The 26 S proteasome is a large eukaryotic protease complex acting in ubiquitin-mediated degradation of abnormal and many short-lived, regulatory proteins. Its cylinder-shaped 20 S proteolytic core consists of two sets, each of seven different alpha and beta-type subunits arranged into two outer alpha-rings surrounding two inner beta-rings. The beta-rings form a central chamber with a total of six proteolytically active centers located in the beta1, beta2 and beta5 subunits. Activation of these subunits occurs during late assembly stages through intramolecular precursor autolysis removing propeptides attached to Thr1, which then serves as N-terminal nucleophile in substrate hydrolysis. This maturation entails intermolecular cleavage of propeptides residing in two of the non-active beta-type subunits, beta6 and beta7. In yeast, deletion of the beta5/Pre2 propeptide was shown to be lethal by preventing assembly of the core particle, while its expression as a separate entity restored growth. We investigated the role of the yeast beta1/Pre3, beta2/Pup1 and beta7/Pre4 propeptides by expressing the mature subunit moieties without propeptides as C-terminal fusions to ubiquitin. In all cases, viable strains could be generated. Deletion of the beta1/Pre3 and beta7/Pre4 propeptides did not affect cell growth, but deletion of the beta2/Pup1 propeptide led to poor growth, which was partially restored by co-expression of the free propeptide. Gain of proteolytic activity of beta1/Pre3 and beta2/Pup1 was abolished or drastically reduced, respectively, if their respective propeptides were not N-terminally bound. We detected N -alpha-acetylation at Thr1 of beta1/Pre3 as cause for its inactivation. Thus, one role for the propeptides of active beta-type subunits might be to protect the mature subunits catalytic Thr1 alpha-amino group from acetylation. The beta2/Pup1 propeptide was, in addition, required for efficient 20 S proteasome maturation, as revealed by the accumulation of beta7/Pre4 precursor and intermediate processing forms upon expression of mature beta2/Pup1. Finally, growth phenotypes resulting from expression of active site mutated beta-type subunits uncoupled from their propeptides allowed us to deduce the hierarchy of the importance of individual subunit activities for proteasomal function as follows: beta5/Pre2>>beta2/Pup1>/=beta1/Pre3.
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