FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2024786692> ?p ?o ?g. }

• W2024786692 endingPage "13411" @default.
• W2024786692 startingPage "13403" @default.
• W2024786692 abstract "The regulatory complex of the 26 S protease contains at least 15 distinct subunits. Six of these subunits (S4, S6, S6′, S7, S8, and S10b) belong to a novel subfamily of presumptive nucleotidases that we call subunit 4 (S4)-like ATPases. Each of these putative ATPases was synthesized in reticulocyte lysate containing [35S]methionine, and the radiolabeled proteins were used in binding studies. S4, S6, S10b, and S6′ displayed specific binding to components of the regulatory complex separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE. S4 bound to S7, and S6 bound two proteins: S8 and centractin, a component of the dynactin complex. S10b bound to S6′ and bound much more weakly to S1 and p50, another component of the dynactin complex. S6′ bound to S10b. Two subunits, S7 and S8, did not bind any components present on nitrocellulose membranes, presumably because S7 and S8 are already oligomeric following synthesis. Co-translation and sucrose gradient sedimentation of 35S-labeled ATPases demonstrated the formation of S6′-S10b dimers in solution but revealed more complex associations, namely the formation of trimers and tetramers, among S4, S6, S7, and S8. Progressive COOH-terminal deletions that removed as much as 300 amino acids from S4 had no effect on the binding of S4 to S7. In striking contrast, truncation of 85 NH2-terminal amino acids from S4 abrogated binding, clearly implicating the NH2 terminus of S4 in its specific interaction with S7. Since S4-like ATPases contain putative coiled-coils within the first 150 NH2-terminal amino acids, we propose that coiled-coil interactions are responsible for the specificity of the observed subunit associations and that these associations are important for self-assembly of the regulatory complex. The regulatory complex of the 26 S protease contains at least 15 distinct subunits. Six of these subunits (S4, S6, S6′, S7, S8, and S10b) belong to a novel subfamily of presumptive nucleotidases that we call subunit 4 (S4)-like ATPases. Each of these putative ATPases was synthesized in reticulocyte lysate containing [35S]methionine, and the radiolabeled proteins were used in binding studies. S4, S6, S10b, and S6′ displayed specific binding to components of the regulatory complex separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) or two-dimensional PAGE. S4 bound to S7, and S6 bound two proteins: S8 and centractin, a component of the dynactin complex. S10b bound to S6′ and bound much more weakly to S1 and p50, another component of the dynactin complex. S6′ bound to S10b. Two subunits, S7 and S8, did not bind any components present on nitrocellulose membranes, presumably because S7 and S8 are already oligomeric following synthesis. Co-translation and sucrose gradient sedimentation of 35S-labeled ATPases demonstrated the formation of S6′-S10b dimers in solution but revealed more complex associations, namely the formation of trimers and tetramers, among S4, S6, S7, and S8. Progressive COOH-terminal deletions that removed as much as 300 amino acids from S4 had no effect on the binding of S4 to S7. In striking contrast, truncation of 85 NH2-terminal amino acids from S4 abrogated binding, clearly implicating the NH2 terminus of S4 in its specific interaction with S7. Since S4-like ATPases contain putative coiled-coils within the first 150 NH2-terminal amino acids, we propose that coiled-coil interactions are responsible for the specificity of the observed subunit associations and that these associations are important for self-assembly of the regulatory complex. The ATP/ubiquitin (Ub) 1The abbreviations used are: Ub, ubiquitin; MCP, multicatalytic protease; RC, regulatory complex; TBP-1,tat-binding protein 1; TBP-7, tat-binding protein 7; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; pBS, pBluescript SK+; TBST, Tris-buffered saline-Tween 20. 1The abbreviations used are: Ub, ubiquitin; MCP, multicatalytic protease; RC, regulatory complex; TBP-1,tat-binding protein 1; TBP-7, tat-binding protein 7; DTT, dithiothreitol; PAGE, polyacrylamide gel electrophoresis; BSA, bovine serum albumin; pBS, pBluescript SK+; TBST, Tris-buffered saline-Tween 20.-dependent proteolytic pathway is involved in a diverse set of cellular processes including cell cycle regulation (1Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 346: 132-138Crossref Scopus (1880) Google Scholar, 2Hershko A. Ganoth D. Pehrson J. Palazzo R.E. Cohen L.H. J. Biol. Chem. 1991; 266: 16376-16379Abstract Full Text PDF PubMed Google Scholar, 3Holloway S.L. Glotzer M. King R.W. Murray A.W. Cell. 1993; 73: 1393-1402Abstract Full Text PDF PubMed Scopus (486) Google Scholar, 4Gordon C. McGurk G. Dillon P. Rosen C. Hastle N.D. Nature. 1993; 366: 355-357Crossref PubMed Scopus (210) Google Scholar, 5Ghislain M. Udvardy A. Mann C. Nature. 1993; 366: 358-362Crossref PubMed Scopus (365) Google Scholar), antigen presentation by major histocompatibility complex class I molecules (6Monaco J.J. Immunol. Today. 1992; 13: 173-178Abstract Full Text PDF PubMed Scopus (393) Google Scholar, 7Michalek M.T. Grant E.P. Gramm C. Goldberg A.L. Rock K.L. Nature. 1993; 363: 552-554Crossref PubMed Scopus (281) Google Scholar, 8Yang Y. Waters J.B. Früh K. Peterson P.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 4928-4932Crossref PubMed Scopus (162) Google Scholar, 9Ustrell V. Realini C. Pratt G. Rechsteiner M. FEBS Lett. 1995; 376: 155-158Crossref PubMed Scopus (30) Google Scholar), and the selective degradation of short lived and abnormal intracellular proteins (10Rechsteiner M. Cell. 1991; 66: 615-618Abstract Full Text PDF PubMed Scopus (205) Google Scholar, 11Hershko A. Ciechanover A. Annu. Rev. Biochem. 1992; 61: 761-807Crossref PubMed Scopus (1190) Google Scholar, 12Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1579) Google Scholar, 13Jentsch S. Schlenker S. Cell. 1995; 82: 881-884Abstract Full Text PDF PubMed Scopus (235) Google Scholar). ATP-dependent conjugation of Ub to ε-amino groups of lysine residues in protein substrates often marks them for degradation by the 26 S protease complex (14Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar, 15Peters J.-M. Trends Biochem. Sci. 1994; 19: 377-382Abstract Full Text PDF PubMed Scopus (297) Google Scholar), which at present is the only cytosolic enzyme known to degrade Ub-conjugated proteins. The 26 S protease is composed of at least 25 protein subunits with molecular masses ranging from 20 to 110 kDa. Considerable evidence shows that the multicatalytic protease (MCP) or proteasome (16Orlowski M. Biochemistry. 1990; 29: 10289-10296Crossref PubMed Scopus (412) Google Scholar) constitutes the proteolytic core of the 26 S enzyme. In the presence of ATP, the multicatalytic protease associates with a 19 S regulatory complex (RC) that confers ATP dependence and Ub recognition to the 26 S protease (17Eytan E. Ganoth D. Armon T. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 7751-7755Crossref PubMed Scopus (308) Google Scholar, 18Driscoll J. Goldberg A.L. J. Biol. Chem. 1990; 265: 4789-4792Abstract Full Text PDF PubMed Google Scholar, 19Peters J.-M. Harris J.R. Kleinschmidt J.A. Eur. J. Cell Biol. 1991; 56: 422-432PubMed Google Scholar, 20Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar, 21DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar). Electron micrographs show that the MCP cylinder is capped at one or both ends by RCs to produce mushroom- or dumbbell-shaped structures (22Peters J.-M. Cejka Z. Harris J.R. Kleinschmidt J.A. Baumeister W. J. Mol. Biol. 1993; 234: 932-937Crossref PubMed Scopus (212) Google Scholar, 23Yoshimura T. Kameyama K. Takagi T. Ikai A. Tokunaga F. Koide T. Tanahashi N. Tamura T. Cejka Z. Baumeister W. Tanaka K. Ichihara A. J. Struct. Biol. 1994; 111: 200-211Crossref Scopus (118) Google Scholar, 24Fujinami K. Tanahashi N. Tanaka K. Ichihara A. Cejka Z. Baumeister W. Miyawaki M. Sato T. Nakagawa H. J. Biol. Chem. 1994; 269: 25905-25910Abstract Full Text PDF PubMed Google Scholar). Among the polypeptides constituting the human RC, subunit 4 (S4) was the first component to be cloned and sequenced (25Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar). The deduced amino acid sequence of S4 predicts a 440-residue protein bearing a putative nucleotide binding site near the center of the sequence (26Walker J.E. Sarasate M. Runswick M.J. Gay N.J. EMBO J. 1982; 1: 945-951Crossref PubMed Scopus (4210) Google Scholar). Comparison of the predicted amino acid sequence of S4 with sequences in the GenBankTM data base reveals significant similarity to a number of proteins originally thought to be components of transcription complexes but found recently to be subunits of the 26 S protease (14Rechsteiner M. Hoffman L. Dubiel W. J. Biol. Chem. 1993; 268: 6065-6068Abstract Full Text PDF PubMed Google Scholar,25Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar). These S4-like ATPases include the human immunodeficiency virustat-binding proteins 1 and 7 (TBP-1 and TBP-7, respectively) (21DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 27Nelbock P. Dillon P.J. Perkins A. Rosen C.A. Science. 1990; 248: 1650-1653Crossref PubMed Scopus (195) Google Scholar, 28Ohana B. Moore P.A. Ruben S.M. Southgate C.D. Green M.R. Rosen C.A. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 138-142Crossref PubMed Scopus (126) Google Scholar, 29Dubiel W. Ferrell K. Rechsteiner M. FEBS Lett. 1993; 323: 276-278Crossref PubMed Scopus (77) Google Scholar); MSS1 (30Shibuya H. Irie K. Ninomiya-Tsuji J. Goebl M. Taniguchi T. Matsumoto K. Nature. 1992; 357: 700-702Crossref PubMed Scopus (140) Google Scholar, 31Dubiel W. Ferrell K. Rechsteiner M. Biol. Chem. Hoppe-Seyler. 1994; 375: 237-240Crossref PubMed Scopus (37) Google Scholar), and the thyroid receptor-interacting protein 1 (Trip1) 2C. Gorbea, L. Hoffman, and M. Rechsteiner, unpublished results. 2C. Gorbea, L. Hoffman, and M. Rechsteiner, unpublished results. (21DeMartino G.N. Moomaw C.R. Zagnitko O.P. Proske R.J. Chu-Ping M. Afendis S.J. Swaffield J.C. Slaughter C.A. J. Biol. Chem. 1994; 269: 20878-20884Abstract Full Text PDF PubMed Google Scholar, 32Lee J.W. Choi H.-S. Gyuris J. Brent R. Moore D.D. Mol. Endocrinol. 1995; 9: 243-354Crossref PubMed Google Scholar, 33Akiyama K.Y. Yokota K.-Y. Kagawa S. Shimbara N. DeMartino G.N. Slaughter C.A. Noda C. Tanaka K. FEBS Lett. 1995; 363: 151-156Crossref PubMed Scopus (59) Google Scholar) or Sug1p (34Swaffield J.C. Bromberg J.F. Johnston S.A. Nature. 1992; 357: 698-700Crossref PubMed Scopus (164) Google Scholar, 35Rubin D.M. Coux O. Wefes I. Hengartner C. Young R.A. Goldberg A.L. Finley D. Nature. 1996; 379: 655-657Crossref PubMed Scopus (143) Google Scholar), all of which are reasonably well conserved in the central ATPase module and at the COOH terminus but diverge considerably over an NH2-terminal region encompassing ∼150 amino acids. In contrast to the substantial amount of information available on the structure of MCP, including the x-ray structure of the enzyme fromThermoplasma acidophilum (36Kopp F. Steiner R. Dahlmann B. Kuehn L. Reinauer H. Biochim. Biophys. Acta. 1986; 872: 253-260Crossref PubMed Scopus (66) Google Scholar, 37Baumeister W. Dahlmann B. Hegerl R. Kopp F. Kuehn L. Pfeifer G. FEBS Lett. 1988; 241: 239-245Crossref PubMed Scopus (119) Google Scholar, 38Grziwa A. Baumeister W. Dahlmann B. Kopp F. FEBS Lett. 1991; 290: 186-190Crossref PubMed Scopus (110) Google Scholar, 39Löwe J. Stock D. Jap B. Zwickl P. Baumeister W. Huber R. Science. 1995; 268: 533-539Crossref PubMed Scopus (1363) Google Scholar, 40Pühler G. Weinkanf S. Bachmann L. Müller S. Engel A. Hegerl R. Baumeister W. EMBO J. 1992; 11: 1607-1616Crossref PubMed Scopus (166) Google Scholar), little is known about the organization of the regulatory complex. In an attempt to determine specific interactions between ATPases of the human RC, we have employed a nitrocellulose filter binding assay. In this report, we show that S4, S6, S10b, and S6′ bind specific subunits within the regulatory complex. The specific interactions observed on filters were confirmed by the co-sedimentation of in vitro-translated ATPases on sucrose gradients. Truncation experiments demonstrate that the NH2terminus of S4 is responsible for its interaction with S7. Based on the presence of putative coiled-coils in the amino termini of the ATPases, we propose that coiled-coil interactions are involved in assembly of the regulatory complex. Human 26 S protease was partially purified from 20 units of outdated blood as described by Dubiel et al. (25Dubiel W. Ferrell K. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22699-22702Abstract Full Text PDF PubMed Google Scholar). Briefly, packed and washed human erythrocytes were lysed with 4 volumes of 1 mmdithiothreitol (DTT) in water. The mixture was stirred for 30 min at 4 °C and centrifuged for 1 h at 10,000 rpm. Glycerol was added to the supernatant fraction to a final concentration of 20%, and the lysate was then mixed overnight with 1.7 liters of Fractogel TSK DEAE 650 m equilibrated in 10 mm Tris-HCl, pH 7.0, containing 1 mm DTT and 20% glycerol. The DEAE/lysate mixture was poured into a 2-liter column and washed with 2 column volumes of 75 mm KCl in TSDG (10 mm Tris-HCl (pH 7.0), 10 mm NaCl, 25 mm KCl, 1.1 mm MgCl2, 1 mm DTT, 0.1 mm EDTA, 20% glycerol) at a flow rate of 2.5 ml/min. Proteins were eluted with a linear gradient of 75–400 mmKCl in TSDG and assayed for ATP-dependent peptidase activity using succinyl-LLVY-aminomethylcoumarin (Sigma) as substrate (41Hough R. Pratt G. Rechsteiner M. J. Biol. Chem. 1987; 262: 8303-8313Abstract Full Text PDF PubMed Google Scholar). Under these conditions, the regulatory complex is eluted last from the column (above 240 mm KCl) relative to MCP and the 26 S protease (20Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar). Active fractions containing 26 S protease or regulatory complexes were pooled and concentrated to 70 ml using an XM300 membrane in a large AmiconTM cell. The concentrates were clarified by centrifugation and applied to a Fractogel TSK HW55 gel filtration column equilibrated in TSDG. Fractions (20 ml) were collected at a flow rate of 2.5 ml/min, assayed for ATP-dependent peptidase activity (26 S protease) as described above, or analyzed by native polyacrylamide gel electrophoresis (regulatory complex) as described by Hoffman et al. (20Hoffman L. Pratt G. Rechsteiner M. J. Biol. Chem. 1992; 267: 22362-22368Abstract Full Text PDF PubMed Google Scholar). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was performed using 10% separating and 4.5% stacking gels as described by Laemmli (42Laemmli U.K. Nature. 1970; 227: 680-685Crossref PubMed Scopus (205492) Google Scholar). Two-dimensional gel electrophoresis was performed by the method of O'Farrell (43O'Farrell P.H. J. Biol. Chem. 1975; 250: 4007-4021Abstract Full Text PDF PubMed Google Scholar) with minor modifications. Briefly, purified 26 S protease was separated in 12.5-cm isoelectric focusing tube gels consisting of 9.2 murea, 4% acrylamide, 2% Nonidet P-40, and 2% ampholines, pH 3–10 (Pharmacia Biotech Inc.). Bottom buffer (anode) consisted of 20 mm phosphoric acid and top buffer (cathode) consisted of 40 mm sodium hydroxide. Proteins were focused for 10,000 V-h after which the gels were layered perpendicularly over an SDS-polyacrylamide slab gel consisting of 4.5% stacking and 10% separating gel. Alternatively, two-dimensional gel electrophoresis of purified 26 S protease was performed in a Bio-Rad mini-Protean®II 2-D cell using 3% ampholines pH 2–11 (Serva) and a mini-Protean®II slab gel system as recommended by the manufacturer. After electrophoresis, proteins were either stained with Coomassie Brilliant Blue or transferred to nitrocellulose (see below). Plasmids pBS-S4 (pBS, pBluescript SK+; Stratagene) and pBS-S8 were used for transcription of S4 and S8, respectively. Plasmids pB1-TBP1 and pB1-TBP7 (gifts from Craig Rosen, Roche Research Center) were used for transcription of S6′ and S6, respectively. A plasmid encoding the sequence of mouse MSS1 (pBS-MSS1) was a gift from Colin Gordon (Medical Research Council, Edinburgh, UK) and was used to transcribe S7. A plasmid, pBS-S10b, encoding the sequence of S10b was a gift from Robert Benezra (Memorial Sloan-Kettering Cancer Center, New York). S4 and S7 transcripts were initiated from the T7 promoter of pBS, whereas transcripts for S6, S8, S10b, and S6′ were initiated from the T3 promoter of pB1 or pBS. Transcription of the NH2-terminal deletion product S4-(85–440) was performed using the plasmid pBS-S4RI generated by subcloning the EcoRI fragment (bp 290–1599) from pBS-S4 into pBS. Nested COOH-terminal deletions of S4 were generated with appropriate restriction endonucleases that cut at unique sites within the S4 sequence as follows: BstXI (base pair 1010) for S4-(1–315),SspI (base pair 689) for S4-(1–208), and BamHI (base pair 562) for S4-(1–167). Linearized plasmid DNA (3 μg) was precipitated with ethanol and used directly for transcription reactions utilizing the Stratagene mRNA Capping Kit® according to the manufacturer's directions. Capped RNA transcripts were extracted with phenol/chloroform, precipitated with ethanol, and used directly for in vitro translation reactions. Translation reactions (50 μl) containing 3 μg ofin vitro transcribed RNA were performed in rabbit reticulocyte lysate (Promega) in the presence of 40 μCi of [35S]methionine (1000 Ci/mmol, Amersham) for 2 h at 30 °C. Alternatively, transcription and translation of RC ATPase subunits were performed in a single reaction using the TNT T7/T3 Coupled Reticulocyte Lysate Systems® according to the directions of the manufacturer (Promega). After incubation, the translation reactions were passed over 0.7–0.9-ml Sephadex-G25 (Sigma) columns equilibrated in 10 mm Tris-HCl, pH 7.4, to remove unincorporated methionine. Fractions (2 drops each) were collected manually and analyzed by SDS-PAGE. Fractions containing full-length proteins were pooled and used directly without further purification in binding or sucrose gradient sedimentation assays. Proteins were transferred to nitrocellulose overnight at 100 mA according to the procedure of Towbin et al. (44Towbin H. Staehelin T. Gordon J. Proc. Natl. Acad. Sci. U. S. A. 1979; 76: 4350-4354Crossref PubMed Scopus (44642) Google Scholar). After transfer, membranes were stained with Ponceau S to visualize protein subunits, cut into strips, and washed with TBST buffer (50 mm Tris-HCl, pH 7.4, 200 mm NaCl, 0.05% Tween 20) for 15 min at 4 °C. The membrane strips were blocked overnight at 4 °C in 10% nonfat dried milk in TBST and stored in this buffer at 4 °C until used. To incubate 26 S subunits with in vitro translated [35S]ATPases, strips were removed from blocking buffer and washed 2 × 10 min with TBST at 4 °C. The membrane strips were then sealed in plastic bags and incubated overnight at 4 °C with 1–2 × 106 cpm of radiolabeled protein in 5% blocking buffer. Following incubation, the strips were washed five times for 5 min in TBST, air-dried, and exposed to a PhosphorImager® screen (Molecular Dynamics) or x-ray film (X-Omat AR, Eastman Kodak Co.) for autoradiography. Polyclonal antiserum against recombinant S4 from Schizosaccharomyces pombe was prepared in a New Zealand White rabbit injected intramuscularly with 63 μg of protein emulsified in TiterMax® adjuvant (Vaxcel). Immune serum was obtained from blood collected 31 days after injection and used for immunoprecipitation of in vitro translated S4 proteins as described by Harlow and Lane (45Harlow E. Lane D. Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York1988: 464-466Google Scholar). Briefly, aliquots (3 μl) of35S-labeled translation products were diluted to 500 μl with radioimmune precipitation buffer (20 mm Tris-HCl, pH 7.4, 150 mm NaCl, 0.5% SDS, 0.5% Nonidet P-40, 1 mm EDTA, 50 μg/ml phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A). S4-specific antiserum (1 μl) was then added to each sample and incubated overnight at 4 °C with continuous mixing. Immune complexes were precipitated by incubating the samples with 75 μl of protein A-Sepharose for 1 h at 4 °C followed by centrifugation. The sedimented beads were washed 5 times with radioimmune precipitation buffer at 4 °C and applied to SDS-PAGE. Proteins were visualized on a PhosphorImager screen or x-ray film. Following transfer of proteins to nitrocellulose and staining with Ponceau S, membranes were blocked for 6 h at 4 °C in TBS containing 10% nonfat dried milk. The blocked filters were then incubated overnight at 4 °C in the presence of anti-S10b (a gift from R. Benezra), anti-centractin (a gift from Sean Clark), or anti-p50 (a gift from Bryce Paschal) antibodies in TBS containing 5% nonfat milk. Antibody binding was visualized by enhanced chemiluminescence (Amersham or NEN DuPont) using secondary antibodies labeled with horseradish peroxidase (Cappel). 35S-Labeled ATPases prepared in rabbit reticulocyte lysate were sedimented through sucrose gradients as described (46Martin R.G. Ames B.N. J. Biol. Chem. 1961; 236: 1372-1379Abstract Full Text PDF PubMed Google Scholar). Samples (1–2 × 106 cpm in a volume of 100 μl) were layered atop 4.9-ml 5–20% sucrose gradients and centrifuged at 39,000 rpm in a Beckman SW 50.1 rotor for 18 h at 4 °C. Fractions (0.25 ml) were collected from the bottom using a peristaltic pump, and 30-μl aliquots were applied to SDS-PAGE. The gels were fixed in 40% methanol, 10% acetic acid in water, dried under vacuum, and exposed to a PhosphorImager screen. The distribution of 35S-labeled ATPases in the gradients was compared with protein standards (catalase, aldolase, and bovine serum albumin (BSA); Pharmacia) to estimate their size. Prediction of coiled-coil regions in S4-like ATPases was carried out as described by Carr and Kim (47Carr C.M. Kim P.S. Cell. 1993; 73: 823-832Abstract Full Text PDF PubMed Scopus (784) Google Scholar) using the computer program described by Lupas et al. (48Lupas A. Van Dyke M. Stock J. Science. 1991; 252: 1162-1164Crossref PubMed Scopus (3441) Google Scholar) (version 2.1) using a window size of 28 residues. The human red blood cell RC is composed of at least 15 distinct subunits with molecular masses between 25 and 110 kDa. A typical two-dimensional map of purified RCs is shown in Fig.1. Using the simple nomenclature introduced by Dubielet al. (49Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar), the regulatory complex subunits are designated S1–S13. The subunits can be classified into ATPases and non-ATPases. Five ATPases are clearly present in our preparations: S4, S6, S7, S8, and S10b (Table I). A sixth nucleotidase, TBP-1, has been indirectly identified in the course of these studies (see below). Since the migration of TBP-1 and S6 is identical on two-dimensional gels, we have designated TBP-1 as S6′. The non-ATPases include S1–S3, S5a, S5b, S9, S10a, and S11-S13 (Table I). One of these proteins, S5a, binds Ub-lysozyme conjugates (50Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar) and inhibits Ub-mediated proteolysis by the 26 S protease (51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). The remaining non-ATPase subunits have unique sequences with limited homology to each other or other classes of proteins (49Dubiel W. Ferrell K. Rechsteiner M. Mol. Biol. Rep. 1995; 21: 27-34Crossref PubMed Scopus (123) Google Scholar).Table ISubunits of human red blood cell regulatory complexComponentS4-like ATPaseIdentificationReferencesS1NoPeptide sequencing51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarS2NoPeptide sequencing51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarS3NoPeptide sequencing51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarS4YesPeptide sequencing, antibody binding, in vitrotranslation25, this studyS5aNoPeptide sequencing, antibody binding, Ub-conjugate binding52Deveraux Q. Jensen Q. Rechsteiner M. J. Biol. Chem. 1995; 270: 23726-23729Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 53Ferrell K. Deveraux Q. van Nocker S. Rechsteiner M. FEBS Lett. 1996; 381: 143-148Crossref PubMed Scopus (68) Google Scholarp50NoAntibody binding54Echeverri C.J. Paschal B.M. Vaughan K.T. Vallee R.B. J. Cell Biol. 1996; 132: 617-633Crossref PubMed Scopus (549) Google ScholarS5bNoPeptide sequencing, antibody binding52Deveraux Q. Jensen Q. Rechsteiner M. J. Biol. Chem. 1995; 270: 23726-23729Abstract Full Text Full Text PDF PubMed Scopus (58) Google ScholarS6 + S6′1-aRefer to legend for Fig. 1.YesPeptide sequencing,in vitro translation, ATPase binding29Dubiel W. Ferrell K. Rechsteiner M. FEBS Lett. 1993; 323: 276-278Crossref PubMed Scopus (77) Google Scholar, this studyS7YesPeptide sequencing, in vitro translation31Dubiel W. Ferrell K. Rechsteiner M. Biol. Chem. Hoppe-Seyler. 1994; 375: 237-240Crossref PubMed Scopus (37) Google Scholar, this studyCentractinNoPeptide sequencing, antibody binding55Clark S.W. Meyer D.I. Nature. 1992; 359: 246-250Crossref PubMed Scopus (136) Google Scholar, this studyS8YesPeptide sequencing, antibody binding, in vitrotranslationThis studyS9NoPeptide sequencing, antibody binding1-bHoffman, L., and Rechsteiner, M. (1997) FEBS Lett. 404,179–184.S10a1-cAn S10 protein different from S10b was identified by peptide sequencing (51). Hence, the more a cidic protein described by Dubiel et al. (51) is referred to as S10a, and the more b asic S4-like ATPase identified in this study is called S10b.NoPeptide sequencing51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google ScholarS10b1-cAn S10 protein different from S10b was identified by peptide sequencing (51). Hence, the more a cidic protein described by Dubiel et al. (51) is referred to as S10a, and the more b asic S4-like ATPase identified in this study is called S10b.YesAntibody bindingThis studyS11NoPeptide sequencing1-dL. Hoffman and M. Rechsteiner, unpublished data.S12NoPeptide sequencing, antibody binding56Dubiel W. Ferrell K. Dumdey R. Standera S. Prehn S. Rechsteiner M. FEBS Lett. 1995; 363: 97-100Crossref PubMed Scopus (31) Google Scholar, this studyS13NoPeptide sequencing51Regulatory complex subunits are designated according to Dubiel et al. (51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar).1-a Refer to legend for Fig. 1.1-b Hoffman, L., and Rechsteiner, M. (1997) FEBS Lett. 404,179–184.1-c An S10 protein different from S10b was identified by peptide sequencing (51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Hence, the more a cidic protein described by Dubiel et al. (51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar) is referred to as S10a, and the more b asic S4-like ATPase identified in this study is called S10b.1-d L. Hoffman and M. Rechsteiner, unpublished data. Open table in a new tab Regulatory complex subunits are designated according to Dubiel et al. (51Deveraux Q. van Nocker S. Mahaffey D. Vierstra R. Rechsteiner M. J. Biol. Chem. 1995; 270: 29660-29663Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). To examine protein-protein interactions between ATPases and other components in the regulatory complex, we translated S4, S6, S6′, S7, S8, and S10b in the presence of [35S]methionine and used the resulting radiolabeled proteins in solid phase binding assays. Fig.2 A shows a 10% SDS-polyacrylamide gel of the translation products used in this study. Each full-length radiolabeled protein migrated on SDS-PAGE with a mobility expected for its molecular mass. The smaller radioactive products are presumably generated by initiation from internal methionines or by proteolysis. Translation of the S6′ sequence consistently produced two bands of approximately 48 and 46 kDa; both products were found to be active in the binding assays (see below). The radiolabeled translation products were also characterized by centrifugation on 5–20% sucrose gradients (Fig. 2 B). About 90% of full-length S6′ and S6, and about 70% of S4 and S10b sedimented slower than BSA, consistent with their being monomers (TableII). By contrast, S7 and S8 sedimented as larger species (Fig. 2 B). S7 partitioned between a f" @default.
• W2024786692 created "2016-06-24" @default.
• W2024786692 creator A5030517769 @default.
• W2024786692 creator A5077603452 @default.
• W2024786692 creator A5089170848 @default.
• W2024786692 date "1997-05-01" @default.
• W2024786692 modified "2023-09-27" @default.
• W2024786692 title "Specific Interactions between ATPase Subunits of the 26 S Protease" @default.
• W2024786692 cites W124504601 @default.
• W2024786692 cites W1252231383 @default.
• W2024786692 cites W1258228527 @default.
• W2024786692 cites W148769332 @default.
• W2024786692 cites W1491324260 @default.
• W2024786692 cites W1496897161 @default.
• W2024786692 cites W1518640128 @default.
• W2024786692 cites W1532285380 @default.
• W2024786692 cites W1532810559 @default.
• W2024786692 cites W1542081345 @default.
• W2024786692 cites W1551702901 @default.
• W2024786692 cites W1606423922 @default.
• W2024786692 cites W1611482599 @default.
• W2024786692 cites W1653065940 @default.
• W2024786692 cites W1964931840 @default.
• W2024786692 cites W1970831375 @default.
• W2024786692 cites W1971192505 @default.
• W2024786692 cites W1972220674 @default.
• W2024786692 cites W1973016706 @default.
• W2024786692 cites W1974100585 @default.
• W2024786692 cites W1975460230 @default.
• W2024786692 cites W1977034044 @default.
• W2024786692 cites W1977610387 @default.
• W2024786692 cites W1980378067 @default.
• W2024786692 cites W1980581336 @default.
• W2024786692 cites W1981772689 @default.
• W2024786692 cites W1985728179 @default.
• W2024786692 cites W1989918211 @default.
• W2024786692 cites W1991101546 @default.
• W2024786692 cites W1992518796 @default.
• W2024786692 cites W1992671721 @default.
• W2024786692 cites W1995596496 @default.
• W2024786692 cites W1996864150 @default.
• W2024786692 cites W1999255494 @default.
• W2024786692 cites W1999741800 @default.
• W2024786692 cites W2003924932 @default.
• W2024786692 cites W2010622646 @default.
• W2024786692 cites W2011851534 @default.
• W2024786692 cites W2012015834 @default.
• W2024786692 cites W2015598117 @default.
• W2024786692 cites W2021838736 @default.
• W2024786692 cites W2022801212 @default.
• W2024786692 cites W2024645073 @default.
• W2024786692 cites W2036356274 @default.
• W2024786692 cites W2050145162 @default.
• W2024786692 cites W2053597753 @default.
• W2024786692 cites W2061601461 @default.
• W2024786692 cites W2071191421 @default.
• W2024786692 cites W2071473164 @default.
• W2024786692 cites W2074662488 @default.
• W2024786692 cites W2077086653 @default.
• W2024786692 cites W2080892506 @default.
• W2024786692 cites W2084375458 @default.
• W2024786692 cites W2088825773 @default.
• W2024786692 cites W2089407625 @default.
• W2024786692 cites W2100158530 @default.
• W2024786692 cites W2100837269 @default.
• W2024786692 cites W2101108802 @default.
• W2024786692 cites W2131544701 @default.
• W2024786692 cites W2157036573 @default.
• W2024786692 cites W2170532025 @default.
• W2024786692 cites W2175093343 @default.
• W2024786692 cites W2318128562 @default.
• W2024786692 cites W4243228481 @default.
• W2024786692 doi "https://doi.org/10.1074/jbc.272.20.13403" @default.
• W2024786692 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/9148964" @default.
• W2024786692 hasPublicationYear "1997" @default.
• W2024786692 type Work @default.
• W2024786692 sameAs 2024786692 @default.
• W2024786692 citedByCount "83" @default.
• W2024786692 countsByYear W20247866922012 @default.
• W2024786692 countsByYear W20247866922013 @default.
• W2024786692 countsByYear W20247866922014 @default.
• W2024786692 countsByYear W20247866922018 @default.
• W2024786692 crossrefType "journal-article" @default.
• W2024786692 hasAuthorship W2024786692A5030517769 @default.
• W2024786692 hasAuthorship W2024786692A5077603452 @default.
• W2024786692 hasAuthorship W2024786692A5089170848 @default.
• W2024786692 hasConcept C12554922 @default.
• W2024786692 hasConcept C181199279 @default.
• W2024786692 hasConcept C185592680 @default.
• W2024786692 hasConcept C23265538 @default.
• W2024786692 hasConcept C2776714187 @default.
• W2024786692 hasConcept C55493867 @default.
• W2024786692 hasConcept C86803240 @default.
• W2024786692 hasConcept C95444343 @default.
• W2024786692 hasConceptScore W2024786692C12554922 @default.
• W2024786692 hasConceptScore W2024786692C181199279 @default.
• W2024786692 hasConceptScore W2024786692C185592680 @default.
• W2024786692 hasConceptScore W2024786692C23265538 @default.

• next