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

Abstract

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 α and β-type subunits arranged into two outer α-rings surrounding two inner β-rings. The β-rings form a central chamber with a total of six proteolytically active centers located in the β1, β2 and β5 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 β-type subunits, β6 and β7. In yeast, deletion of the β5/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 β1/Pre3, β2/Pup1 and β7/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 β1/Pre3 and β7/Pre4 propeptides did not affect cell growth, but deletion of the β2/Pup1 propeptide led to poor growth, which was partially restored by co-expression of the free propeptide. Gain of proteolytic activity of β1/Pre3 and β2/Pup1 was abolished or drastically reduced, respectively, if their respective propeptides were not N-terminally bound. We detected N-α-acetylation at Thr1 of β1/Pre3 as cause for its inactivation. Thus, one role for the propeptides of active β-type subunits might be to protect the mature subunits catalytic Thr1 α-amino group from acetylation. The β2/Pup1 propeptide was, in addition, required for efficient 20 S proteasome maturation, as revealed by the accumulation of β7/Pre4 precursor and intermediate processing forms upon expression of mature β2/Pup1. Finally, growth phenotypes resulting from expression of active site mutated β-type subunits uncoupled from their propeptides allowed us to deduce the hierarchy of the importance of individual subunit activities for proteasomal function as follows: β5/Pre2⪢β2/Pup1⩾β1/Pre3.