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The eukaryotic 20 S proteasome is formed by dimerization of two precursor complexes containing the maturation factor Ump1. Beta7/Pre4 is the only one of the 14 subunits forming the 20 S proteasome that is absent from these precursor complexes in Saccharomyces cerevisiae. Increased expression of Pre4 leads to a reduction in the level of precursor complex, indicating that Pre4 incorporation into these complexes is rate-limiting for their dimerization. When we purified these precursor complexes, we observed co-purification of Blm10, a large protein known to attach to the alpha ring surface of proteasomes. In contrast to single mutants lacking either Blm10 or the C-terminal extension of Pre4, a mutant lacking both grew extremely poorly, accumulated very high levels of precursor complexes, and was impaired in beta subunit maturation. The effect of blm10Delta on proteasome biogenesis is modest, apparently because the 19 S regulatory particle is capable of substituting for Blm10, as long as precursor complex dimers are stabilized by the Pre4 C terminus. We found that a mutation (sen3/rpn2) affecting the Rpn2 subunit inhibits attachment of the 19 S activator to the 20 S particle or its precursors. Although the sen3 mutation alone had no apparent effect on precursor complex dimerization and active site maturation, the sen3 blm10 double mutant was impaired in these processes. Together these data demonstrate that Blm10 and the 19 S activator have a partially redundant function in stabilizing nascent 20 S proteasomes and in promoting their activation.

A close inspection of the crystal structure of the yeast 20 S proteasome revealed that a prominent connection between the two beta-rings is mediated by the subunit beta7/Pre4. Its C-terminal extension intercalates between the beta1/Pre3 and beta2/Pup1 subunits on the opposite ring. We show that the interactions promoted by the beta7/Pre4 tail are important to facilitate the formation of 20 S particles from two half-proteasome precursor complexes and/or to stabilize mature 20 S proteasomes. The deletion of 19 residues from the beta7/Pre4 C terminus leads to an accumulation of half-proteasome precursor complexes containing the maturation factor Ump1. The C-terminal extension of beta7/Pre4, which forms several hydrogen bonds with beta1/Pre3, is in addition required for the post-acidic activity mediated by the latter subunit. Deletion of the C-terminal tail of beta7/Pre4 results in an inhibition of beta1/Pre3 propeptide processing and abrogation of post-acidic activity. Our data obtained with yeast strains that expressed the mature form of Pre3 lacking its propeptide suggest that interactions between the Pre4 C terminus and Pre3 stabilize a conformation of its active site, which is essential for post-acidic activity. Deletion of the C-terminal extension of beta2/Pup1, which wraps around beta3/Pup3 within the same beta-ring, is lethal, indicating that this extension serves an essential function in proteasome assembly or stability.