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• W2074424881 abstract "A novel member of the ubiquitin carrier protein family, designated E2EPF, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2EPF catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2EPF was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2EPF involving auto- and multiubiquitination. A novel member of the ubiquitin carrier protein family, designated E2EPF, has been cloned by our laboratory and expressed in a bacterial system in an active form. Ubiquitin carrier proteins, or E2s, catalyze one step in a multistep process that leads to the covalent conjugation of ubiquitin to substrate proteins. In this paper, we show that recombinant E2EPF catalyzes auto/multiubiquitination, the conjugation of multiple ubiquitin molecules to itself. Multiubiquitination has been shown previously to be required for targeting of a substrate protein for rapid degradation. Using a rabbit reticulocyte lysate system, E2EPF was shown to support the degradation of a model substrate in an ATP- and ubiquitin-dependent fashion. In contrast to a previous study which showed that selective protein degradation in one system is dependent upon multiubiquitination via the lysine 48 residue of ubiquitin, multiubiquitination, and proteolytic targeting by E2EPF was shown here to be independent of the lysine 48 multiubiquitin linkage. This functional characterization of E2EPF revealed a combination of features that distinguishes this enzyme from all previously characterized members of the ubiquitin carrier protein family. These results also suggest several possible autoregulatory models for E2EPF involving auto- and multiubiquitination. Ubiquitin, a highly conserved 76-amino acid polypeptide, is present in all eukaryotic cells either in a free state or covalently conjugated to various cellular proteins(1.Hershko J. J. Biol. Chem. 1988; 263: 15237-15240Abstract Full Text PDF PubMed Google Scholar, 2.Jentsch S. Seufert W. Sommer T. Reins H. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Ub-protein conjugates are formed through an isopeptide linkage between the carboxyl-terminal glycine residue of ubiquitin and ε-amino groups of substrate proteins (3.Ciechanover A. Heller H. Elias S. Haas A.L. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1365-1368Crossref PubMed Scopus (375) Google Scholar, 4.Hershko A. Heller H. Eytan E. Kaklij G. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 7021-7025Crossref PubMed Scopus (158) Google Scholar). Ub-protein conjugates typically account for about 50% of total cellular ubiquitin (5.Haas A.L. Rechsteiner M. Ubiquitin. Plenum Publishing Corp., New York1988: 173-206Crossref Google Scholar) and have been localized to various cellular compartments including the cytosol(6.Haas A.L. Bright P.M. J. Biol. Chem. 1985; 260: 12464-12473Abstract Full Text PDF PubMed Google Scholar), nucleus(7.Busch H. Goldknopt I.L. Mol. Cell. Biochem. 1981; 840: 173-187Google Scholar), cell surface(8.St. John T. Gallatin W.M. Siegelman M. Smith H.T. Fried V.A. Weissman I.L. Science. 1986; 231: 845-850Crossref PubMed Scopus (110) Google Scholar), and mitochondrion(6.Haas A.L. Bright P.M. J. Biol. Chem. 1985; 260: 12464-12473Abstract Full Text PDF PubMed Google Scholar). Ubiquitin conjugation has been implicated in a variety of cellular functions. The best understood of these is the targeting of the substrate protein for selective degradation via a nonlysosomal pathway(9.Hershko A. Ciechanover A. Heller H. Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1783-1786Crossref PubMed Scopus (497) Google Scholar). Other cellular processes mediated by the ubiquitin conjugation system include DNA repair(10.Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (545) Google Scholar), cell cycle progression(11.Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar), regulation of chromatin structure(12.Goldknopt I.L. Busch H. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 864-868Crossref PubMed Scopus (308) Google Scholar), cell surface recognition(13.Siegelman M. Bond M.W. Gallatin W.M. St. John T. Smith H.T. Fried V.A. Weissman I.L. Science. 1986; 231: 823-829Crossref PubMed Scopus (205) Google Scholar), and regulation of transcription factors, e.g. NFκB(14.Palombella V.J. Rando O.J. Goldberg A.L. Maniatis T. Cell. 1994; 78: 773-785Abstract Full Text PDF PubMed Scopus (1915) Google Scholar). The formation of ubiquitin-protein conjugates involves a three-step process(15.Haas A.L. Warms J.V.B. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 16.Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). The first step is the ATP-dependent activation of ubiquitin by the 105-kDa ubiquitin activating (E1) ( 1The abbreviations used are: E1ubiquitin activating enzymeE2ubiquitin carrier proteinE3ubiquitin-protein isopeptide ligaseEPFendemic pemphigus foliaceusGSTglutathione S-transferaseDTTdithiothreitolPAGEpolyacrylamide gel electrophoresisFPLCfast protein liquid chromatographyUbubiquitinBSAbovine serum albuminrcmBSAreduced, carboxymethylated form of BSArmUbreductively methylated form of Ub.) enzyme (15.Haas A.L. Warms J.V.B. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar, 17.Ciechanover A. Heller H. Katz-Etzion R. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 761-765Crossref PubMed Scopus (158) Google Scholar) involving the formation of a thiol ester linkage between the ubiquitin carboxyl terminus and a thiol group of E1. In the second step, ubiquitin is transferred to the active site cysteine residue of a ubiquitin carrier protein (or E2). In the last step, an isopeptide bond is formed between the carboxyl terminus of ubiquitin and a lysyl ε-amino group within a substrate protein, a reaction catalyzed either directly by the E2 enzyme or via a third enzyme designated isopeptide ligase (E3). Enzymes designated ubiquitin isopeptidases have also been described which deubiquitinate conjugates(18.Wilkinson K.D. Lee K. Deshpande S. Duerksen-Hughes P. Boss J.M. Pohl J. Science. 1989; 246: 670-673Crossref PubMed Scopus (760) Google Scholar), consistent with evidence for the dynamic balance governing ubiquitin adduct pools(5.Haas A.L. Rechsteiner M. Ubiquitin. Plenum Publishing Corp., New York1988: 173-206Crossref Google Scholar). ubiquitin activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase endemic pemphigus foliaceus glutathione S-transferase dithiothreitol polyacrylamide gel electrophoresis fast protein liquid chromatography ubiquitin bovine serum albumin reduced, carboxymethylated form of BSA reductively methylated form of Ub. E2s exist as a family of isozymes that exhibit variability in terms of molecular weight, physiological function, substrate specificity, and dependence on E3 in in vitro systems(2.Jentsch S. Seufert W. Sommer T. Reins H. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 19.Pickart C.M. Rose I.A. J. Biol. Chem. 1985; 260: 1573-1581Abstract Full Text PDF PubMed Google Scholar, 20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). For example, in the yeast Saccharomyces cerevisiae, eight genes (designated UBC1-8) encoding distinct E2s have been described(2.Jentsch S. Seufert W. Sommer T. Reins H. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar, 21.Qin S. Nakajima B. Normura M. Arfin S. J. Biol. Chem. 1991; 266: 15549-15554Abstract Full Text PDF PubMed Google Scholar). The RAD6 (UBC2) protein functions in DNA repair, sporulation, and induced mutagenesis(10.Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (545) Google Scholar). CDC34 (UBC3) is an E2 of 24 kDa that is essential for G1/S transition during mitosis(11.Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (321) Google Scholar). The basic structure of E2s consists of a 153-amino acid core domain containing an active site cysteine within a highly conserved random coil segment. Additional carboxyl-terminal extension domains present on many isozymes are often acidic and generally show a high degree of sequence divergence, suggesting that they may play a role in substrate specificity during E3-independent conjugation or may contribute to the specificity of binding to cognate E3 isoforms. A subset of E2s has been shown to support multiubiquitination, a process in which successive ubiquitin molecules are linked by an isopeptide bond between a side chain amino group of one ubiquitin and the terminal carboxyl group of a second ubiquitin molecule. In vitro studies have demonstrated that E3-independent multiubiquitination by several E2 isozymes is sufficient for degradative targeting by the 26 S multicatalytic protease complex(22.Haas A.L. Reback P.M. Pratt G. Rechsteiner M. J. Biol. Chem. 1990; 265: 21664-21669Abstract Full Text PDF PubMed Google Scholar). In contrast, polyubiquitination refers to the conjugation of multiple ubiquitin molecules directly to different lysine residues within a single substrate molecule. Studies show that RAD6, CDC34, and the rabbit reticulocyte E232K catalyze E3-indepedent multiubiquitination and support E3-dependent ubiquitin conjugation; however, these enzymes differ in their linkage specificity for multiubiquitination, with CDC34 and E232K using Lys-48 (20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar) and RAD6 utilizing Lys-6(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). ( 2O. V. Baboshina and A. L. Haas, manuscript in preparation.) Multiubiquitination via lysine 48 of ubiquitin has also been demonstrated in the E220K of wheat germ (23.Nocker S.V. Vierstra R.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10297-10301Crossref PubMed Scopus (40) Google Scholar) and E225K from calf thymus(24.Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar). Formation of branched, multiubiquitin adducts have been shown to be involved in targeting of the substrate protein for selective degradation by an ATP-dependent protease complex(25.Mattews W. Tanaka K. Driscoll J. Ichihara A. Goldberg A.L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 2597-2601Crossref PubMed Scopus (141) Google Scholar). Chau and co-workers (26.Chau V. Tobias J.W. Bachmain A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1108) Google Scholar, 27.Gregori L. Poosch M.S. Cousin G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar) have shown that proteolytic targeting can be inhibited by preventing multiubiquitination via the lysine 48 residue of ubiquitin. A novel member of the E2 protein family, E2EPF, has recently been cloned from human keratinocytes(28.Liu Z. Diaz L.A. Haas A.L. Giudice G.J. J. Biol. Chem. 1992; 267: 15829-15835Abstract Full Text PDF PubMed Google Scholar). E2EPF is unique among E2s in that it contains a highly basic carboxyl-terminal extension domain. The E2EPF transcript was also shown to encode an antigenic polypeptide recognized by autoantibodies from pemphigus foliaceus patients. E2EPF is therefore the first member of the E2 enzyme family to be implicated in a disease process. The present paper reports the detailed enzymatic characterization of recombinant E2EPF, revealing a set of functional properties that distinguishes this isozyme from all other characterized E2s. E2EPF is shown to exhibit auto- and multiubiquitination activities. We further show that this E2 supports the ubiquitin-dependent protein degradation pathway in the absence of Lys-48 multiubiquitination. The latter observation indicates that multiubiquitination by linkage other than Lys-48 are competent degradative intermediates, supporting a role for subpopulations of ubiquitin having different linkage specificities within the overall pathway of ATP, ubiquitin-dependent protein degradation. Ubiquitin, rcmBSA (reduced, carboxymethylated form of BSA), UbK48R (site-directed mutant of Ub in which the lysine residue at position 48 has been replaced by arginine), and rmUb (reductively methylated form of ubiquitin in which all free amino groups are blocked by methyl groups) were prepared as described previously(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). Rabbit reticulocyte E1 and E214K were purified to homogeneity by a combination of affinity and high performance chromatography and then quantitated by stoichiometric activity assays(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Recombinant yeast RAD6 was expressed and purified as described previously(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). Protein concentrations were determined by the Bio-Rad dye binding assay using BSA as a standard. All other proteins and reagents were purchased from Sigma unless otherwise indicated. The removal of endogenous ubiquitin, E1, and various E2 isozymes from rabbit reticulocyte lysate was accomplished using previously described procedures(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 30.Ciechanover A. Hod Y. Hershko A. Biochem. Biophys. Res. Commun. 1978; 81: 1100-1105Crossref PubMed Scopus (422) Google Scholar). Rabbit reticulocyte lysate was depleted of ubiquitin according to a modified procedure for fraction II(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 30.Ciechanover A. Hod Y. Hershko A. Biochem. Biophys. Res. Commun. 1978; 81: 1100-1105Crossref PubMed Scopus (422) Google Scholar) followed by covalent affinity depletion of E1 and E2s as described previously(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). The resulting unadsorbed fraction from the ubiquitin affinity column (depleted fraction II) was determined to be quantitatively depleted of ubiquitin, E1, and E2 isozymes by its inability to support 125I-ubiquitin conjugation without supplementation by exogenous E1 and E214K and inability to support 125I-rcmBSA degradation without further addition of ubiquitin. Cloned E2EPF was expressed in Escherichia coli DH5α harboring the pGEXEPF5-ORF2B expression construct(28.Liu Z. Diaz L.A. Haas A.L. Giudice G.J. J. Biol. Chem. 1992; 267: 15829-15835Abstract Full Text PDF PubMed Google Scholar). Cells were grown at 37°C in LB medium containing 50 μg/ml ampicillin. When the culture reached A600 of 0.5, isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 0.5 mM. After an additional 3-h incubation, the cells were harvested by centrifugation and resuspended in 0.04 volume of solution TD (50 mM Tris-Cl (pH 7.5) and 1 mM DTT). The cells were lysed by passing the suspension twice through a French press at 1000 p.s.i. The cell lysate containing the glutathione S-transferase (GST)-E2EPF fusion protein was clarified by centrifugation at 105× g for 60 min at 4°C. The fusion protein was purified by glutathione-agarose affinity chromatography as described previously (31.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5043) Google Scholar). Ten ml of the clarified cell extract was mixed with 10 ml of 50% (v/v) glutathione-agarose (Sigma) suspension (in solution TD) in a 50-ml capped tube and rocked at 4°C for 30 min. The agarose beads were washed 5 times with 30 ml of solution TD. The washed beads were then poured into a column and washed again with 5 bed volumes of solution TD. The bound fusion protein was eluted with 10 mM glutathione (Sigma) in solution TD. The capacity of the glutathione-agarose affinity column was calculated to be approximately 1 mg of fusion protein per mg of the glutathione-agarose matrix under the conditions described. Isolation of E2EPF (cDNA-encoded polypeptide without the amino-terminal GST moiety) was accomplished as follows. The immobilized fusion protein was incubated with a highly purified preparation of thrombin (4000 NIH units/mg of protein; Sigma) at a concentration of 30 NIH units/ml in TD buffer resulting in elution of the E2EPF while the GST moiety of the fusion protein remained bound to the column. Optimal digestion conditions were 1 unit of thrombin per 5 μg of fusion protein at 25°C for 1-2 h. Longer incubations resulted in partial degradation of E2EPF (data not shown). To remove minor contaminants including thrombin, undigested fusion protein, and GST, the eluted fraction was subjected to further purification by anion exchange chromatography(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Prior to chromatography, the sample was filtered through a 0.2-μm membrane. The filtrate was chromatographed at 4°C on a Mono Q HR 5/10 anion exchange column equilibrated with solution TD using a Pharmacia Biotech Inc. fast protein liquid chromatography system(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Flow rate was maintained at 1 ml/min during sample loading and gradient elution. After sample injection, the column was washed with solution TD and eluted with a linear 0-0.5 M NaCl gradient having a slope of 12.5 mM/min. Eluted fractions were assayed by both SDS-PAGE and ubiquitin thiol ester formation(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). E2EPF consistently eluted as a single peak at 125 mM NaCl. A typical yield of E2EPF based on the thiol ester formation assay was approximately 10 nmol/liter bacterial culture. Purified E2EPF was stored at −80°C. Under these conditions, E2EPF retained full activity for over 8 months and after two cycles of freeze thawing. Purified E2EPF was assayed for the ability to form a thiol ester linkage with ubiquitin, according to the procedure of Haas and Bright(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Briefly, E2EPF was incubated with 125I-ubiquitin (5 μM, 5-10 × 104 cpm/pmol) in the presence of 10 nM purified reticulocyte E1, 20 IU/ml inorganic pyrophosphatase, 2 mM ATP, 5 mM MgCl2, 0.5 mM DTT, and 50 mM Tris-Cl (pH 7.5). After a 1-min incubation at 37°C, the reaction was stopped by addition of an equal volume of 2 × SDS-PAGE sample buffer without 2-mercaptoethanol. Replicate-quenched samples were boiled for 1 min in the presence of 5% 2-mercaptoethanol to cleave the 125I-ubiquitin thiol ester(16.Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar). All samples were then resolved by SDS-PAGE (12% acrylamide), and 125I-ubiquitin-containing bands were visualized by autoradiography(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Quantification of E2EPF activity was accomplished using a modification of the above thiol ester assay. One-minute incubations containing different amounts of E2EPF were analyzed by electrophoresis and autoradiography. Thiol ester bands were cut from the gel and quantified by γ counting on an automatic γ counter (Micromedic 4/600 Plus, ICN Micromedic Systems Inc.). The absolute content of thiol ester was calculated based on the specific activity of 125I-ubiquitin(32.Haas A.L. Rose A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). Conjugation assays for measuring steady state levels of conjugates were performed at 37°C for 60 min as described previously(29.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar, 33.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). The assay mixtures contained 50 mM Tris-Cl (pH 7.5), 2 mM ATP, 0.5 mM DTT, 10 mM MgCl2, 20 IU/ml inorganic pyrophosphatase, various concentrations of 125I-ubiquitin (native, UbK48R, or rmUb), purified rabbit reticulocyte E1, and purified E2 (E214K, E2EPF, or CDC34). The reaction products were resolved by SDS-PAGE (12% acrylamide) and visualized by autoradiography. A similar approach was used to assay the conjugation of ubiquitin to endogenous reticulocyte proteins. Assays contained 50 mM Tris-Cl (pH 7.5), 2 mM ATP, 10 mM MgCl2, 0.5 mM DTT, 20 IU/ml inorganic pyrophosphatase, 5 μM125I-ubiquitin (4-8 × 104 cpm/pmol), 10 nM E1, indicated concentrations of E2s, and the depleted fraction II (158 μg) as a source of both E3 and substrates. Degradation of reduced, carboxymethylated BSA (125I-rcmBSA) was quantified by generation of trichloroacetic acid-soluble radioactivity similar to that described previously(9.Hershko A. Ciechanover A. Heller H. Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1980; 77: 1783-1786Crossref PubMed Scopus (497) Google Scholar). Briefly, the reactions of 50 μl total volume containing 50 mM Tris-Cl (pH 7.5), 0.5 mM DTT, 10 mM MgCl2, 2 mM ATP, 20 IU/ml creatine phosphokinase, 5 μM ubiquitin, 4 μM125I-rcmBSA (4 × 104cpm/pmol), 10 nM E1, the indicated concentrations of E2s, and 158 μg of depleted fraction II were incubated at 37°C for 60 min. The reactions were stopped by addition of 100 μl of 5 mg of BSA/ml as carrier followed by 150 μl of ice-cold 16% trichloroacetic acid. Following a 10-min precipitation on ice, the samples were centrifuged at 4°C for 5 min in an Eppendorf microcentrifuge. The level of radioactivity in each 200 μl of supernatant was determined by γ counting using a Micromedic 4/600 Plus Automatic Gamma Counter (ICN Micromedic Systems, Inc.). Acid-soluble radioactivity present in the blank was subtracted from each reading, and the results were expressed as the net counts/min. The coding region of the E2EPF cDNA was subcloned into the prokaryotic expression vector, pGEX-2T (Pharmacia), to generate construct pGEXEPF5-ORF2B as described previously(5.Haas A.L. Rechsteiner M. Ubiquitin. Plenum Publishing Corp., New York1988: 173-206Crossref Google Scholar). The fusion protein encoded by this construct consists of the full-length 24-kDa E2EPF fused to the carboxyl terminus of a 26-kDa segment of GST. The recombinant protein was purified using the glutathione-agarose affinity chromatography procedure essentially as described by Smith and Johnson(31.Smith D.B. Johnson K.S. Gene (Amst.). 1988; 67: 31-40Crossref PubMed Scopus (5043) Google Scholar). As shown in Fig. 1, lanes 1 and 2, this single step affinity purification yielded an electrophoretically homogeneous preparation of the GST-E2EPF fusion protein. Separation of the GST and E2EPF moieties of the fusion protein was accomplished by thrombin digestion followed by Mono Q anion exchange fast protein liquid chromatography as described under “Materials and Methods.” The purity of the resulting E2EPF polypeptide was demonstrated by electrophoretic analysis (Fig. 1, lane 3). The apparent molecular weight of the E2EPF polypeptide agreed with the size predicted by sequence analysis. The Mono Q chromatography step resolved intact E2 from minor fragmentation products generated during thrombin processing (not shown). Note that E2EPF exhibits a more intense level of staining by the silver technique relative to that of an equal mass of GST-E2EPF fusion protein (Fig. 1, lanes 2 and 3). This is attributable to differences in mole amounts between the two proteins when normalized for equal mass and a greater mole fraction of lysine for E2EPF compared to GST, since lysine serves as the site of silver deposition. The GST-E2EPF fusion protein is catalytically active in forming the corresponding 125I-ubiquitin thiol ester in the presence of E1(28.Liu Z. Diaz L.A. Haas A.L. Giudice G.J. J. Biol. Chem. 1992; 267: 15829-15835Abstract Full Text PDF PubMed Google Scholar). Fig. 2 shows that recombinant E2EPF processed from GST by thrombin and subsequently purified by Mono Q FPLC is also active in ubiquitin thiol ester formation (lane 5). The amount of thiol ester formed to free E2EPF, determined by quantification of 125I radioactivity associated with the corresponding band in lane 5 of Fig. 2(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar), agreed with that predicted from the mass of E2EPF protein determined as described under “Materials and Methods.” The 125I-ubiquitin thiol ester adducts of both E2EPF and GST-E2EPF formed in incubations parallel to those of Fig. 2 were quantitatively labile to reducing conditions in the presence of 2-mercaptoethanol (not shown), confirming that the associated 125I-ubiquitin was in thiol ester linkage(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). We consistently noted that, at equimolar concentrations, free E2EPF formed more ubiquitin thiol ester than did the GST-E2EPF fusion protein (Fig. 2, lanes 4 and 5). The amount of thiol ester formed to the fusion protein did not increase on longer incubation and thus did not result from a lower rate of transthiolation from the E1 ternary complex (not shown). The lower level of GST-E2EPF thiol ester also did not result from inactivation since the predicted amount of free E2EPF thiol ester, based on protein determination, was observed if the fusion protein was first incubated with thrombin (not shown). These results indicate that the presence of GST at the amino terminus of E2EPF sterically alters the equilibrium constant rather than the rate of thiol ester formation from the E1 ternary complex. The results of Fig. 2 demonstrate that transthiolation from the E1 ternary complex to either free E2EPF or the GST fusion is complete within the 1.5-min incubation time employed, as is typical of the analogous loading reaction with other members of the E2 family(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). On longer incubation with free E2EPF, a series of higher molecular weight 125I-ubiquitin adducts was consistently observed on nonreducing thiol ester gels (Fig. 3A). Unlike the 125I-ubiquitin-E2EPF thiol ester formed following short incubation (Fig. 2), the higher molecular weight bands formed during prolonged incubation were stable to reducing conditions, Fig. 3B, indicating that they represented peptide conjugates to radioiodinated ubiquitin. In addition, a small fraction of the monoubiquitin adduct was also stable to reducing conditions and showed a time-dependent accumulation on prolonged incubation. Therefore, the E2-Ub1 band in Fig. 3 represents a mixture of 125I-ubiquitin thiol ester and conjugate adducts at long incubation times but principally only thiol esters at short times. The relative molecular weights of the ladder of ubiquitin conjugates corresponded to integer multiples of the molecular weight for ubiquitin ligated to free E2EPF. The bands present under reducing conditions in Fig. 3B were confirmed as E2EPF-ubiquitin conjugates by comigration of identical bands when similar incubations containing unlabeled ubiquitin were analyzed on immunoblots probed with either anti-E2EPF or antiubiquitin antibodies (not shown). Thus, E2EPF catalyzes an E3-independent autoubiquitination reaction resulting in the formation of multiubiquitin homopolymer chains. Accumulation of autoubiquitinated E2EPF adducts of molecular weights greater than that predicted for monoubiquitination of all 17 lysines within E2EPF (Fig. 3) indicates that some or all of the attached ubiquitin moieties are present as multiubiquitin chains. To demonstrate this conclusively, E2EPF autoubiquitination was carried out in the presence of 125I-rmUb which is incapable of supporting chain elongation due to the absence of primary lysyl ε-amines(20.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar, 33.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). Inability to form adducts above E2EPF-Ub2 (Fig. 4, lane 2), under conditions for which there is accumulation of these products with wild-type ubiquitin (lane 1), demonstrates that ubiquitin moieties ligated to E2EPF can" @default.
• W2074424881 created "2016-06-24" @default.
• W2074424881 creator A5002217144 @default.
• W2074424881 creator A5030741486 @default.
• W2074424881 creator A5032889074 @default.
• W2074424881 creator A5073051739 @default.
• W2074424881 creator A5081765476 @default.
• W2074424881 date "1996-02-01" @default.
• W2074424881 modified "2023-09-28" @default.
• W2074424881 title "Characterization of a Novel Keratinocyte Ubiquitin Carrier Protein" @default.
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