Mass spectrometry reveals the missing links in the assembly pathway of the bacterial 20 S proteasome.
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.
Sharon
Michal
M
Departments of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom.
Witt
Susanne
S
Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried Germany.
Glasmacher
Elke
E
Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried Germany.
Baumeister
Wolfgang
W
Department of Molecular Structural Biology, Max-Planck-Institute of Biochemistry, Am Klopferspitz 18, D-82152 Martinsried Germany. Electronic address: baumeist@biochem.mpg.de.
Robinson
Carol V
CV
Departments of Chemistry, University of Cambridge, Lensfield Road, Cambridge, CB2 1EW, United Kingdom. Electronic address: cvr24@cam.ac.uk.
D000595:Amino Acid Sequence; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D046911:Macromolecular Substances; D013058:Mass Spectrometry / Q000379:methods; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D016296:Mutagenesis; D010455:Peptides / Q000737:chemistry; D046988:Proteasome Endopeptidase Complex / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D012240:Rhodococcus / Q000737:chemistry / Q000378:metabolism*; D021241:Spectrometry, Mass, Electrospray Ionization
D046911:Macromolecular Substances; D010455:Peptides; D046988:Proteasome Endopeptidase Complex