Mapping subunit contacts in the regulatory complex of the 26 S proteasome. S2 and S5b form a tetramer with ATPase subunits S4 and S7.
The 19 S regulatory complex (RC) of the 26 S proteasome is composed of at least 18 different subunits, including six ATPases that form specific pairs S4-S7, S6-S8, and S6'-S10b in vitro. One of the largest regulatory complex subunits, S2, was translated in reticulocyte lysate containing [(35)S]methionine and used to probe membranes containing SDS-polyacrylamide gel electrophoresis separated RC subunits. S2 bound to two ATPases, S4 and S7. Association of S2 with regulatory complex subunits was also assayed by co-translation and sedimentation. S2 formed an immunoprecipitable heterotrimer upon co-translation with S4 and S7. The non-ATPase S5b also formed a ternary complex with S4 and S7 and the three proteins assembled into a tetramer with S2. Neither S2 nor S5b formed complexes with S6'-S10b dimers or with S6-S8 oligomers. The use of chimeric ATPases demonstrated that S2 binds the NH(2)-terminal region of S4 and the COOH-terminal two-thirds of S7. Conversely, S5b binds the COOH-terminal two-thirds of S4 and to S7's NH(2)-terminal region. The demonstrated association of S2 with ATPases in the mammalian 19 S regulatory complex is consistent with and extends the recent finding that the yeast RC is composed of two subcomplexes, the lid and the base (Glickman, M. H., Rubin, D. M., Coux, O., Wefes, I., Pfeifer, G., Cejka, Z., Baumeister, W., Fried, V. A., and Finley, D. (1998) Cell 94, 615-623).
Gorbea
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Department of Biochemistry, University of Utah School of Medicine, Salt Lake City, Utah 84132, USA.
Taillandier
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Rechsteiner
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D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000378:metabolism; D000818:Animals; D002417:Cattle; D002474:Cell-Free System; D002499:Centrifugation, Density Gradient; D004591:Electrophoresis, Polyacrylamide Gel; D004912:Erythrocytes / Q000201:enzymology; D006801:Humans; D046911:Macromolecular Substances; D008958:Models, Molecular; D010447:Peptide Hydrolases / Q000097:blood / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D046988:Proteasome Endopeptidase Complex; D014176:Protein Biosynthesis; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012156:Reticulocytes / Q000378:metabolism
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease; D000251:Adenosine Triphosphatases