Distinct Elements in the Proteasomal β5 Subunit Propeptide Required for Autocatalytic Processing and Proteasome Assembly

Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (beta 1, beta 2, and beta 5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, beta 5 (Doa3/Pre2) has a 75-residue propeptide, beta 5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the beta 5 propeptide nonetheless causes substantial defects in proteasome maturation. Sequences closer to the cleavage site have critical but redundant roles in both assembly and self-cleavage. A conserved histidine two residues upstream of the autocleavage site strongly promotes processing. Surprisingly, although beta 5pro is functionally linked to the Ump1 assembly factor, trans-expressed beta 5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed beta 5pro mutants; the strongest encoded the beta 7 proteasome subunit. Previous data suggested that beta 7 and beta 5pro have overlapping roles in bringing together two half-proteasomes, but the timing of beta 7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind beta 7 incorporation into the half-mer. Our results suggest that beta 7 insertion precedes half-mer dimerization, and the beta 7 tail and beta 5 propeptide have unequal roles in half-mer joining.