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• W1994608712 abstract "Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with 125I-Nedd8 (k cat = 3.5 ± 0.2 s–1), yielding K m values for ATP (103 ± 12 μm), 125I-Nedd8 (0.95 ± 0.18 μm), and HsUbc12 (43 ± 13 nm) similar to those for ubiquitin activation by Uba1. Wild type 125I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of 125I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a K d > 300 μm for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because 125I-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields K m = 20 ± 9 μm and k cat = 0.9 ± 0.3 s–1. These observations demonstrate remarkable conservation in the mechanism of AppBp1-Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme. Human Nedd8-activating enzyme AppBp1-Uba3 was purified to apparent homogeneity from erythrocytes. In the presence of [2,8-3H]ATP and 125I-Nedd8, heterodimer rapidly forms a stable stoichiometric ternary complex composed of tightly bound Nedd8 [3H]adenylate and Uba3-125I-Nedd8 thiol ester. Isotope exchange kinetics show that the heterodimer follows a pseudo-ordered mechanism with ATP the leading and Nedd8 the trailing substrate. Human AppBp1-Uba3 follows hyperbolic kinetics for HsUbc12 transthiolation with 125I-Nedd8 (k cat = 3.5 ± 0.2 s–1), yielding K m values for ATP (103 ± 12 μm), 125I-Nedd8 (0.95 ± 0.18 μm), and HsUbc12 (43 ± 13 nm) similar to those for ubiquitin activation by Uba1. Wild type 125I-ubiquitin fails to support AppBp1-Uba3 catalyzed activation or HsUbc12 transthiolation. However, modest inhibition of 125I-Nedd8 ternary complex formation by unlabeled ubiquitin suggests a K d > 300 μm for ubiquitin. Alanine 72 of Nedd8 is a critical specificity determinant for AppBp1-Uba3 binding because 125I-UbR72L undergoes heterodimer-catalyzed hyperbolic HsUbc12 transthiolation and yields K m = 20 ± 9 μm and k cat = 0.9 ± 0.3 s–1. These observations demonstrate remarkable conservation in the mechanism of AppBp1-Uba3 that mirrors its sequence conservation with the Uba1 ubiquitin-activating enzyme. Class I ubiquitin-like proteins exert their biological effects through covalent conjugation to their respective target proteins via distinct ligation pathways that function in parallel to those of ubiquitination. The ubiquitin-like proteins include Sumo (1Johnson E.S. Schwienhorst R.J. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (445) Google Scholar, 2Okuma T. Honda R. Ichikawa G. Tsumagari N. Yasuda H. Biochem. Biophys. Res. Commun. 1999; 254: 693-698Crossref PubMed Scopus (184) Google Scholar), Nedd8 (3Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (307) Google Scholar, 4Osaka F. Kawasaki H. Aida N. Saeki M. Chiba T. Kawashima S. Tanaka K. Kato S. Genes Dev. 1998; 12: 2263-2268Crossref PubMed Scopus (227) Google Scholar), Hub1 (5Dittmar G.A. Wilkinson C.R. Jedrzejewski P.T. Finley D. Science. 2002; 295: 2442-2446Crossref PubMed Scopus (77) Google Scholar), ISG15 (6Yuan W. Krug R.M. EMBO J. 2001; 20: 362-371Crossref PubMed Scopus (422) Google Scholar), FAT10 (7Liu Y.C. Pan J. Zhang C. Fan W. Collinge M. Bender J.R. Weissman S.M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4313-4318Crossref PubMed Scopus (144) Google Scholar, 8Raasi S. Schmidtke G. Groettrup M. J. Biol. Chem. 2001; 276: 35334-35343Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar), and Apg12 (9Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (327) Google Scholar) among others, reviewed in Ref. 10Larsen C.N. Wang H. J. Proteome Res. 2002; 1: 411-419Crossref PubMed Scopus (23) Google Scholar. Generally, conjugation of ubiquitin-like proteins modulates the protein-ligand interactions of their target rather than committing the target protein to degradation, the role most associated with ubiquitin ligation. Among the ubiquitin-like proteins, Nedd8 is the most closely related to ubiquitin with 58% identity between human paralogs (10Larsen C.N. Wang H. J. Proteome Res. 2002; 1: 411-419Crossref PubMed Scopus (23) Google Scholar). Nedd8 and its plant ortholog Rub1 are conjugated to Cdc53/Cul1 (11Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (279) Google Scholar, 12Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (331) Google Scholar) and other members of the Cullin family of proteins (13Hori T. Osaka F. Chiba T. Miyamoto C. Okabayashi K. Shimbara N. Kato S. Tanaka K. Oncogene. 1999; 18: 6829-6834Crossref PubMed Scopus (246) Google Scholar). Cullins are essential structural subunits of the Skp1-based (SCF, Skp1, Cul1, F-box) and elongin B/C-based families of ubiquitin protein ligases (E3), 1The abbreviations used are: E3, ubiquitin-protein ligase; E2, ubiquitin carrier protein or ubiquitin-conjugating enzyme (Ubc); GST, glutathione S-transferase; DTT, dithiothreitol; Ub, ubiquitin. reviewed in Refs. 14Jackson P.K. Eldridge A.G. Freed E. Furstenthal L. Hsu J.Y. Kaiser B.K. Reimann J.D. Trends Cell Biol. 2000; 10: 429-439Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar and 15Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (348) Google Scholar. Conjugation of Nedd8 to Cul1 and Cul2 requires the Ring finger protein Roc1/Rbx1/Hrt1, which serves as a docking adapter (16Tan P. Fuchs S.Y. Chen A. Wu K. Gomez C. Ronai Z. Pan Z.Q. Mol. Cell. 1999; 3: 527-533Abstract Full Text Full Text PDF PubMed Scopus (303) Google Scholar, 17Furukawa M. Ohta T. Xiong Y. J. Biol. Chem. 2002; 277: 15758-15765Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The attachment of Nedd8 to Cullin isoforms is not required for the intrinsic ubiquitin ligase activity of the SCF complex; however, it enhances ubiquitin chain formation through activation of the cognate E2 ubiquitin-conjugating enzyme (12Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (331) Google Scholar, 18Wu K. Chen A. Pan Z.Q. J. Biol. Chem. 2000; 275: 32317-32324Abstract Full Text Full Text PDF PubMed Scopus (172) Google Scholar). Because of the central role of SCF and elongin B/C ubiquitin ligases in critical regulatory processes within eukaryotes, Nedd8 conjugation is an essential post-translational modification that is subject to considerable recent interest (12Read M.A. Brownell J.E. Gladysheva T.B. Hottelet M. Parent L.A. Coggins M.B. Pierce J.W. Podust V.N. Luo R.S. Chau V. Palombella V.J. Mol. Cell. Biol. 2000; 20: 2326-2333Crossref PubMed Scopus (331) Google Scholar, 15Conaway R.C. Brower C.S. Conaway J.W. Science. 2002; 296: 1254-1258Crossref PubMed Scopus (348) Google Scholar). The ATP-coupled activation of Nedd8 that is required for subsequent charging of the Nedd8-specific Ubc12-conjugating enzyme is catalyzed by heterodimeric AppBp1-Uba3 in humans (4Osaka F. Kawasaki H. Aida N. Saeki M. Chiba T. Kawashima S. Tanaka K. Kato S. Genes Dev. 1998; 12: 2263-2268Crossref PubMed Scopus (227) Google Scholar, 19Gong L. Yeh E.T. J. Biol. Chem. 1999; 274: 12036-12042Abstract Full Text Full Text PDF PubMed Scopus (217) Google Scholar). Human Uba3 shows 43% homology to the carboxyl-terminal 500 residues of the human ubiquitin-activating enzyme HsUba1 and encompasses the putative ubiquitin adenylate active site identified by homology to the MoeB subunit of molybdopterin synthase (20Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar, 21Lake M.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nature. 2001; 414: 325-329Crossref PubMed Scopus (206) Google Scholar). Deletion of Uba3 is embryonic lethal in mice, arising from a mitotic defect in the G1/G0 transition and the resulting accumulation of cyclin E and β-catenin (22Latres E. Chiaur D.S. Pagano M. Oncogene. 1999; 18: 849-854Crossref PubMed Scopus (377) Google Scholar, 23Tateishi K. Omata M. Tanaka K. Chiba T. J. Cell Biol. 2001; 155: 571-579Crossref PubMed Scopus (12) Google Scholar), both targets of SCF ligases (24Deshaies R.J. Annu. Rev. Cell Dev. Biol. 1999; 15: 435-467Crossref PubMed Scopus (1083) Google Scholar). However, the Uba3-catalyzed activation of Nedd8 exhibits an absolute requirement for AppBp1, a protein first identified by its interaction with the carboxyl terminus of amyloid precursor protein and that has marked homology to the amino-terminal half of Uba1 (20Rudolph M.J. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nat. Struct. Biol. 2001; 8: 42-46Crossref PubMed Scopus (178) Google Scholar, 21Lake M.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nature. 2001; 414: 325-329Crossref PubMed Scopus (206) Google Scholar, 25Chow N. Korenberg J.R. Chen X.N. Neve R.L. J. Biol. Chem. 1996; 271: 11339-11346Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar). Overexpression of AppBp1 rescues the temperature-sensitive ts41 mutation of Chinese hamster lung cells by driving S-M checkpoint progression through Nedd8 conjugation (26Chen Y. McPhie D.L. Hirschberg J. Neve R.L. J. Biol. Chem. 2000; 275: 8929-8935Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The recent 2.6 Å structure of human AppBp1-Uba3 confirms that AppBp1 is required in part to contribute a short conserved active site segment first identified in the mechanistically related MoeB subunit of molybdopterin synthase (21Lake M.W. Wuebbens M.M. Rajagopalan K.V. Schindelin H. Nature. 2001; 414: 325-329Crossref PubMed Scopus (206) Google Scholar, 27Walden H. Podgorski M.S. Schulman B.A. Nature. 2003; 422: 330-334Crossref PubMed Scopus (180) Google Scholar). In the activation of ubiquitin, Uba1 forms a ternary complex composed of 1 eq each of a tightly bound ubiquitin adenylate and a covalent Uba1-ubiquitin thiol ester to a conserved active site, Cys632 (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar), HsUba1a numbering. Early ATP:PPi exchange studies demonstrated that rabbit Uba1 catalyzes an absolutely ordered mechanism in which ATP binding precedes that of ubiquitin prior to the first catalytic step of ubiquitin adenylate formation (29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). The activated ubiquitin moiety is subsequently transferred to the thiol ester site comprising Cys632 prior to formation of a second ubiquitin adenylate, to generate the final ternary complex (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). Marked conservation among the activating enzymes for ubiquitin and ubiquitin-like proteins argues that they proceed by similar mechanisms. However, the apparently substoichiometric formation of the predicted Nedd8 adenylate intermediate catalyzed by the reconstituted plant ortholog of AppBp1-Uba3 suggests that the catalytic cycle for Nedd8 activation may exhibit some differences from that of ubiquitin (30del Pozo J.C. Estelle M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 15342-15347Crossref PubMed Scopus (138) Google Scholar). The latter observation is significant because the presence of enzyme-bound ubiquitin adenylate is required for ubiquitin transthiolation from the E1 ternary complex to E2 carrier proteins, even though this intermediate is not the immediate donor of activated polypeptide (31Pickart C.M. Kasperek E.M. Beal R. Kim A. J. Biol. Chem. 1994; 269: 7115-7123Abstract Full Text PDF PubMed Google Scholar). To date, the enzymes involved in the activation of ubiquitin-like proteins have not been mechanistically characterized in sufficient detail to resolve these and related questions. In the present studies, we have found that human erythrocytes represent an excellent source of active AppBp1-Uba3 heterodimer and have utilized covalent affinity purified AppBp1-Uba3 and recombinant human Ubc12 in the first mechanistic studies of Nedd8 activation. The results demonstrate marked conservation between the mechanisms for Nedd8 and ubiquitin activation and identify a critical specificity determinant for polypeptide recognition by their respective activating enzymes. Bovine ubiquitin was purchased from Sigma and purified to homogeneity as described previously (32Baboshina O.V. Haas A.L. J. Biol. Chem. 1996; 271: 2823-2831Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar). Homogeneous wild-type ubiquitin, the recombinant ubiquitin mutant UbR72L (33Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (79) Google Scholar), and recombinant human Nedd8 were radiolabeled by the chloramine-T method using carrier-free Na125I obtained from PerkinElmer Life Sciences (34Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (107) Google Scholar). The [2,8-3H]ATP and Na4[32PPi] used in Nedd8 [3H]adenylate quantitation and ATP:32PPi exchange kinetics, respectively, were obtained from PerkinElmer Life Sciences. Cloning, Expression, and Purification of Human Recombinant Nedd8 —Nedd8 was cloned from a HeLa cell cDNA library by PCR amplification using 5′ and 3′ primers flanking the coding sequence that contained NdeI or EcoRI restriction sites, respectively. The PCR product was ligated directly into pGEM-T (Promega) for amplification and purification. The resulting pGEM-Nedd8 construct was digested with NdeI/EcoRI and ligated into similarly restricted pPLhUb to yield pPLNedd8 (33Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (79) Google Scholar). The Nedd8 coding sequence was verified by sequencing the entire coding region by automated sequencing in The Protein and Nucleic Acid Core Facility at the Medical College of Wisconsin. The pPLNedd8 plasmid was transformed into AR58 Escherichia coli cells constitutively expressing a temperature-sensitive [lamda] repressor protein (35Ecker D.J. Khan M.I. Marsh J. Butt T.R. Crooke S.T. J. Biol. Chem. 1987; 262: 3524-3527Abstract Full Text PDF PubMed Google Scholar). Protein expression was induced by rapidly increasing the cultures to the non-permissive temperature of 42 °C (32Baboshina O.V. Haas A.L. J. Biol. Chem. 1996; 271: 2823-2831Abstract Full Text Full Text PDF PubMed Scopus (183) Google Scholar, 33Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (79) Google Scholar, 35Ecker D.J. Khan M.I. Marsh J. Butt T.R. Crooke S.T. J. Biol. Chem. 1987; 262: 3524-3527Abstract Full Text PDF PubMed Google Scholar). After 2 h of induction at 42 °C, cells from 2 liters of LB culture containing 100 μg/ml ampicillin were collected by centrifugation at 5,000 × g for 15 min then lysed by passage through a French press. All subsequent steps were conducted at 4 °C. The lysate was centrifuged for 30 min at 30,000 × g. Recombinant Nedd8 protein occurred nearly quantitatively within bacterial inclusion bodies present in the 30,000 × g pellet but could be extracted and refolded using a modification of a protocol previously described for the isolation of unstable recombinant ubiquitin mutants (33Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (79) Google Scholar). The pellet containing the Nedd8-associated inclusion bodies was collected and resuspended to the original lysate volume in 50 mm Tris acetate (pH 7.5) then centrifuged again. The resulting pellet was solubilized in 30 ml of Tris acetate buffer (pH 7.5) containing 6 m urea. The mixture was allowed to stir at room temperature for 30 min, then the urea was slowly removed by dialysis (3.5-kDa exclusion limit dialysis tubing) overnight against 2 × 4 liters of 50 mm Tris acetate buffer (pH 7.5). Insoluble protein was removed by centrifugation at 5,000 × g for 15 min. The supernatant was batch adsorbed onto a 250-ml bed volume of DEAE cellulose (Whatman) equilibrated with 50 mm Tris acetate (pH 7.5). The unadsorbed fraction from the DEAE cellulose was collected and titrated to pH 4.5 with acetic acid before being applied to a 2.5 × 10-cm column of CM52 cellulose (Whatman) equilibrated with 25 mm ammonium acetate (pH 4.5) (36Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). Bound protein was eluted stepwise with 50 mm ammonium acetate (pH 5.5). Fractions containing Nedd8 protein were pooled and dialyzed against distilled water. The pH of the dialyzed solution was adjusted to 4.5 with acetic acid before being applied to a Pharmacia Mono S 5/5 column equilibrated with 25 mm acetic acid titrated to pH 4.5 with ammonium acetate (36Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). The bound protein was eluted with a linear 0–0.5 m NaCl gradient (10 mm/min) at 1 ml/min flow rate. This procedure generally yielded 2–5 mg of protein per liter of E. coli culture. The protein was greater than 99% homogeneous as assessed by Coomassie Brilliant Blue staining following resolution by 14% (w/v) polyacrylamide SDS-PAGE. Absolute protein concentration was determine spectrophotometrically using the empirical 280 nm ubiquitin extinction coefficient of 0.16 (mg/ml)–1 (36Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). 2The identical aromatic amino acid content for ubiquitin and human Nedd8 allowed us to use the empirical extinction coefficient of the former polypeptide in this spectrophotometric assay. Cloning and Expression of Human AppBp1 and Uba3—The full-length coding sequence of Uba3 was cloned by PCR from human fetal brain expressed sequence tagged I.M.A.G.E. Consortium Clone 45573 (American Type Culture Collection). Flanking primers containing complimentary 5′ and 3′ coding sequences and either SalI or EcoRI restriction sites, respectively, were used to amplify the cDNA. The resulting PCR product was ligated into pGEM-T (Promega) to yield pGEMT-Uba3 for subsequent amplification, purification, and sequencing. The complete Uba3 coding sequence was then subcloned into pGEX4T-1 (Amersham Biosciences) using SalI and EcoRI restriction sites to yield pGEX-Uba3. AppBp1 was cloned by PCR from a HeLa cDNA library using flanking primers that contained complimentary 5′ and 3′ coding sequences and either NdeI or EcoRI restriction sites, respectively. The PCR product was ligated directly into pGEM-T. The resulting pGEMT-AppBp1 clone was digested with NdeI and EcoRI, then subcloned into complimentary digested pGEX4T-1 to yield pGEX-AppBp1. The AppBp1 sequence was verified by sequencing the entire insert. The glutathione S-transferase fusion proteins GST-Uba3 and GST-AppBp1 were expressed in E. coli BL21 cultures and purified from refolded inclusion bodies by glutathione affinity chromatography. Briefly, bacteria were grown to an A 600 of 0.6 at 30 °C and induced by the addition of 0.1 mm isopropyl-1-thio-β-d-galactopyranoside. After 2 h of induction, the cells were collect by centrifugation and lysed by passage through a French press. The lysate was centrifuged at 30,000 × g for 30 min. The resulting pellets were washed with buffer containing 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, and 1 mm DTT, then resuspended to the original lysate volume in the same buffer containing 6 m urea. After being allowed to stand on ice for 30 min, the urea was removed by dialysis (3.5-kDa exclusion limit dialysis tubing) against 2× 4 liters of 50 mm Tris-HCl (pH 7.5) containing 1 mm DTT. Insoluble protein was removed by centrifugation prior to applying the dialysate to a 5-ml glutathione-agarose column. Unbound protein was removed by washing the column with 5 bed volumes of 50 mm Tris-HCl (pH 7.5). Bound protein was eluted with 5 bed volumes of 50 mm Tris-HCl (pH 7.5) containing 20 mm glutathione, then concentrated with a Millipore Ultrafree BioMax-5K centrifugal filter. The resulting fusion protein was cleaved by digestion with 10 units of thrombin (Amersham Biosciences) per milligram of recombinant protein according to the manufacturer's recommendations. Processed AppBp1 or Uba3 were resolved from GST and thrombin by fast protein liquid chromatography using a Pharmacia Mono Q 5/5 column equilibrated with 50 mm Tris-HCl (pH 7.5) containing 1 mm DTT. Both AppBp1 and Uba3 eluted between 0.31 and 0.35 m within a linear 0–0.5 m NaCl gradient (12.5 mm/min) at 1 ml/min flow rate. Recombinant AppBp1 and Uba3 proteins were greater than 80% pure, as assessed by Coomassie Brilliant Blue staining of samples resolved by 10% (w/v) SDS-PAGE, and were used without further purification. Protein concentrations for AppBp1 and Uba3 were estimated densitometrically by comparing Coomassie-stained bands to bovine serum albumin standards. Cloning and Expression of HsUbc12—Human Ubc12 was cloned by PCR from a HeLa cell cDNA library using 5′ and 3′ primers immediately flanking the HsUbc12 coding sequences that contained SalI and EcoRI restriction sites, respectively. The PCR product was subsequently ligated into pGEM-T (Promega) for amplification and sequencing. The resulting construct was digested with SalI and EcoRI, then the HsUbc12 coding sequence was isolated and ligated into similarly restricted pGEX4T-1 (Amersham Biosciences) to yield pGEX-Ubc12. The HsUbc12 coding sequence was verified by sequencing the entire insert. The GST-HsUbc12 fusion protein was expressed in E. coli BL21 cells and purified by glutathione affinity chromatography. Bacterial cells transformed with pGEX-Ubc12 were grown at 37 °C to an A 600 of 0.6, then protein expression was induced by the addition of isopropyl-1-thio-β-d-galactopyranoside to a final concentration of 0.1 mm. After 2 h of induction, cells were collected by centrifugation, resuspended in buffer containing 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, and 1 mm DTT, then lysed by passage through a French press. The lysate was clarified by centrifugation at 30,000 × g for 30 min and the resulting supernatant was applied to a glutathione-agarose column. Unbound protein was removed by washing the column with 5 bed volumes of 50 mm Tris-HCl (pH 7.5), 2 mm EDTA, and 1 mm DTT. Bound protein was eluted with 50 mm Tris-HCl (pH 7.5) containing 20 mm glutathione, then concentrated using a Millipore Ultrafree BioMax-5K centrifugal filter. Following processing of the fusion protein with thrombin (Amersham Biosciences) at 10 units per mg of fusion protein, free HsUbc12 was further purified by fast protein liquid chromatography using a Pharmacia Mono Q 5/5 column equilibrated with 50 mm Tris-HCl (pH 7.0) containing 1 mm DTT. The HsUbc12 protein was eluted at 0.26 m within a linear 0–0.5 m NaCl gradient (12.5 mm/min) at 1 ml/min. The protein was greater than 95% pure as assessed by Coomassie Blue Brilliant staining. The concentration of active protein was determined by an end point thiol ester assay using 125I-Nedd8 (about 10,000 cpm/pmol) and affinity purified AppBp1-Uba3 heterodimer (37Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Purified protein was flash frozen and stored at –80 °C for several months without loss of activity. Affinity Purification of Human AppBp1-Uba3 Heterodimer from Human Erythrocytes—The AppBp1-Uba3 complex was isolated from human red blood cell Fraction II using Nedd8 affinity chromatography by adapting earlier methods for the isolation of Uba1 (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 37Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Recombinant Nedd8 was coupled to Affi-Gel 10 (Bio-Rad) at ∼0.5 mg of Nedd8/ml of resin for a final concentration of 60 μm Nedd8 (37Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Five units of outdated whole blood was obtained from the Blood Center of Southeastern Wisconsin and used to prepare Fraction II as described previously (37Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Erythrocyte Fraction II was supplemented with a final concentration of 2 mm ATP, 10 mm MgCl2, 10 mm creatine phosphate, and 1 IU/ml creatine phosphokinase, then applied to the Nedd8 affinity column (10 ml bed volume) previously equilibrated with 50 mm Tris-HCl (pH 7.5), 2 mm ATP, and 2 mm MgCl2. The column was washed successively with 2 bed volumes of 50 mm Tris-HCl (pH 7.5), 3 bed volumes of 50 mm Tris-HCl (pH 7.5) containing 0.25 m KCl, and 2 bed volumes of 50 mm Tris-Cl (pH 7.5). Bound protein was eluted with 2 bed volumes of 50 mm Tris-HCl (pH 7.5) containing 2 mm AMP and 2 mm PPi followed by 2 bed volumes of 0.1 m Tris-HCl (pH 9.0) containing 10 mm DTT. The latter eluate was adjusted to pH 7.5 immediately following elution. The proteins from the two elutions were separately concentrated using a Millipore Ultrafree BioMax-5K centrifugal filter then dialyzed against 50 mm Tris-HCl (pH 7.5) containing 1 mm DTT and used without further purification. The concentration of active AppBp1-Uba3 heterodimer was determined by measuring the stoichiometric formation of Uba3-125I-Nedd8 thiol ester (below). Stoichiometric Assays for Active AppBp1-Uba3 Heterodimer—The stoichiometric formation of covalent Nedd8 [3H]adenylate was measured directly using [2,8-3H]ATP as described previously for ubiquitin-activating enzyme (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). Fifty-μl assays containing 50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 5 μm Nedd8, 2 mg/ml carrier bovine serum albumin, 0.5 IU inorganic pyrophosphatase, 0.5 μm [2,8-3H]ATP (35,200 cpm/pmol), and about 1 pmol of AppBp1-Uba3 heterodimer were incubated at 37 °C for 5 min. The reactions were quenched by the addition of 400 μl of 20% (w/v) trichloroacetic acid containing 4 mm ATP and allowed to stand on ice for 10 min. Precipitated protein was collected by centrifugation at 12,000 × g for 10 min, then the resulting pellets were washed with 1 ml of 2% (w/v) trichloroacetic acid. The pellets were then resuspended in 0.1 ml of 0.2 m triethanolamine-HCl (pH 8.0). The amount of Nedd8 [3H]adenylate formed was determined by liquid scintillation counting. The stoichiometric formation of Uba3-125I-Nedd8 thiol ester was determined as previously described for Uba1 using radioiodinated protein (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). Briefly, 25-μl reactions containing 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 2 mm ATP, 1 mm DTT, 5 μm125I-Nedd8 (about 10,000 cpm/pmol), 0.5 IU inorganic pyrophosphatase, and the indicated amounts of AppBp1-Uba3 were incubated at 37 °C for 3 min. The reactions were quenched by the addition of an equal volume of 4% SDS-sample buffer and the proteins were resolved by non-reducing SDS-PAGE on 12% (w/v) acrylamide gels. The gels were dried and the thiol esters were visualized by autoradiography, then excised and quantified by γ counting (28Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). ATP:PP i Isotope Exchange Kinetic Assays—Initial rates of ATP:32PPi isotope exchange were measured as previously described for the ubiquitin-activating enzyme (29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). Fifty-μl reactions contained 50 mm Tris-HCl (pH 7.5), 10 mm MgCl2, 1 mm DTT, 1 mm Na32PPi (25–50 cpm/pmol), 10 nm human erythrocyte AppBp1-Uba3 heterodimer, and the indicated concentrations of ATP, Nedd8, and 32PPi. After starting the reactions by addition of Nedd8, the assays were incubated at 37 °C for 20 min, then quenched by the addition of 0.5 ml of 5% (w/v) trichloroacetic acid containing 4 mm NaPPi followed by 300 μl of a 10% (w/v) charcoal slurry in 2% (w/v) trichloroacetic acid. The assays were centrifuged at 14,000 × g for 5 min, then the supernatant was removed by aspiration. The charcoal pellet was washed three times with 1 ml of 2% (w/v) trichloroacetic acid prior to quantitation of 32P radioactivity incorporated into ATP by Cerenkov radiation (29Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). HsUbc12 Transthiolation Kinetic Assays—Initial rates of AppBp1-Uba3 heterodimer-catalyzed transthiolation were assayed by monitoring formation of HsUbc12-125I-Nedd8 as describ" @default.
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• W1994608712 title "Conservation in the Mechanism of Nedd8 Activation by the Human AppBp1-Uba3 Heterodimer" @default.
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