Intermediates in the formation of mouse 20S proteasomes: implications for the assembly of precursor beta subunits.
The assembly of individual proteasome subunits into catalytically active mammalian 20S proteasomes is not well understood. Using subunit-specific antibodies, we characterized both precursor and mature proteasome complexes. Antibodies to PSMA4 (C9) immunoprecipitated complexes composed of alpha, precursor beta and processed beta subunits. However, antibodies to PSMA3 (C8) and PSMB9 (LMP2) immunoprecipitated complexes made up of alpha and precursor beta but no processed beta subunits. These complexes possess short half-lives, are enzymatically inactive and their molecular weight is approximately 300 kDa. Radioactivity chases from these complexes into mature, long-lived approximately 700 kDa proteasomes. Therefore, these structures represent precursor proteasomes and are probably made up of two rings: one containing alpha subunits and the other, precursor beta subunits. The assembly of precursor proteasomes occurs in at least two stages, with precursor beta subunits PSMB2 (C7-I), PSMB3 (C10-II), PSMB7 (Z), PSMB9 (LMP2) and PSMB10 (LMP10) being incorporated before others [PSMB1 (C5), PSMB6 (delta), and PSMB8 (LMP7)]. Proteasome maturation (processing of the beta subunits and juxtaposition of the two beta rings) is accompanied by conformational changes in the (outer) alpha rings, and may be inefficient. Finally, interferon-gamma had no significant effect on the half-lives or total amounts of precursor or mature proteasomes.
Nandi
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Howard Hughes Medical Institute and Department of Molecular Genetics, University of Cincinnati, 231 Bethesda Avenue, Cincinnati, OH 45267-0524, USA.
Woodward
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Ginsburg
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Monaco
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D000818:Animals; D019843:Archaeal Proteins; D002478:Cells, Cultured; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000187:drug effects / Q000276:immunology; D007371:Interferon-gamma / Q000494:pharmacology; D008099:Liver / Q000737:chemistry / Q000166:cytology; D008264:Macrophages / Q000166:cytology; D051379:Mice; D008954:Models, Biological; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000187:drug effects / Q000276:immunology; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D011498:Protein Precursors / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000276:immunology; D013154:Spleen / Q000737:chemistry; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured
D019843:Archaeal Proteins; D009097:Multienzyme Complexes; D011498:Protein Precursors; D011506:Proteins; C071313:LMP-2 protein; D007371:Interferon-gamma; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C097046:PsmA protein, Methanosarcina thermophila; C097047:PsmB protein, Methanosarcina thermophila