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• W2036727875 abstract "Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys → Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd≤ 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase. Targeting of substrates for degradation by the ATP, ubiquitin-dependent pathway requires formation of multiubiquitin chains in which the 8.6-kDa polypeptide is linked by isopeptide bonds between carboxyl termini and Lys-48 residues of successive monomers. Binding of Lys-48-linked chains by subunit 5 of the 26 S proteasome regulatory complex commits the attached target protein to degradation with concomitant release of free ubiquitin monomers following disassembly of the chains. Point mutants of ubiquitin (Lys → Arg) were used to map the linkage specificity for ubiquitin-conjugating enzymes previously demonstrated to form novel multiubiquitin chains not attached through Lys-48. Recombinant human E2EPF catalyzed multiubiquitin chain formation exclusively through Lys-11 of ubiquitin while recombinant yeast RAD6 formed chains linked only through Lys-6. Multiubiquitin chains linked through Lys-6, Lys-11, or Lys-48 each bound to subunit 5 of partially purified human 26 S proteasome with comparable affinities. Since chains bearing different linkages are expected to pack into distinct structures, competition between Lys-11 and Lys-48 chains for binding to subunit 5 demonstrates that the latter possesses determinants for recognizing alternatively linked chains and precludes the existence of subunit 5 isoforms recognizing distinct structures. In addition, competition studies provided an estimate of Kd≤ 18 nM for the intrinsic binding of Lys-48-linked chains of linkage number n > 4. This result suggests that the principal mechanistic advantage of multiubiquitin chain formation is to enhance the affinity of the associated substrate for the 26 S complex relative to that of unconjugated target protein. Complementation studies with E1/E2-depleted rabbit reticulocyte extract demonstrated RAD6 supported isopeptide ligase-dependent degradation only through Lys-48-linked chains, while E2EPF retained the ability to target a model radiolabeled substrate through Lys-11-linked chains. Therefore, the linkage specificity exhibited by these E2 isozymes depends on their catalytic context with respect to isopeptide ligase. ATP-dependent conjugation of ubiquitin to protein targets is currently recognized to mediate a variety of cellular processes by signaling selective degradation of the latter through the 26 S proteasome pathway, reviewed most recently in (1.Driscoll J. Histol. Histopathol. 1994; 9: 197-202PubMed Google Scholar) and (2.Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1580) Google Scholar). Among the cellular targets serving as substrates for this unique post-translational modification are various proteins exhibiting either constitutive or conditional short half-lives including cyclins(3.Glotzer M. Murray A.W. Kirschner M.W. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1881) Google Scholar, 4.Hershko A. Ganoth D. Sudakin V. Dahan A. Cohen L.H. Luca F.C. Ruderman J.V. Eytan E. J. Biol. Chem. 1994; 269: 4940-4946Abstract Full Text PDF PubMed Google Scholar, 5.Deshaies R.J. Chau V. Kirschner M. 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Oncogene. 1995; 10: 1849-1854PubMed Google Scholar), and proteins of abnormal structure(19.Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (400) Google Scholar, 20.Ciechanover A. Finley D. Varshavsky A. Cell. 1984; 37: 57-66Abstract Full Text PDF PubMed Scopus (373) Google Scholar, 21.Hershko A. Eytan E. Ciechanover A. Haas A.L. J. Biol. Chem. 1982; 257: 13964-13970Abstract Full Text PDF PubMed Google Scholar). In all cases, the signal for ubiquitination probably requires transient exposure of one or more lysines that can serve as sites for recognition and attachment of the polypeptide. For certain targets, enhanced steric accessibility of sensitive lysines arising by minute conformational changes (22.Dunten R.L. Cohen R.E. Gregori L. Chau V. J. Biol. Chem. 1991; 266: 3260-3267Abstract Full Text PDF PubMed Google Scholar, 23.Sokolik C.W. Cohen R.E. J. Biol. Chem. 1991; 266: 9100-9107Abstract Full Text PDF PubMed Google Scholar, 24.Hill C.P. Johnston N.L. Cohen R.E. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4136-4140Crossref PubMed Scopus (35) Google Scholar) or more global folding transitions (21.Hershko A. Eytan E. Ciechanover A. Haas A.L. J. Biol. Chem. 1982; 257: 13964-13970Abstract Full Text PDF PubMed Google Scholar, 25.Dunten R.L. Cohen R.E. J. Biol. Chem. 1989; 264: 16739-16747Abstract Full Text PDF PubMed Google Scholar) may be accompanied by unmasking of specific amino-terminal residues that dispose the protein to recognition by relevant isopeptide ligases (E3) that confer specificity(4.Hershko A. Ganoth D. Sudakin V. Dahan A. Cohen L.H. Luca F.C. Ruderman J.V. Eytan E. J. Biol. Chem. 1994; 269: 4940-4946Abstract Full Text PDF PubMed Google Scholar, 26.Reiss Y. Hershko A. J. Biol. Chem. 1990; 265: 3685-3690Abstract Full Text PDF PubMed Google Scholar, 27.Heller H. Hershko A. J. Biol. Chem. 1990; 265: 6532-6535Abstract Full Text PDF PubMed Google Scholar, 28.Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar, 29.Raboy B. Kulka R.G. Eur. J. Biochem. 1994; 221: 247-251Crossref PubMed Scopus (8) Google Scholar, 30.Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1939) Google Scholar). In the case of cyclins, discrete recognition signals are conserved among related isoforms and within unrelated proteins(6.Yaglom J. Linskens M.H. Sadis S. Rubin D.M. Futcher B. Finley D. Mol. Cell. Biol. 1995; 15: 731-741Crossref PubMed Google Scholar, 31.Galan J.M. Volland C. Urban-Grimal D. Haguenauer-Tsapis R. Biochem. Biophys. Res. Commun. 1994; 201: 769-775Crossref PubMed Scopus (37) Google Scholar), although the precise mechanism by which these sequences contribute to substrate recognition by relevant conjugating enzymes has not been well elucidated. Attachment of single ubiquitin moieties to target proteins effects a modest rate of degradation by the 26 S proteasome(32.Haas A. Reback P.M. Pratt G. Rechsteiner M. J. Biol. Chem. 1990; 265: 21664-21669Abstract Full Text PDF PubMed Google Scholar, 33.Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 34.Finley D. Sadis S. Monia B.P. Boucher P. Ecker D.J. Crooke S.T. Chau V. Mol. Cell. Biol. 1994; 14: 5501-5509Crossref PubMed Scopus (299) Google Scholar); however, more robust signals for degradative targeting require subsequent formation of multiubiquitin homopolymers by chain elongation from the initial polypeptide conjugate(32.Haas A. Reback P.M. Pratt G. Rechsteiner M. J. Biol. Chem. 1990; 265: 21664-21669Abstract Full Text PDF PubMed Google Scholar, 33.Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 35.Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1096) Google Scholar). Considerable recent work has demonstrated that these multiubiquitin chains are formed by a repeating structure in which the carboxyl terminus of each ubiquitin is linked to Lys-48 of the preceding ubiquitin(33.Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 35.Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1096) Google Scholar). The crystal structure of the resulting multiubiquitin chain exhibits considerable packing order and symmetry that is thought essential for recognition by the S5 subunit of the regulatory complex capping the 26 S proteasome (36.Cook W.J. Jeffrey L.C. Kasperek E. Pickart C.M. J. Mol. Biol. 1994; 236: 601-609Crossref PubMed Scopus (129) Google Scholar, 37.Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). This model is supported by mutagenesis studies identifying essential ubiquitin residues required for both multiubiquitin chain binding to S5 and for subsequent degradative targeting(38.Pickart C. Beal R. Deveraux Q. Xia G. Rechsteiner M. FASEB J. 1995; 9: 1473Crossref PubMed Google Scholar). Ubiquitin-mediated proteolysis has been most extensively studied in yeast and rabbit reticulocytes. Within these systems, the almost quantitative inhibition of ATP-dependent degradation accompanying substitution of rmUb ( 1The abbreviations used are: rmUbreductively methylated ubiquitinBSAbovine serum albuminE1ubiquitin activating enzymeE2ubiquitin carrier protein (subscript denotes relative molecular weight or isozyme)E3ubiquitin:protein isopeptide ligasercmBSAreduced carboxymethylated BSAPAGEpolyacrylamide gel electrophoresisUbubiquitin.) or UbK48R for wild type polypeptide demonstrates that a significant fraction of degradative flux proceeds through conjugated intermediates bearing Lys-48-linked multiubiquitin chains since neither rmUb nor UbK48R supports chain elongation(33.Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 35.Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1096) Google Scholar). However, mounting evidence supports the existence of multiubiquitin chains bearing linkage specificities distinct from Lys-48. Purified recombinant yeast RAD6, a member of the ubiquitin carrier protein (E2) isozyme family, catalyzes multiubiquitin chain formation to core histones in the presence of only ubiquitin activating enzyme (E1) to maintain the E2 active site cysteine charged with ubiquitin thiolester(39.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). The linkage specificity for these chains does not require Lys-48 since rmUb but not UbK48R blocks the characteristic ladder of conjugates revealed by SDS-PAGE(39.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). Similar results supporting Lys-48-independent chains have more recently been obtained with recombinant E2EPF, an isoform cloned from human keratinocytes using autoantibodies obtained from pemphigous foliaceus patients(64.Liu Z. Haas A.L. Conrad C.A. Diaz L.A. Giudice G.J. J. Biol. Chem. 1996; 271: 2817-2822Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar). Finally, stable Lys-63-linked chains requiring participation of RAD6 (UBC2) have been observed in yeast and proposed to account in part for the DNA repair function of this E2 isoform(40.Spence J. Sadis S. Haas A.L. Finley D. Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar). Since RAD6 normally supports Lys-48 chain-dependent degradation in yeast within the N-end rule pathway(41.Dohmen R.J. Madura K. Bartel B. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 7351-7355Crossref PubMed Scopus (202) Google Scholar, 42.Blumenfeld N. Gonen H. Mayer A. Smith C.E. Siegel N.R. Schwartz A.L. Ciechanover A. J. Biol. Chem. 1994; 269: 9574-9581Abstract Full Text PDF PubMed Google Scholar), the latter observation suggests linkage specificity may be context specific and depend on either the target protein or, more likely, the cognate E3 required for conjugation. reductively methylated ubiquitin bovine serum albumin ubiquitin activating enzyme ubiquitin carrier protein (subscript denotes relative molecular weight or isozyme) ubiquitin:protein isopeptide ligase reduced carboxymethylated BSA polyacrylamide gel electrophoresis ubiquitin. The currently accepted paradigm for target protein conjugation requires ubiquitin activating enzyme, a ubiquitin carrier protein, and ubiquitin:protein isopeptide ligase. The metabolic significance of E3-independent conjugation by certain members of the E2 family remains uncertain, although conjugates formed in the absence of E3 by yeast RAD6 and CDC34 as well as the rabbit reticulocyte isoform E232k are substrates for 26 S proteasome-mediated degradation(32.Haas A. Reback P.M. Pratt G. Rechsteiner M. J. Biol. Chem. 1990; 265: 21664-21669Abstract Full Text PDF PubMed Google Scholar). In the present studies, mutagenesis of the lysine residues present on ubiquitin have allowed the assignment of linkage specificity for multiubiquitin chain formation by RAD6 and E2EPF. Other results indicate that these alternatively linked chains are recognized by subunit 5 of the 26 S proteasome, suggesting target proteins marked by such homopolymer structures may be degradative intermediates. Finally, reconstitution experiments with rabbit reticulocyte extracts demonstrate that alternatively linked chains are competent in the overall degradative pathway. These results define a functional role for alternatively linked chains and serve as a basis for future mechanistic studies with the cognate E3 isoforms. Inorganic pyrophosphatase (high pressure liquid chromatography purified) was obtained from Sigma. Carrier-free Na125I and [2,8-3H]ATP were purchased from DuPont NEN. Lysine 48-linked diubiquitin generated by recombinant E225K(43.Chen Z. Pickart C.M. J. Biol. Chem. 1990; 265: 21835-21842Abstract Full Text PDF PubMed Google Scholar) was the generous gift of Dr. Cecile Pickart (Johns Hopkins University). The monomer concentration of diubiquitin was determined spectrophotometrically from the extinction coefficient of free polypeptide(44.Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). Homogeneous wild type, di-, and mutant ubiquitins were radioiodinated by the chloramine-T procedure(45.Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (106) Google Scholar). Recrystallized BSA was obtained from Pentex and used for the preparation of 125I-rcmBSA(46.Evans Jr., A.C. Wilkinson K.D. Biochemistry. 1985; 24: 2915-2923Crossref PubMed Scopus (25) Google Scholar). Rabbit reticulocyte-rich whole blood was generated by phenylhydrazine induction and used to generate fraction II (45.Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (106) Google Scholar). A portion of fraction II was used to prepare apparently homogeneous E232k and E1/E2-depleted fraction II(39.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar, 47.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar). Rabbit liver E1 was purified to apparent homogeneity by adapting reported affinity chromatography/fast protein liquid chromatography methods reported previously (47.Haas A.L. Bright P.M. J. Biol. Chem. 1988; 263: 13258-13267Abstract Full Text PDF PubMed Google Scholar) and quantitated by the stoichiometric formation of ubiquitin [3H]adenylate(48.Haas A.L. Warms J.V. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar). Homogeneous native histone H2B (generous gift of Dr. Vaughn Jackson, Medical College of Wisconsin) and recombinant yeast CDC34 (UBC3) and RAD6 (UBC2) were those reported previously(49.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). Bovine erythrocyte ubiquitin was purchased from Sigma as a lyophilized powder. Although previous lots of this polypeptide were sufficiently homogeneous for use without additional purification, recent lots have consistently contained several contaminating proteins that preclude accurate spectrophotometric quantitation of ubiquitin and obviate its direct use following radioiodination. Therefore, commercial preparations were additionally purified by modification of published procedures(44.Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). Commercial ubiquitin was dissolved in water to a final concentration of 5 mg/ml and then titrated to pH 4.5 (4°C) with glacial acetic acid. Aliquots were applied to an HR 10/10 Mono S cation exchange fast protein liquid chromatography column (Pharmacia) equilibrated in 25 mM ammonium acetate (pH 4.5). Ubiquitin eluted as a single, symmetric peak at 0.29 M NaCl within a linear gradient of 5 mM/ml (2 ml/min). Ubiquitin-containing fractions were pooled and dialyzed overnight against distilled water using tubing having an exclusion limit of 3.5 kDa. The resulting sample was concentrated by lyophilization and then dissolved in a minimum amount of distilled water. Ubiquitin (>99% pure) was quantitated spectrophotometrically using an empirically determined 280-nm extinction coefficient of 0.16 ml/mg•cm(44.Haas A.L. Wilkinson K.D. Prep. Biochem. 1985; 15: 49-60PubMed Google Scholar). A portion of the homogeneous ubiquitin was used to prepare rmUb as described previously (49.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). Single-site mutagenesis of each lysine residue present within ubiquitin was accomplished by the polymerase chain reaction-based overlap extension method of Ho et al.(50.Ho S.N. Hunt H.D. Horton R.M. Pullen J.K. Pease L.R. Gene (Amst.). 1989; 77: 51-59Crossref PubMed Scopus (6771) Google Scholar) using the pPLhUb mutagenesis/expression plasmid described earlier(51.Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (78) Google Scholar). Following the final amplification step, the mutant polymerase chain reaction product was restricted with NdeI/SapI then ligated into NdeI/SapI-restricted pPLhUb. Generation of the predicted mutants was confirmed by dideoxy sequencing the complete ubiquitin coding region of the resulting pPLhUb constructs. Mutant ubiquitins were expressed in the Escherichia coli AR58 strain by heat induction and purified to apparent homogeneity without modification(51.Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (78) Google Scholar). Ubiquitin concentrations were determined spectrophotometrically as for wild type polypeptide. Typical yields were in the range of 10 mg/liter of culture for all mutants except UbK29R, which gave a consistent yield of approximately 0.5 mg/liter of culture. In all subsequent applications, all mutants displayed stabilities comparable to wild type polypeptide. In addition, all mutants exhibited CD spectra between 190 and 260 nm, indistinguishable from that of wild type ubiquitin (not shown). Initial rates of histone H2B monoubiquitination were measured for wild type and mutant ubiquitins as described(51.Burch T.J. Haas A.L. Biochemistry. 1994; 33: 7300-7308Crossref PubMed Scopus (78) Google Scholar). Briefly, various concentrations of wild type or mutant radioiodinated ubiquitin (ca. 2-4 × 103 cpm/pmol) were incubated at 37°C in reactions of 25 μl, final volume, containing 50 mM Tris-Cl (pH 8.0), 2 mM ATP, 10 mM MgCl2, 1 mM dithiothreitol, 10 mM creatine phosphate, 15 μM H2B, 1 IU creatine phosphokinase, 1 IU inorganic pyrophosphatase, 1 nM E1, and 20 nM E232k. In addition, all reactions contained 0.5 mg/ml BSA as a carrier protein to prevent adsorption of the enzymes to the reaction tubes. Reactions were quenched by addition of 25 μl of SDS sample buffer containing 3% (v/v) β-mercaptoethanol and then boiled for 5 min. Following SDS-PAGE resolution, the monoubiquitinated H2B band was excised and associated radioactivity was determined by γ counting(45.Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (106) Google Scholar). Data were corrected for radioactivity present in an identical section of a control lane derived from an incubation performed in the absence of E1 and E232k. The incubation conditions were chosen to be E1 limiting, indicated by a linear dependence of initial rate on [E1], to kinetically isolate the ubiquitin-dependent activation step. The linkage specificity for multiubiquitin chain formation catalyzed by recombinant CDC34, RAD6, and E2EPF was determined in incubations similar to those for the kinetic assays with the exception that E1 and E2 concentrations were empirically adjusted to be rate-limiting with respect to E2 to kinetically isolate the step of multiubiquitin chain elongation, and histone was present only in the experiment testing RAD6 to serve as a substrate for conjugation(39.Haas A.L. Reback P.B. Chau V. J. Biol. Chem. 1991; 266: 5104-5112Abstract Full Text PDF PubMed Google Scholar). Each incubation was conducted under initial velocity conditions with 5 μM radioiodinated wild type or mutant ubiquitin. Following resolution by 12% SDS-PAGE, the pattern of conjugates was visualized by autoradiography. Correction for slight differences in the specific activities of the radioiodinated proteins was achieved by either normalizing for exposure at constant sample volume (Fig. 1) or adjusting the sample volume at constant exposure (Figure 2:, Figure 3:, Figure 4:).Figure 4:Subunit 5 of the 26 S proteasome binds alternatively linked multiubiquitin chains. Aliquots of partially purified 26 S proteasome (25 μg) were resolved by 10% SDS-PAGE and either stained with Coomassie Blue (left lane) or transferred to nitrocellulose and incubated with radiolabeled chains of the indicated linkage type as described under “Materials and Methods.” Positions of subunit 5 and the 100-kDa putative isopeptidase-T bands are indicated to the left. Positions of molecular weight markers are shown to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 3:Determination of RAD6 linkage specificity. The linkage specificity for RAD6-catalyzed ubiquitination of histone H2B was examined in incubations similar to those described in the legend to Fig. 1 except that reactions were for 20 min in the presence of 10 nM E1, 20 nM recombinant RAD6, and 12 μM H2B. Linkage number for multiubiquitin chains is shown to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 2:Determination of E2EPF linkage specificity. The linkage specificity for E2EPF-catalyzed autoubiquitination was examined in incubations similar to those described in the legend to Fig. 1 except that reactions were for 20 min in the presence of 20 nM E1 and 30 nM recombinant E2EPF. Linkage number for multiubiquitin chains is shown to the right.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The 26 S proteasome was partially purified from human erythrocytes by the method of Hough et al.(52.Hough R. Pratt G. Rechsteiner M. J. Biol. Chem. 1987; 262: 8303-8313Abstract Full Text PDF PubMed Google Scholar). Proteolytic activity was monitored with the fluorogenic peptide N-succinyl-leucyl-leucyl-valyl-tyrosyl-7-amido-4-methylcoumarin. Fractions exhibiting ATP-dependent hydrolysis of the peptide substrate were pooled and used in the direct conjugate binding assay of Deveraux et al.(37.Deveraux Q. Ustrell V. Pickart C. Rechsteiner M. J. Biol. Chem. 1994; 269: 7059-7061Abstract Full Text PDF PubMed Google Scholar). Briefly, 25 μg of purified 26 S proteasome per lane was resolved by 10% SDS-PAGE and then electrophoretically transferred to BA83 nitrocellulose (Schleicher and Schuell)(53.Haas A.L. Bright P.M. J. Biol. Chem. 1985; 260: 12464-12473Abstract Full Text PDF PubMed Google Scholar). Excess nitrocellulose binding sites were blocked by incubation for 1 h with 50 mM Tris-Cl (pH 7.5) containing 0.15 M NaCl and 5% (w/v) powdered milk. The blots were then incubated for 1.5 h in 50 mM Tris-Cl (pH 7.5) containing 0.15 M NaCl, 25 mg/ml BSA, and 3 × 104 cpm/ml multiubiquitin chain prepared using 125I-ubiquitin and the indicated E2 isozyme. Nonspecifically bound label was removed by four successive 5-min washes in 50 mM Tris-Cl (pH 7.5) containing 0.15 M NaCl, the second and third of which additionally contained 0.05% (v/v) Triton X-100. Specifically bound label was visualized by autoradiography. Binding quantitation was achieved by excising the S5 band and determining associated 125I label by γ counting. Radioactivity was corrected for nonspecifically bound label associated with an equally sized portion of the blot not containing the S5 band. The ability of wild type or mutant ubiquitins to support ATP, ubiquitin-dependent degradation of 125I-rcmBSA in the presence of the three E2 isozymes was assayed using E1/E2-depleted rabbit reticulocyte fraction II(45.Haas A.L. Rose I.A. Proc. Natl. Acad. Sci. U. S. A. 1981; 78: 6845-6848Crossref PubMed Scopus (106) Google Scholar, 49.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). Fraction II was depleted of endogenous E1 and E2 isozymes by passage through an Affi-Gel 10 affinity column containing 5 mg of ubiquitin/ml bed volume (49.Haas A.L. Bright P.M. Jackson V.E. J. Biol. Chem. 1988; 263: 13268-13275Abstract Full Text PDF PubMed Google Scholar). Initial rates of degradation were assayed in triplicate for 1 h at 37°C in a final volume of 50 μl containing 50 mM Tris-Cl (pH 8.0), 2 mM ATP, 10 mM MgCl2, 1 mM dithiothreitol, 1 IU creatine phosphokinase, 10 mM creatine phosphate, 300 μg of depleted fraction II, 1 μM125I-rcmBSA (3 × 105 cpm), 20 μM wild type or mutant ubiquitin, 40 nM E1, and the indicated concentration of E2 isozyme. Depleted fraction II exhibited negligible ubiquitin-dependent degradation of 125I-rcmBSA in the absence of added affinity-" @default.
• W2036727875 created "2016-06-24" @default.
• W2036727875 creator A5030741486 @default.
• W2036727875 creator A5044714245 @default.
• W2036727875 date "1996-02-01" @default.
• W2036727875 modified "2023-10-14" @default.
• W2036727875 title "Novel Multiubiquitin Chain Linkages Catalyzed by the Conjugating Enzymes E2EPF and RAD6 Are Recognized by 26 S Proteasome Subunit 5" @default.
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