ complexes. We have previously shown that a highly conserved loop in the REGα crystal structure is critical for proteasome activation. We now show that hetero-oligomers formed from REGα activation loop mutants and wild-type REGβ or vice versa are partially active. By contrast, heterooligomers bearing mutations in the activation loops of REGα and REGβ subunits are inactive, demonstrating that both α and β subunits contribute to proteasome activation. We have also characterized REG proteins with mutations near or at their C termini. Partially active REGα(Y249C) and REGα(M247V) and an inactive REGα subunit hearing five additional C-terminal amino acids formed active hetero-oligomers with REGβ. REGβ subunits lacking 1, 2, or 9 C-terminal amino acids did not bind or activate the proteasome, but each of these mutants formed partially active hetero-oligomers with the monomer REGα(N50Y). However, hetero- oligomers formed from REG subunits lacking the last 14 amino acids were unable to bind the proteasome. Thus, C-terminal regions of both α and β subunits are required for hetero-oligomers to bind the proteasome.