Proteasome β-type subunits:: Unequal roles of propeptides in core particle maturation and a hierarchy of active site function

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 hive 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 beta 1, beta 2 and beta 5 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, beta 6 and beta 7. In yeast, deletion of the beta 5/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 beta 1/Pre3, beta 2/Pup1 and beta 7/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 beta 1/Pre3 and beta 7/Pre4 propeptides did not affect cell growth, but deletion of the beta 2/Pup1 propeptide led to poor growth, which was partially restored by co-expression of the free propeptide. Gain of proteolytic activity of beta 1/Pre3 and beta 2/Pup1 was abolished or drastically reduced, respectively, if their respective propeptides were not N-terminally bound. We detected N-alpha-acetylation at Thr1 of beta 1/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 beta 2/Pup1 propeptide was, in addition, required for efficient 20 S proteasome maturation, as revealed by the accumulation of beta 7/Pre4 precursor and intermediate processing forms upon expression of mature beta 2/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: beta 5/Pre2 much greater than beta 2/Pup1 greater than or equal to beta 1/Pre3. (C) 1999 Academic Press.