Abstract
Eukaryotic 20S proteasome assembly remains poorly understood. The subunits stack into four heteroheptameric rings; three inner-ring subunits (β1, β2, and β5) bear the protease catalytic residues and are synthesized with N-terminal propeptides. These propeptides are removed autocatalytically late in assembly. In Saccharomyces cerevisiae, β5 (Doa3/Pre2) has a 75-residue propeptide, β5pro, that is essential for proteasome assembly and can work in trans. We show that deletion of the poorly conserved N-terminal half of the β5 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 β5pro is functionally linked to the Ump1 assembly factor, trans-expressed β5pro associates only weakly with Ump1-containing precursors. Several genes were identified as dosage suppressors of trans-expressed β5pro mutants; the strongest encoded the β7 proteasome subunit. Previous data suggested that β7 and β5pro have overlapping roles in bringing together two half-proteasomes, but the timing of β7 addition relative to half-mer joining was unclear. Here we report conditions where dimerization lags behind β7 incorporation into the half-mer. Our results suggest that β7 insertion precedes half-mer dimerization, and the β7 tail and β5 propeptide have unequal roles in half-mer joining.
Highlights
The degradation rates of intracellular proteins can vary over many orders of magnitude, with half-lives ranging from seconds to years [1,2,3,4]
Conserved Elements in the 5 Propeptide—Across all eukaryotic species, 5pro is the longest of the active subunit propeptides (ϳ50 – 85 residues), and studies in different eukaryotes have shown that it has a major role in 20S proteasome assembly [36, 50, 51]
Intermediates generated by processing within the proteasome as demonstrated by the increased mass resulting from mutation of the 2 (Pup1) active site Thr to Ala (T30A)
Summary
Yeast and Bacterial Media and Methods—Yeast rich (YPD) and minimal (SD) media were prepared as described, and standard methods were used for genetic manipulation of yeast [38]. In two separate reactions, the 5Ј outer primer from the CUP1 promoter region was used together with a long antisense primer containing the desired mutation(s), whereas the other half of the gene was amplified with the 3Ј outer primer and a sense primer exactly complementary to the mutant antisense primer, each using pTRANS as template. The mutant sequences were subcloned into pCIS and pTRANS using an EcoRI site at the 3Ј end of the CUP1 promoter (from YATAG200) and the StyI site introduced into the end of the 5pro sequence and verified by DNA sequencing. Internal deletions of codons 42–55 or 56 –73 within the N-terminal propeptide coding region of 5 were constructed by a two-stage PCR protocol followed by QuikChange mutagenesis [40] using YCplac22-DOA3-His as the template. Reactions were carried out at 30 °C for 1 h with 50 l of gradient fraction, 100 l of gel filtration fraction, or 6 g of affinitypurified proteasomes
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