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The 20 S proteasome is an essential proteolytic particle, responsible for degrading short-lived and abnormal intracellular proteins. The 700-kDa assembly is comprised of 14 alpha-type and 14 beta-type subunits, which form a cylindrical architecture composed of four stacked heptameric rings (alpha7beta7beta7alpha7). The formation of the 20 S proteasome is a complex process that involves a cascade of folding, assembly, and processing events. To date, the understanding of the assembly pathway is incomplete due to the experimental challenges of capturing short-lived intermediates. In this study, we have applied a real-time mass spectrometry approach to capture transient species along the assembly pathway of the 20 S proteasome from Rhodococcus erythropolis. In the course of assembly, we observed formation of an early alpha/beta-heterodimer as well as an unprocessed half-proteasome particle. Formation of mature holoproteasomes occurred in concert with the disappearance of half-proteasomes. We also analyzed the beta-subunits before and during assembly and reveal that those with longer propeptides are incorporated into half- and full proteasomes more rapidly than those that are heavily truncated. To characterize the preholoproteasome, formed by docking of two unprocessed half-proteasomes and not observed during assembly of wild type subunits, we trapped this intermediate using a beta-subunit mutational variant. In summary, this study provides evidence for transient intermediates in the assembly pathway and reveals detailed insight into the cleavage sites of the propeptide.

Original publication

DOI

10.1074/jbc.M701534200

Type

Journal article

Journal

J Biol Chem

Publication Date

22/06/2007

Volume

282

Pages

18448 - 18457

Keywords

Amino Acid Sequence, Dimerization, Electrophoresis, Polyacrylamide Gel, Macromolecular Substances, Mass Spectrometry, Models, Molecular, Molecular Conformation, Molecular Sequence Data, Mutagenesis, Peptides, Proteasome Endopeptidase Complex, Protein Conformation, Protein Structure, Tertiary, Rhodococcus, Spectrometry, Mass, Electrospray Ionization