Title: Identification of Amino Acid Residues in a Class I Ubiquitin-conjugating Enzyme Involved in Determining Specificity of Conjugation of Ubiquitin to Proteins
Abstract: The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities. The ubiquitin pathway is a major system for selective proteolysis in eukaryotes. However, the mechanisms underlying substrate selectivity by the ubiquitin system remain unclear. We previously identified isoforms of a rat ubiquitin-conjugating enzyme (E2) homologous to the Saccharomyces cerevisiae class I E2 genes, UBC4/UBC5. Two isoforms, although 93% identical, show distinct features. UBC4-1 is expressed ubiquitously, whereas UBC4-testis is expressed in spermatids. Interestingly, although these isoforms interacted similarly with some ubiquitin-protein ligases (E3s) such as E6-AP and rat p100 and an E3 that conjugates ubiquitin to histone H2A, they also supported conjugation of ubiquitin to distinct subsets of testis proteins. UBC4-1 showed an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction. Site-directed mutagenesis of the UBC4-testis isoform was undertaken to identify regions of the molecule responsible for the observed difference in substrate specificity. Four residues (Gln-15, Ala-49, Ser-107, and Gln-125) scattered on surfaces away from the active site appeared necessary and sufficient for UBC4-1-like conjugation. These four residues identify a large surface of the E2 core domain that may represent an area of binding to E3s or substrates. These findings demonstrate that a limited number of amino acid substitutions in E2s can dictate conjugation of ubiquitin to different proteins and indicate a mechanism by which small E2 molecules can encode a wide range of substrate specificities. The ubiquitin system is implicated in an ever expanding array of cellular processes that now range from DNA repair to cell cycle progression and muscle protein degradation (reviewed in Refs. 1Ciechanover A. Cell. 1994; 79: 13-21Abstract Full Text PDF PubMed Scopus (1602) Google Scholar, 2Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1461) Google Scholar, 3Coux O. Tanaka K. Goldberg A.L. Annu. Rev. Biochem. 1996; 65: 801-847Crossref PubMed Scopus (2239) Google Scholar). Ubiquitin is a highly conserved 76-residue protein whose many cellular functions are mediated by its covalent ligation to other proteins. Most of these functions arise from the ability of ubiquitination to lead to degradation of the selected protein. Indeed, ubiquitin-mediated proteolysis is responsible for the turnover of key regulatory proteins, including mitotic cyclins (cyclin B) (4Hershko A. Ganoth D. Pehrson J. Palazzo R.E. Cohen L.H. J. Biol. Chem. 1991; 266: 16376-16379Abstract Full Text PDF PubMed Google Scholar, 5King R.W. Peters J.-M. Tugendreich S. Rolfe M. Hieter P. Kirschner M.W. Cell. 1995; 81: 279-288Abstract Full Text PDF PubMed Scopus (831) Google Scholar), cyclin-dependent kinases (Sic1 and p27) (6Schwob E. Boehm T. Mendenhall M.D. Nasmyth K. Cell. 1994; 79: 233-244Abstract Full Text PDF PubMed Scopus (771) Google Scholar, 7Sheaff R.J. Groudine M. Gordon M. Roberts J.M. Clurman B.E. Genes Dev. 1997; 11: 1464-1478Crossref PubMed Scopus (797) Google Scholar), and transcription factors (Matα2, c-Jun, and p53) (8Chen P. Johnson P. Sommer T. Jentsch S. Hochstrasser M. Cell. 1993; 74: 357-369Abstract Full Text PDF PubMed Scopus (355) Google Scholar, 9Treier M. Staszewski L.M. Bohmann D. Cell. 1994; 78: 787-798Abstract Full Text PDF PubMed Scopus (847) Google Scholar, 10Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text PDF PubMed Scopus (3484) Google Scholar). Recognition of specific substrates occurs at the level of conjugation, which is a multistep process involving three types of enzymes (11Hershko A. Heller H. Elias S. Ciechanover A. J. Biol. Chem. 1983; 258: 8206-8214Abstract Full Text PDF PubMed Google Scholar): a ubiquitin-activating enzyme (E1), 1The abbreviations used are: E1, ubiquitin-activating enzyme; E2 and UBC, ubiquitin-conjugating enzyme; E3, ubiquitin-protein ligase; PCR, polymerase chain reaction; GST, glutathione S-transferase; DTT, dithiothreitol; AMP-PNP, 5′-adenylyl imidodiphosphate; RM-Ub, reductively methylated ubiquitin. ubiquitin-conjugating enzymes (UBCs or E2s), and, in many cases, ubiquitin-protein ligases (E3s). Initially, ubiquitin is activated by E1 through the ATP-dependent formation of a thiol ester bond between ubiquitin and E1 (12Haas A.L. Warms J.V.B. Hershko A. Rose I.A. J. Biol. Chem. 1982; 257: 2543-2548Abstract Full Text PDF PubMed Google Scholar). The activated ubiquitin is then transferred via a thiol ester linkage to a cysteine residue of an E2 (reviewed in Ref. 13Haas A.L. Siepmann T.J. FASEB J. 1997; 11: 1257-1268Crossref PubMed Scopus (277) Google Scholar). Finally, the E2 itself, or more commonly in concert with an E3, ligates the ubiquitin via its carboxyl terminus to lysine residues of a protein substrate. Successive ubiquitin molecules may be added to lysine residues of the previous ubiquitin to produce a multi-ubiquitin chain. Although the biochemical mechanisms of the pathway are becoming well defined, the molecular mechanisms by which substrates are selected by the ubiquitin-conjugating apparatus remain unclear. E3s are important for recognition and binding of the substrate (14Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar). E3s may serve as docking proteins that bind both specific substrates and E2s (14Reiss Y. Heller H. Hershko A. J. Biol. Chem. 1989; 264: 10378-10383Abstract Full Text PDF PubMed Google Scholar), thereby permitting the transfer of ubiquitin from an E2 to a substrate. For example, the E3 SCFCdc4 binds the E2 molecule Cdc34 and a specific substrate, Sic1, simultaneously, thereby facilitating the transfer of ubiquitin from Cdc34 to Sic1, an inhibitor of the yeast S-phase cyclin-dependent kinase Cln1-Cdc28 (15Skowra D. Craig K.L. Tyers M. Elledge J. Harper J.W. Cell. 1997; 91: 209-219Abstract Full Text Full Text PDF PubMed Scopus (1032) Google Scholar, 16Feldman R.M.R. Correll C.C. Kaplan K.B. Deshaies R.J. Cell. 1997; 91: 221-230Abstract Full Text Full Text PDF PubMed Scopus (716) Google Scholar). Alternatively, E3s may function as the final intermediate in the ubiquitin thiol ester cascade (17Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (756) Google Scholar). The E3 E6-AP (E6-associated protein) forms a thiol ester linkage with ubiquitin prior to catalyzing the ubiquitination of p53 in the presence of the viral E6 protein (17Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (756) Google Scholar). The catalytically active cysteine in E6-AP is found within its carboxyl terminus domain, and a number of putative E3s have been identified based on the presence of such HECT (homology toE6-AP carboxyl terminus) domains (18Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (705) Google Scholar). Although E3s bind substrates, E2s may also be involved in substrate recognition either by conjugating substrates directly or probably more commonly by interacting only with specific E3s. Indeed, yeast genetic studies have revealed a variety of functions for different E2s indicating that they can direct conjugation of ubiquitin to specific substrates. For example, UBC2 (RAD6) is required for DNA repair (19Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (547) Google Scholar), whereas UBC4/UBC5 are required for the degradation of short-lived and abnormal proteins (20Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (408) Google Scholar). Differences in E2 function evidently reflect differences in E2 structure. E2 enzymes have been divided into four structural classes based on amino acid sequence comparison (21Jentsch S. Seufert W. Sommer T. Reins H.A. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar). Class I enzymes (e.g. Ubc4 and Ubc5) (20Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (408) Google Scholar) consist of a conserved catalytic core domain of ∼150 amino acids that contains the active-site cysteine involved in ubiquitin transfer. Class II enzymes (e.g. Ubc2/Rad6 and Ubc3/Cdc34) (19Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (547) Google Scholar, 22Goebl M.G. Yochem J. Jentsch S. McGrath J.P. Varshavsky A. Byers B. Science. 1988; 241: 1331-1335Crossref PubMed Scopus (323) Google Scholar) have extra C-terminal extensions or tails attached to the core domain, whereas class III enzymes (e.g. UbcH6 and UbcD2) (23Nuber U. Schwarz S. Kaiser P. Schneider R. Scheffner M. J. Biol. Chem. 1996; 271: 2795-2800Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 24Matuschewski K. Hauser H.-P. Treier M. Jentsch S. J. Biol. Chem. 1996; 271: 2789-2794Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar) have attached N-terminal tails. Finally, class IV enzymes (e.g.E2-C) (25Aristarkhov A. Eytan E. Moghe A. Admon A. Hershko A. Ruderman J.V. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4294-4299Crossref PubMed Scopus (121) Google Scholar) possess both C- and N-terminal extensions. Jentsch et al. (21Jentsch S. Seufert W. Sommer T. Reins H.A. Trends Biochem. Sci. 1990; 15: 195-198Abstract Full Text PDF PubMed Scopus (124) Google Scholar) speculated that E2 extensions play either a direct role in substrate recognition or else an indirect role through their interaction with E3s. While C- and N-terminal extensions may participate in specifying the interaction of E2s with E3s and/or substrates, a number of studies have indicated that specificity elements also reside within the E2 core. For example, the class II enzyme RAD6 is required for DNA repair, induced mutagenesis, and sporulation in yeast (19Jentsch S. McGrath J.P. Varshavsky A. Nature. 1987; 329: 131-134Crossref PubMed Scopus (547) Google Scholar) and is capable of polyubiquitinating histones in vitro (26Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (143) Google Scholar). However, removal of the polyacidic tail of Rad6 results only in loss of its sporulation function (27Morrison A. Miller E.J. Prakash L. Mol. Cell. Biol. 1988; 8: 1179-1185Crossref PubMed Scopus (78) Google Scholar) and histone-polyubiquitinating activity (26Sung P. Prakash S. Prakash L. Genes Dev. 1988; 2: 1476-1485Crossref PubMed Scopus (143) Google Scholar). The core domain is sufficient for performing its DNA repair function. Since this truncated Rad6 containing only the core domain exhibits a distinct phenotype from the class I (core domain only) E2s, Ubc4 and Ubc5, which function in the turnover of short-lived and abnormal proteins (20Seufert W. Jentsch S. EMBO J. 1990; 9: 543-550Crossref PubMed Scopus (408) Google Scholar), the core domain must possess determinants of function and by inference substrate specificity. More recently, the C-terminal tail of E2–25K was found to be necessary, but not sufficient, for some of its E2 functions, indicating that the tail depends on structural features in the core for its function (28Haldeman M.T. Xia G. Kasperek E.M. Pickart C.M. Biochemistry. 1997; 36: 10526-10537Crossref PubMed Scopus (123) Google Scholar). These and other results suggest that although E2 core domains are highly conserved, they possess unique structural features that are critical for individual E2 function and specificity. Recently, we cloned and characterized a family of mammalian class I E2s homologous to S. cerevisiae Ubc4/Ubc5 (29Wing S. Jain P. Biochem. J. 1995; 305: 125-132Crossref PubMed Scopus (58) Google Scholar, 30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). Two isoforms of rat UBC4, 2Previously, the rat homologue of yeast Ubc4 was referred to as E217KB and included isoforms 2E and 8A (29Wing S. Jain P. Biochem. J. 1995; 305: 125-132Crossref PubMed Scopus (58) Google Scholar,30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). For purposes of clarity and to conform to a trend by workers in the field to name E2s after their apparent yeast homologues, isoform 2E will henceforth be referred to as rat UBC4-1, and isoform 8A as rat UBC4-testis. The nucleotide sequences of UBC4-1 and UBC4-testis have been submitted to GenBankTM with accession numbers U13177and U56407. although 93% identical, show distinct features. Rat UBC4-testis possesses an acidic pI and shows testis-specific RNA expression that is specifically induced in the developing spermatids (30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar), whereas rat UBC4-1 has a basic pI and is expressed ubiquitously (29Wing S. Jain P. Biochem. J. 1995; 305: 125-132Crossref PubMed Scopus (58) Google Scholar). Therefore, although the high degree of sequence similarity might suggest that these isoforms are redundant, the highly regulated and cell-specific expression suggested a unique role for the UBC4-testis isoform. Therefore, we characterized carefully the abilities of rat UBC4-1 and UBC4-testis to support conjugation of ubiquitin in vitro to different subsets of testis proteins. Rat UBC4-1 shows an 11-fold greater ability to support conjugation of ubiquitin to endogenous substrates present in a testis nuclear fraction (30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). We also determined whether these two isoforms interact differentially with other E3s. In addition, since rat UBC4-1 and UBC4-testis differ by only 11 amino acids (Fig. 1) and they are highly similar to yeast Ubc4, whose crystal structure has been solved (31Cook W.J. Jeffrey L.C. Xu Y. Chau V. Biochemistry. 1993; 32: 13809-13817Crossref PubMed Scopus (83) Google Scholar), this provided a unique opportunity to identify, by site-directed mutagenesis of UBC4-testis, regions of the E2 core domain responsible for the observed difference in substrate specificity. pET-11d (Novagen)-basedEscherichia coli expression plasmids encoding rat UBC4-1 or UBC4-testis have been described (29Wing S. Jain P. Biochem. J. 1995; 305: 125-132Crossref PubMed Scopus (58) Google Scholar, 30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). Mutagenesis of selected residues in UBC4-testis to those in UBC4-1 was performed using the Chameleon double-stranded site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. Briefly, separate mutagenic primers 3Oligonucleotide sequences and detailed PCR conditions are available on request. encoding site-specific mutations in UBC4-testis and a selection primer were annealed to denatured UBC4-testis-containing pET-11d plasmids, and the mutant DNA strand was extended with T7 DNA polymerase and ligated with T4 DNA ligase. The selection primer, located ∼2 kilobases from the mutagenic primers, changed the unique AccI restriction site on pET-11d to a unique KpnI restriction site on the mutant plasmid strand, thereby permitting selection of intact mutant plasmids by digestion with AccI. Initially, separate mutant UBC4-testis constructs with a D55H, E68A, or double D55H/E68A substitution were generated. Similarly, subsequent mutations were added separately or in combination onto the initial UBC4-testis D55H/E68A double mutant in the following order: Gln-15, Gln-125, Ala-49, or Ser-107. The intact mutant plasmids were transformed into E. coli XL1-Blue cells (Stratagene). All plasmids were sequenced to confirm the presence of the desired mutation using thefmolTM DNA sequencing System (Promega). Subtractive mutagenesis was then performed in a similar manner to define the minimal UBC4-1 residues on UBC4-testis that were necessary and sufficient for the UBC4-1-like conjugating activity. The converse mutagenesis of the four critical residues (Arg-15, Val-49, Cys-107, and Arg-125) of UBC4-1 to those of UBC4-testis was performed via PCR amplification (32Higuchi R. Krummel B. Saiki R.K. Nucleic Acids Res. 1988; 16: 7351-7367Crossref PubMed Scopus (2104) Google Scholar). Mutant UBC4-1 fragments were generated using UBC4-1-containing pET-11d as a template, primers bearing the relevant base substitutions, and sense or antisense oligonucleotides encoding the amino and carboxyl termini of the protein as well as restriction sites to permit cloning into the NcoI andBamHI sites of pET-11d. The mutant UBC4-1 fragments were then purified by agarose gel electrophoresis and incorporated into full-length mutant UBC4-1 inserts via a second round of PCR amplification using the separate mutant UBC4-1 fragments as a template and both the 5′-NcoI and the 3′-BamHI primers. The PCR products were purified on a QIAquick PCR purification column (QIAGEN Inc.), digested with NcoI and BamHI, and then ligated into a pET-11d vector that had been digested with the same enzymes. Purified plasmids were transformed into XL1-Blue cells, and individual positive clones were sequenced using thefmolTM DNA sequencing system to confirm the presence of the desired mutation. The pGEX-ubiquitin plasmid encoding the GST-ubiquitin fusion protein (33Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1993) Google Scholar) was expressed in E. coli strain DH5α and induced with 0.1 mmisopropyl-β-d-thiogalactopyranoside for 2 h at 37 °C. Bacterial cell pellets resuspended in phosphate-buffered saline and 1% Triton X-100 were lysed by sonication and clarified by centrifugation at 12,000 × g. Glutathione-Sepharose (Amersham Pharmacia Biotech) was added to the supernatant, and the mixture was rotated overnight at 4 °C. Beads were washed in phosphate-buffered saline and 1% Triton X-100 and eluted in 20 mm glutathione in phosphate-buffered saline for 20 min at 25 °C. The GST-ubiquitin fusion protein content was estimated to be 1 mg/ml by Coomassie Blue staining. E1 was prepared from rabbit liver. Bacterially expressed recombinant UBC4-1 and UBC4-testis proteins were also purified as described previously (29Wing S. Jain P. Biochem. J. 1995; 305: 125-132Crossref PubMed Scopus (58) Google Scholar, 30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). The E1, UBC4-1, and UBC4-testis enzymes were quantified by measuring the initial release of radioactive pyrophosphate following incubation in the presence of [γ-32P]ATP and ubiquitin (34Haas A.L. Rose I.A. J. Biol. Chem. 1982; 257: 10329-10337Abstract Full Text PDF PubMed Google Scholar). The purified pET-11d-based plasmids containing the mutant UBC4-1 and UBC4-testis genes were transformed into E. coli BL21 (DE3) (Novagen), and induction of the recombinant proteins with 1 mm isopropyl-β-d-thiogalactopyranoside was carried out for 2 h at 30 °C. The cells were pelleted and resuspended in 0.1 volume and then lysed by sonication in 50 mm Tris, pH 7.5, and 1 mm DTT. Cellular debris was removed by centrifugation at 12,000 × g. The enzymatic activities of the mutant E2-containing bacterial lysates relative to the purified recombinant UBC4-1 and UBC4-testis enzymes were determined by thiol ester assays, as described below. The [35S]methionine-labeled E6-AP (35Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 775-784Crossref PubMed Scopus (471) Google Scholar) or rat p100 (36Muller D. Rehbein M. Baumeister H. Richter D. Nucleic Acids Res. 1992; 20: 1471-1475Crossref PubMed Scopus (21) Google Scholar) proteins were synthesized separately in vitro using a coupled transcription/translation kit (wheat germ extract TNT, Promega) with T7 polymerase. The translation reactions (150 μl) were partially purified with DEAE-cellulose resin (Whatman DE52; 200 mg of wet resin/TNT reaction) using a batch elution procedure to remove ubiquitin, E1, and E2s homologous to rat UBC4 that were present in the break-though fraction. Briefly, the translation reactions and resin were incubated in 4.5 ml of loading buffer (50 mm Tris-HCl, pH 7.5, and 1 mm DTT) for 1 h at 4 °C, washed two times, and eluted in loading buffer containing 0.5 m NaCl. The E3-containing eluates (2 ml) were then concentrated 10-fold by ultrafiltration with Centricon-30 concentrators (Amicon, Inc.), and 2 μl of each E3 preparation were utilized in each thiol ester assay. The testis cytosolic E3 and nuclear fractions eluting from a MonoQ anion-exchange column (Amersham Pharmacia Biotech) at 0.4 and 0.05m NaCl, respectively, were isolated as described previously (30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar). In some preparations, the nuclear fraction activity was found in the flow-through fraction instead of eluting at 0.05 mNaCl; however, it behaved identically to the original preparation eluting at 0.05 m NaCl. This nuclear fraction was concentrated 4-fold using a Centricon-10 concentrator (Amicon, Inc.). The testis cytosolic E3 activity was further purified by chromatography on a Superdex 200 gel filtration column (Amersham Pharmacia Inc.). The chloramine-T method was used to label bovine ubiquitin with Na125I to a specific radioactivity of 3000 cpm/pmol (37Ciechanover A. Heller H. Elias S. Haas A.L. Hershko A. Proc. Natl. Acad. Sci. U. S. A. 1980; 87: 1365-1368Crossref Scopus (378) Google Scholar) and histone H2A (Boehringer Mannheim) to a specific radioactivity of 375,000 cpm/μg. Unincorporated 125I was removed by passing the reaction products over a Sephadex G-25 column. The relative enzymatic activities of the purified recombinant rat UBC4-1 and UBC4-testis proteins and of the mutant E2-containing bacterial lysates were determined by incubating the following in a total volume of 10 μl: 50 mm Tris-HCl, pH 7.5, 1 mm DTT, 2 mm MgCl2, 2 mm ATP, 100 nm E1, 20 units/ml inorganic pyrophosphatase, 5 μm125I-ubiquitin (3000 cpm/pmol), and 2.5 pmol of the purified E2s (5 pmol/μl) or various amounts of the mutant E2 lysates. After incubation at 37 °C for 1 min, the reaction was stopped with Laemmli sample buffer without β-mercaptoethanol and resolved by 12.5% SDS-polyacrylamide gel electrophoresis at 4 °C followed by autoradiography. The thiol ester bands were excised from the gels to measure the incorporated radioactivity and thereby estimate the mutant E2 enzymatic activities relative to those of the purified native E2s (5 pmol/μl). Assaying dilutions of the E2-containing extracts confirmed linearity of the assays. The [35S]methionine-labeled E3s E6-AP (35Huibregtse J.M. Scheffner M. Howley P.M. Mol. Cell. Biol. 1993; 13: 775-784Crossref PubMed Scopus (471) Google Scholar) and rat p100 (36Muller D. Rehbein M. Baumeister H. Richter D. Nucleic Acids Res. 1992; 20: 1471-1475Crossref PubMed Scopus (21) Google Scholar), covalently bound to GST-ubiquitin fusion protein in thiol ester linkages (17Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (756) Google Scholar, 18Huibregtse J.M. Scheffner M. Beaudenon S. Howley P.M. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 2563-2567Crossref PubMed Scopus (705) Google Scholar), were detected by incubating the enzymes in the presence of 50 mm Tris-HCl, pH 7.5, 1 mm DTT, 2 mm MgCl2, 2 mm ATP, 50 nm E1, 20 units/ml inorganic pyrophosphatase, 500 nm E2, and 2 μl of each partially purified and concentrated E3 preparation. The reaction mixtures were preincubated at 25 °C for 3 min, and then the reaction was initiated with 1 μg of GST-ubiquitin fusion protein (33Scheffner M. Huibregtse J.M. Vierstra R.D. Howley P.M. Cell. 1993; 75: 495-505Abstract Full Text PDF PubMed Scopus (1993) Google Scholar), incubated at 25 °C for 5 min, and stopped with Laemmli sample buffer with or without β-mercaptoethanol. The reactions were resolved at 4 °C on an SDS-12.5% polyacrylamide gel, which was then soaked in ENHANCE (NEN Life Science Products) and autoradiographed. For the conjugation of ubiquitin to endogenous substrates present in the testis nuclear fraction, the reaction mixture contained the following in a final volume of 20 μl: 10 μl of the 0.05 m NaCl nuclear fraction, 50 mm Tris-HCl, pH 7.5, 1 mm DTT, 2 mmMgCl2, 2 mm AMP-PNP, 5 μm125I-ubiquitin (3000 cpm/pmol), 50 nm E1, and 250 nm E2s. The ubiquitination rate for the nuclear fraction was linear for 1 h at 37 °C, and this assay was performed in the presence or absence of 0.5 μg of ubiquitin aldehyde, an isopeptidase inhibitor, or 40 μm MG132 (Proscript), a proteasome inhibitor. For the conjugation of ubiquitin to the exogenous substrate histone H2A, mediated by the testis cytosolic E3, the reaction mixture contained the following in a final volume of 20 μl: 3 μl of the cytosolic E3 Superdex 200 fraction, 50 mm Tris-HCl, pH 7.5, 1 mm DTT, 2 mm MgCl2, 2 mm ATP, 0.5 units pyrophosphatase, 12.5 mmphosphocreatine, 2.5 units of creatine kinase, 250 nm E1,125I-histone H2A (specific activity of 375,000 cpm/μg), and varied concentrations of E2 as indicated. Reactions were initiated with 25 μm reductively methylated ubiquitin (RM-Ub), prepared, and quantified as described (38Hershko A. Heller H. Biochem. Biophys. Res. Commun. 1985; 128: 1079-1086Crossref PubMed Scopus (194) Google Scholar). Since histone H2A contains a number of lysine residues, mono- to penta-RM-Ub conjugates were formed, and the rate of formation of these conjugates was found to be linear for 10 min at 30 °C, permitting their quantification. To test whether the structural differences between rat UBC4-1 and UBC4-testis conferred different abilities to conjugate ubiquitin to proteins, ubiquitination assays were performed using testis extracts fractionated on a MonoQ anion-exchange column. As shown previously (30Wing S.S. Bedard N. Morales C. Hingamp P. Trasler J. Mol. Cell. Biol. 1996; 16: 4064-4072Crossref PubMed Scopus (50) Google Scholar), a nuclear fraction eluting at 0.05 m NaCl supported conjugation of ubiquitin to proteins essentially only with the UBC4-1 isoform (Fig. 2 A). This indicated that these two isoforms showed differential substrate specificity and suggested an enhanced ability of the ubiquitous isoform to conjugate ubiquitin to endogenous proteins in this fraction. To evaluate the possibility that UBC4-testis was conjugating ubiquitin to proteins that are preferentially de-ubiquitinated by a co-purifying isopeptidase activity, the conjugation assay was performed in the presence of the isopeptidase inhibitor ubiquitin aldehyde. Notably, the level of UBC4-testis-dependent conjugation was not increased by the addition of this reagent, rendering unlikely the possibility of a co-purifying interfering isopeptidase. Likewise, to rule out the possibility of enhanced proteasomal degradation of the UBC4-testis-dependent conjugates, the conjugation assay was performed in the presence of the proteasomal inhibitor MG132. Similarly, MG132 did not increase the levels of UBC4-testis-dependent conjugates. Significantly, the observed difference in conjugating ability between UBC4-1 and UBC4-testis was not due to a difference in the ability of these E2s to accept ubiquitin from E1 because UBC4-1 and UBC4-testis formed similar amounts of ubiquitin thiol esters (Fig. 2 B). These thiol ester assays are end-point assays, and the results cannot exclude the possibility of different affinities of these two isoforms for E1. However, more detailed thiol ester-based enzyme kinetic studies suggest that UBC4-1 and UBC4-testis show less than a 2-fold difference in their affinities for E1 (data not shown). Since the observed difference in conjugation by rat UBC4-1 and UBC4-testis was found not to be due to significant differences in their interaction with E1, this suggested that the specificity might arise at the E2-E3 level. We therefore tested the abilities of rat UBC4-1 and UBC4-testis to interact with some well defined E3s, E6-AP (10Scheffner M. Werness B.A. Huibregtse J.M. Levine A.J. Howley P.M. Cell. 1990; 63: 1129-1136Abstract Full Text