title: 26 S proteasomes function as stable entities.
abstract: Most proteins in eukaryotic cells are degraded by 26-S proteasomes, usually after being conjugated to ubiquitin. In the absence of ATP, 26-S proteasomes fall apart into their two sub-complexes, 20-S proteasomes and PA700, which reassemble upon addition of ATP. Conceivably, 26-S proteasomes dissociate and reassemble during initiation of protein degradation in a ternary complex with the substrate, as in the dissociation-reassembly cycles found for ribosomes and the chaperonin GroEL/GroES. Here we followed disassembly and assembly of 26-S proteasomes in cell extracts as the exchange of PA700 subunits between mouse and human 26-S proteasomes. Compared to the rate of proteolysis in the same extract, the disassembly-reassembly cycle was much too slow to present an obligatory step in a degradation cycle. It has been suggested that subunit S5a (Mcb1, Rpn10), which binds poly-ubiquitin substrates, shuttles between a free state and the 26-S proteasome, bringing substrate to the complex. However, S5a was not found in the free state in HeLa cells. Besides, all subunits in PA700, including S5a, exchanged at similar low rates. It therefore seems that 26-S proteasomes function as stable entities during degradation of proteins.
authors.affiliation: August Krogh Institute, Copenhagen O, DK-2100, Denmark. KBHendil@aki.ku.dk
authors.forename: Klavs B
authors.initials: KB
authors.lastname: Hendil
authors.forename: Rasmus
authors.initials: R
authors.lastname: Hartmann-Petersen
authors.forename: Keiji
authors.initials: K
authors.lastname: Tanaka
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D002457:Cell Extracts; D002460:Cell Line; D002850:Chromatography, Gel; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D004795:Enzyme Stability; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D051379:Mice; D009097:Multienzyme Complexes / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D011233:Precipitin Tests; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D025801:Ubiquitin / Q000378:metabolism
chemical_list: D000911:Antibodies, Monoclonal; D002457:Cell Extracts; D009097:Multienzyme Complexes; D021122:Protein Subunits; D025801:Ubiquitin; D010447:Peptide Hydrolases; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease