Title: Certain Pairs of Ubiquitin-conjugating Enzymes (E2s) and Ubiquitin-Protein Ligases (E3s) Synthesize Nondegradable Forked Ubiquitin Chains Containing All Possible Isopeptide Linkages
Abstract: It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys48, but not through Lys63, target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys48, Lys63, and Lys11 linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys6 + Lys11, Lys27 + Lys29, or Lys29 + Lys33 on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys48 chains (E6AP) or Lys63 chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys63 chains, but with UbcH1 (E2–25K), MuRF1 synthesizes Lys48 chains on the substrate.We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys48-Ub chain or, surprisingly, to a Lys63-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys63 chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys63-Ub chains from proteasomal degradation. It is generally assumed that a specific ubiquitin ligase (E3) links protein substrates to polyubiquitin chains containing a single type of isopeptide linkage, and that chains composed of linkages through Lys48, but not through Lys63, target proteins for proteasomal degradation. However, when we carried out a systematic analysis of the types of ubiquitin (Ub) chains formed by different purified E3s and Ub-conjugating enzymes (E2s), we found, using Ub mutants and mass spectrometry, that the U-box E3, CHIP, and Ring finger E3s, MuRF1 and Mdm2, with the E2, UbcH5, form a novel type of Ub chain that contains all seven possible linkages, but predominantly Lys48, Lys63, and Lys11 linkages. Also, these heterogeneous chains contain forks (bifurcations), where two Ub molecules are linked to the adjacent lysines at Lys6 + Lys11, Lys27 + Lys29, or Lys29 + Lys33 on the preceding Ub molecule. However, the HECT domain E3s, E6AP and Nedd4, with the same E2, UbcH5, form homogeneous chains exclusively, either Lys48 chains (E6AP) or Lys63 chains (Nedd4). Furthermore, with other families of E2s, CHIP and MuRF1 synthesize homogeneous Ub chains on the substrates. Using the dimeric E2, UbcH13/Uev1a, they attach Lys63 chains, but with UbcH1 (E2–25K), MuRF1 synthesizes Lys48 chains on the substrate. We then compared the capacity of the forked heterogeneous chains and homogeneous chains to support proteasomal degradation. When troponin I was linked by MuRF1 to a Lys48-Ub chain or, surprisingly, to a Lys63-Ub chain, troponin I was degraded rapidly by pure 26S proteasomes. However, when linked to the mixed forked chains, troponin I was degraded quite poorly, and its polyUb chain, especially the forked linkages, was disassembled slowly by proteasome-associated isopeptidases. Because these Ring finger and U-box E3s with UbcH5 target proteins for degradation in vivo, but Lys63 chains do not, cells probably contain additional factors that prevent formation of such nondegradable Ub-conjugates and that protect proteins linked to Lys63-Ub chains from proteasomal degradation. In eukaryotic cells, ubiquitination serves to target regulatory and misfolded proteins for rapid degradation by proteasomes (1Goldberg A.L. Nature. 2003; 426: 895-899Crossref PubMed Scopus (1662) Google Scholar, 2Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6879) Google Scholar, 3Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (605) Google Scholar), to trigger endocytosis of membrane proteins (4Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (960) Google Scholar), and also to allow specific protein-protein associations important in signal transduction, DNA repair, and gene transcription (5Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1505) Google Scholar, 6Hoege C. Pfander B. Moldovan G.L. Pyrowolakis G. Jentsch S. Nature. 2002; 419: 135-141Crossref PubMed Scopus (1734) Google Scholar, 7Spence J. Sadis S. Haas A.L. Finley D. Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar, 8Lipford J.R. Smith G.T. Chi Y. Deshaies R.J. Nature. 2005; 438: 113-116Crossref PubMed Scopus (158) Google Scholar). Protein ubiquitination involves formation of isopeptide linkages between the C-terminal carboxyl group of a ubiquitin (Ub) 4The abbreviations used are: Ub, ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring. 4The abbreviations used are: Ub, ubiquitin; UPKn, ubiquitin peptide modified at lysine n by ubiquitination; Forked chain, Ub chain in which two Ub chains are linked to the adjacent lysines on the preceding Ub; E1, Ub-activating enzyme; E2, Ub-conjugating enzyme; E3, ubiquitin-protein isopeptide ligase; LC-MSMS liquid chromatography-tandem mass spectrometry; SIM, selective ion monitoring. and an ∈-amino group on a lysine on the protein substrate or a preceding Ub to form a polyUb chain. To synthesize such linkages, the C-terminal carboxyl group of a Ub is first activated by formation of a thioester bond with a cysteine on the Ub-activating enzyme (E1), and the activated Ub is then transferred as a thioester to one of the 20–40 Ub-conjugating enzymes (E2) of the cell. The formation of a Ub chain on the substrate is then catalyzed by a Ub ligase (E3), which binds the substrate and an E2. Several families of E3s exist that differ in structure and mechanism. If ubiquitination is catalyzed by a member of the Ring finger or the U-box E3 family, the activated Ub is transferred from the E2 directly to a lysine on the protein substrate or to a preceding Ub. The abundant Ring finger and the related U-box families are small monomeric proteins that bind the substrate at one end and then in that vicinity release the reactive Ub from the E2-Ub thioester (9Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar, 10Furukawa M. Ohta T. Xiong Y. J. Biol. Chem. 2002; 277: 15758-15765Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). If ubiquitination is catalyzed by an E3 of the HECT domain family, the activated Ub is transferred from the E2 first to a cysteine on the E3 to form another thioester bond and then to the substrate or to a preceding Ub (11Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (743) Google Scholar). The much larger multicomponent SCF complexes, SOCS E3s and APC/C, contain a substrate-binding subunit and a distinct Ring finger subunit, which catalyzes direct addition of a Ub from the E2 to the substrate. Because a Ub molecule contains seven lysines (Lys6, Lys11, Lys27, Lys29, Lys33, Lys48, and Lys63), there are seven different possible types of isopeptide linkage. It is generally assumed that Ub chains contain only one type of isopeptide linkage (12Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1116) Google Scholar, 13Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 14Wu-Baer F. Lagrazon K. Yuan W. Baer R. J. Biol. Chem. 2003; 278: 34743-34746Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar), and that the nature of the Ub linkage determines the specific fate of the protein. Most proteins degraded by 26S proteasomes are believed to be linked to a homogeneous polyUb chain, in which the Ubs are coupled through isopeptide linkages to Lys48 or Lys29 on the preceding Ub (12Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1116) Google Scholar, 13Gregori L. Poosch M.S. Cousins G. Chau V. J. Biol. Chem. 1990; 265: 8354-8357Abstract Full Text PDF PubMed Google Scholar, 15Johnson E.S. Ma P.C. Ota I.M. Varshavsky A. J. Biol. Chem. 1995; 270: 17442-17456Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar). PolyUb chains in which the Ubs are linked through other lysines are believed to serve roles unrelated to proteolysis (3Pickart C.M. Cohen R.E. Nat. Rev. Mol. Cell. Biol. 2004; 5: 177-187Crossref PubMed Scopus (605) Google Scholar, 4Hicke L. Dunn R. Annu. Rev. Cell Dev. Biol. 2003; 19: 141-172Crossref PubMed Scopus (960) Google Scholar, 5Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1505) Google Scholar, 7Spence J. Sadis S. Haas A.L. Finley D. Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar). For example, Lys63 chains have been shown to function in DNA repair (7Spence J. Sadis S. Haas A.L. Finley D. Mol. Cell. Biol. 1995; 15: 1265-1273Crossref PubMed Google Scholar) and signal transduction (5Deng L. Wang C. Spencer E. Yang L. Braun A. You J. Slaughter C. Pickart C. Chen Z.J. Cell. 2000; 103: 351-361Abstract Full Text Full Text PDF PubMed Scopus (1505) Google Scholar). However, the isopeptide linkages in polyUb chains formed by different types of purified ubiquitinating enzymes have not been studied systematically. Most conclusions about the nature of the Ub chain have been based on qualitative studies using various Ub mutants in vivo or in vitro, and in only a few cases has the validity of these conclusions been confirmed by mass spectrometry (16Nishikawa H. Ooka S. Sato K. Arima K. Okamoto J. Klevit R.E. Fukuda M. Ohta T. J. Biol. Chem. 2004; 279: 3916-3924Abstract Full Text Full Text PDF PubMed Scopus (191) Google Scholar, 17Wang M. Cheng D. Peng J. Pickart C.M. EMBO J. 2006; 25: 1710-1719Crossref PubMed Scopus (71) Google Scholar). In fact, it has not yet been established for most E3s whether they synthesize Ub chains composed of single or multiple types of linkages or whether the nature of the Ub linkage is determined by the E2 or the E3. Such information is essential to understand the functions of the many different types of Ub ligases of the cells and the consequences of protein ubiquitination. In this study, we examined systematically the nature of the isopeptide linkages in the polyUb chains formed by different types of E2s and E3s that are known to target proteins for degradation. These initial studies uncovered several unexpected features of the ubiquitination process as follows: 1) the type of isopeptide linkages formed by U-box or Ring finger E3s is determined by the E2, and a single E3 can form different types of chains depending on the E2; 2) the small Ring finger and U-box E3s, using UbcH5 as the E2, form novel types of Ub chains that contain all seven type of isopeptide linkages and that are also forked (i.e. they contain two Ubs linked to a preceding Ub); 3) by contrast, different HECT domain E3s using this same E2, UbcH5, can form homogeneous chains composed of different linkages. These findings enabled us to investigate the capacity of these different types of conjugates to be degraded by pure 26S proteasomes. We found that purified 26S proteasomes degrade very poorly the proteins linked to these novel, forked Ub chains composed of mixed linkages, although they efficiently degrade proteins linked to Lys48 chains and also surprisingly to Lys63 chains. These observations demonstrate important differences between the functioning of different E2-E3 pairs, and they clearly do not support certain widely accepted conclusions about the nature of Ub chains capable of supporting protein degradation by the Ub-proteasome pathway. Ubiquitination and Proteasomal Degradation—Ubiquitination of luciferase was assayed by a modification of the method of Murata et al. (18Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (464) Google Scholar). Luciferase (75 nm) was heat-treated to 43 °C for 10 min in the presence of Hsp70 (150 nm) and then cooled on ice for 5 min. The Hsp70-luciferase complex was added to the ubiquitination mixture containing His6-E1 (5 nm), E2 (750 nm), His6-CHIP (300 nm), bovine ubiquitin (58 μm) in the buffer containing 20 mm Tris-Cl (pH 7.6), 20 mm KCl, 5 mm MgCl2, 2 mm ATP, and 1 mm dithiothreitol. The reactions were run for 60 min at 37 °C. Ubiquitination of troponin I by MuRF1, HHR23A by E6AP, and autoubiquitination of GST-MuRF1, Mdm2, GST-E6AP, or GST-Nedd4 were carried out under these same conditions but without the heat treatment or Hsp70. The autoubiquitination of Cdc34 was carried out with yeast E1 and Ub in the same buffer at 30 °C. For the ubiquitination of Sic1, 0.5 pmol of SCFCDC4 and 70 pmol of Cdc34Δ were incubated with 5 pmol of Sic1 at 37 °C for 1 h. Ub-luciferase conjugates were resolved on 4–12% gradient SDS-PAGE and detected by PhosphorImager. Ubiquitinated MuRF1, Mdm2, E6AP, Nedd4, and Cdc34 were detected by Coomassie Blue staining. Also ubiquitinated Sic1 was detected by silver staining. Proteasomal degradation of ubiquitinated troponin I was assayed by adding purified rabbit muscle 26S proteasomes (4 nm) to the reaction mixture at the onset of the ubiquitination. Data shown are the average of three separate experiments. Mass Spectrometry—Ubiquitinated luciferase or HHR23A were immunoprecipitated with antibody, resolved on 4–12% SDS-PAGE, and stained with Coomassie Blue. Ubiquitinated MuRF1, Mdm2, E6AP, HHR23A, Nedd4, Cdc34, and Sic1 were resolved on SDS-PAGE without immunoprecipitation. Ub-conjugate bands larger than 188 kDa were excised from the SDS-polyacrylamide gels and digested with sequencing grade trypsin (Promega). When required, sequencing grade Arg-C, Glu-C, and Asp-N were used instead of trypsin. Digested samples were loaded onto a fused silica microcapillary C18 column (Magic: Michrom BioReseources, Auburn, CA) prepared in-house (75 μm inner diameter, 10 cm long). An Agilent 1100 high pressure liquid chromatography system was used to deliver a gradient across a flow splitter to the column over 40 min. The eluent was directed into an LCQ-Deca electrospray ion-trap mass spectrometer (ThermoFinnigan), and the eluent peptides were dynamically selected for fragmentation by the operating software. The MS-MS data were analyzed using SEQUEST data base search tool. All matched peptides were confirmed by visual examination of the spectra, and all spectra were searched against the Ub peptide data base. Modification was permitted to allow for the detection of the following (mass shift shown in daltons): oxidized methionine (+16) and ubiquitinated lysine (+114). All data shown were obtained in at least two separate experiments. We defined specific UPKs as highly abundant when the frequency of identification of the UPK was higher than 10% of the total identified UPKs, and if their abundance was less than 10%, they were defined as of low abundance. Ubiquitin Peptide Enrichment Analysis—Luciferase was ubiquitinated as described above and then purified using the antibody against luciferase. One-half was kept as a control, and the other half was incubated with 26S proteasomes. To analyze deubiquitination, the ubiquitinated luciferase attached to the bead was incubated with 0.45 μg of rabbit muscle 26S proteasomes (19Kisselev A.F. Kaganovich D. Goldberg A.L. J. Biol. Chem. 2002; 277: 22260-22270Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar) for 1 h at 37 °C. The quantity of each UPK was analyzed by nano-SIM, and the data from three separate experiments were averaged. CHIP and Monomeric Ring Finger E3s Form Ub Chains Containing All Possible Isopeptide Linkages—We initially studied the nature of the isopeptide linkages formed by CHIP, a U-box E3, which functions with Hsp70 in the selective degradation of misfolded proteins (20Cyr D.M. Hohfeld J. Patterson C. Trends Biochem. Sci. 2002; 27: 368-375Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar), including several proteins important in human disease (e.g. cystic fibrosis transmembrane regulator (21Meacham G.C. Patterson C. Zhang W. Younger J.M. Cyr D.M. Nat. Cell Biol. 2001; 3: 100-105Crossref PubMed Scopus (704) Google Scholar) and phosphorylated Tau (22Shimura H. Schwartz D. Gygi S.P. Kosik K.S. J. Biol. Chem. 2004; 279: 4869-4876Abstract Full Text Full Text PDF PubMed Scopus (415) Google Scholar)). With E1, UbcH5 as the E2, Hsp70, and Ub, CHIP catalyzed ligation of multiple Ub molecules to the model substrate, heat-denatured luciferase (18Murata S. Minami Y. Minami M. Chiba T. Tanaka K. EMBO Rep. 2001; 2: 1133-1138Crossref PubMed Scopus (464) Google Scholar) (Fig. 1A). To identify the isopeptide linkages in these chains, we used various single-lysine Ub mutants, each of which contained only one lysine residue with the six other lysines replaced by arginines (12Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1116) Google Scholar). Surprisingly, all seven mutant Ubs supported the formation of a polyUb chain, although with quite different efficiencies (Fig. 1A). Wild type Ub supported the synthesis of the longest polyUb chain, but long chains were also formed with Ub mutants containing a single-lysine at Lys27, Lys29, Lys33, Lys48, or Lys63. Thus CHIP can form all seven possible isopeptide linkages (in sharp contrast to a prior report (23Alberti S. Demand J. Esser C. Emmerich N. Schild H. Hohfeld J. J. Biol. Chem. 2002; 277: 45920-45927Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar)). Because luciferase has multiple lysine residues, a large shift in molecular weight upon Ub conjugation can occur through ligation of multiple Ub molecules to different sites on the substrate or by formation of a single long polyUb chain. However, CHIP linked to luciferase only with a single methylated Ub, which lacks free amino groups and cannot form a Ub chain (Fig. 1B). Therefore, CHIP can attach a Ub to only a single site on luciferase, and the large conjugates formed with wild type Ub are because of a single long polyUb chain on that site. To test whether other monomeric E3s can also form all types of isopeptide linkages, we used single-lysine and K48R substitution Ub mutants and assayed chain formation during autoubiquitination by two medically important members of the large Ring finger family of E3s, MuRF1, the muscle-specific E3 critical in muscle atrophy (24Bodine S.C. Latres E. Baumhueter S. Lai V.K. Nunez L. Clarke B.A. Poueymirou W.T. Panaro F.J. Na E. Dharmarajan K. Pan Z.Q. Valenzuela D.M. DeChiara T.M. Stitt T.N. Yancopoulos G.D. Glass D.J. Science. 2001; 294: 1704-1708Crossref PubMed Scopus (2633) Google Scholar), and Mdm2, the oncoprotein that catalyzes ubiquitination of the tumor suppressor p53 (25Honda R. Yasuda H. Oncogene. 2000; 19: 1473-1476Crossref PubMed Scopus (313) Google Scholar). Like CHIP, MuRF1 (Fig. 1C) and Mdm2 (data not shown) with UbcH5 as the E2 could synthesize polyUb chains using any of the single-lysine Ub mutants. Thus, they can use any lysine residue in chain formation. These Ring finger E3s also showed no requirement for the Lys48, which is widely believed to be essential for proteasomal degradation. In fact, with the K48R mutant, MuRF1 formed long polyUb chains (Fig. 1C, 8th lane), similar in length to those formed with wild type Ub (1st lane). To test whether MuRF1 and UbcH5 also ubiquitinated exogenous substrates, we assayed the ubiquitination of troponin I using all seven types of isopeptide linkages (26Kedar V. McDonough H. Arya R. Li H.H. Rockman H.A. Patterson C. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 18135-18140Crossref PubMed Scopus (271) Google Scholar) (Fig. 1D). All seven single-substitution Ub mutants, each of which contained one substitution of lysine with arginine, like wild type Ub, supported the polyubiquitination of troponin I (as was also found with autoubiquitination of MuRF1). Thus, with UbcH5 two types of ubiquitinating enzymes can form polyUb chains containing all possible isopeptide linkages and do not show any preference for Lys48 linkages. Mass Spectrometry Confirms Multiple Types of Isopeptide Linkages in These PolyUb Chains—To eliminate the possibility that these various Ub mutants alter the types of linkages formed, we investigated the types of isopeptide linkages formed with wild type Ub by nano-LC-MSMS (27Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1307) Google Scholar). After ubiquitination of luciferase by CHIP with wild type Ub, the Ub-conjugates were purified with an anti-luciferase antibody and subjected to exhaustive tryptic digestion. Cleavage by trypsin released from the Ub chains a large variety of peptides that were identified by mass spectrometry. Each type of isopeptide linkage yields a distinct peptide (termed a UPK) composed of the C-terminal Gly-Gly of one Ub linked to the ∈-amino group of a lysine plus the neighboring residues in the proximal Ub (Fig. 2A and supplemental Fig. S3). Mass spectrometry of these UPKs also demonstrated that CHIP formed polyUb chains containing all seven possible isopeptide linkages (Table 1), as suggested by the observations with Ub mutants (Fig. 1A).TABLE 1Demonstration by mass spectrometry of heterogeneous and forked isopeptide linkages in Ub chains formed by U-box or Ring finger E3s with UbcH5Ub peptideE3 and substratesCHIPMuRF1Mdm2LuciferaseLuciferaseaDigestion was by a mixture of endopeptidases of Arg-C, Glu-C, and Asp-N. All other conjugates were digested with trypsin.Linear UPK6++++ UPK11++++++++++++ UPK48++++++++++++ UPK63++++++++++++Forked UPK6/11++++ UPK27/29NQbNQ indicates present but not quantifiable. UPK27/29 and UPK29/33 were present but could not be quantitated reliably because of incomplete release from ubiquitin conjugates by trypsin.+NQNQ UPK29/33NQ+NQNQa Digestion was by a mixture of endopeptidases of Arg-C, Glu-C, and Asp-N. All other conjugates were digested with trypsin.b NQ indicates present but not quantifiable. UPK27/29 and UPK29/33 were present but could not be quantitated reliably because of incomplete release from ubiquitin conjugates by trypsin. Open table in a new tab The relative amounts of UPKs indicative of linkages to Lys6, Lys11, Lys48, or Lys63 were determined based on the number of spectra assigned to the corresponding UPKs (not on their peak intensities). This spectral sampling is indicative of the relative abundance of peptides (28Liu H. Sadygov R.G. Yates 3rd, J.R. Anal. Chem. 2004; 76: 4193-4201Crossref PubMed Scopus (2066) Google Scholar). UPK48 and UPK63 were found most frequently, whereas UPK6 was rarely found. It is noteworthy that the most abundant linkages (UPK11, UPK48, and UPK63) were reported as the most abundant UPKs in analysis of the total Ub-conjugates in growing yeast (27Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1307) Google Scholar). In our experiments, UPK27, UPK29, and UPK33 were also formed but could not be quantitated reliably by mass spectrometry, because the sequences from which they were derived were digested only partially, even after exhaustive trypsinization. To verify the findings obtained with trypsin, the ubiquitinated luciferase was incubated with a mixture of other endoproteases (Arg-C, Glu-C, and Asp-N), and the Ub peptides were identified by nano-LC-MSMS (Table 1). Again peptides corresponding to all seven possible isopeptide linkages were identified, but they were generated with lower efficiencies than with trypsin. However, with the multiple protease treatment, we were able to identify reliably UPK27, UPK29, and UPK33. Interestingly with both protease treatments, no peptide was found indicating N-terminal ubiquitination. We then tested whether the Ring finger E3s with UbcH5 and wild type Ub formed Ub chains containing all possible isopeptide linkages. After autoubiquitination, the MuRF1 and Mdm2 conjugates were treated with trypsin, and the released peptides were analyzed by nano-LC-MSMS. Again, all seven possible UPKs were found, and their relative abundance appeared similar in the Mdm2 and MuRF1 conjugates and resembled that in luciferase ubiquitinated by CHIP (Table 1). Mass Spectrometry Reveals “Forked Chains” with Two Ubs Linked to Adjacent Lysines—Analysis by mass spectrometry of the Ub chains formed by CHIP, MuRF1, and Mdm2 revealed an even more surprising feature. In the tryptic digests of luciferase ubiquitinated by CHIP, we initially identified a novel peptide that indicated that two Ub molecules were linked covalently to adjacent lysines on the proximal Ub molecule at Lys6 and Lys11 (UPK6/11) (Fig. 2B and supplemental Fig. S3). We termed these novel structures forked chains, because the Ub chain forks or bifurcates at these places. The abundance of this novel UPK indicative of forked chains was lower than that of the standard UPKs (Table 1). Moreover, analysis by tryptic digestion and mass spectrometry of the polyUb chains formed by MuRF1 and Mdm2 during autoubiquitination (Table 1) also revealed the presence of UPK6/11 forked peptides, whose relative abundance resembled that in luciferase ubiquitinated by CHIP. In addition, when the conjugates produced by CHIP, MuRF1, and Mdm2 were digested by trypsin, these analyses demonstrated peptides indicative of additional types of Ub forks, in which two Ubs are linked to the proximal Ub through the adjacent lysines at Lys27 + Lys29 and Lys29 + Lys33 (Table 1 and supplemental Fig. S3). The formation of UPK6/11, UPK27/29, and UPK29/33 was verified by mass spectrometry after the polyUb chains were digested by the mixture of proteases, Arg-C, Glu-C, and Asp-N (Table 1). However, quantitation of UPK27/29 and UPK29/33 was less reproducible than of UPK6/11, because these forked peptides are not released quantitatively upon tryptic digestion. It is noteworthy that the UPK29/33 fork was previously detected in tryptic digests of Ub-conjugates in growing yeast (27Peng J. Schwartz D. Elias J.E. Thoreen C.C. Cheng D. Marsischky G. Roelofs J. Finley D. Gygi S.P. Nat. Biotechnol. 2003; 21: 921-926Crossref PubMed Scopus (1307) Google Scholar). Although real quantitation of these forks is not yet possible, it is clear that CHIP, MuRF1, Mdm2, and presumably other U-box and Ring finger E3s with UbcH5 as the E2 synthesize novel Ub chains that are composed of diverse types of linkages and bifurcations (Table 1 and Fig. 2C). Moreover, because these E3s with UbcH5 form three types of Ub forks (UPK6/11, UPK27/29, and UPK29/33), it seems likely that they also form forked chains with two Ubs linked to nonadjacent lysines on the proximal Ub (e.g. to Lys48 and Lys63). Thus, these novel chains may even be arborized and contain multiple forks in tree-like structures. However, the present approaches cannot test if two Ubs are linked to nonadjacent lysines on the proximal Ub, because the treatment with trypsin (or the other protease mixture) would have cleaved between these two lysines. CHIP and MuRF1 Form Homogeneous Lys63 Chains When They Act with the E2, UbcH13/Uev1a—Additional experiments tested whether the formation of forked chains containing all linkages was related to the E2, UbcH5. Recently CHIP, with the dimeric E2, UbcH13/Uev1a, was reported to form free polyUb chains that were not linked to a substrate (9Zhang M. Windheim M. Roe S.M. Peggie M. Cohen P. Prodromou C. Pearl L.H. Mol. Cell. 2005; 20: 525-538Abstract Full Text Full Text PDF PubMed Scopus (336) Google Scholar). We therefore tested whether CHIP may also ubiquitinate luciferase with this E2 (in place of UbcH5) together with E1, Hsp70, and ATP (supplemental Fig. 1A and B). As shown with its yeast homolog Ubc13/Mms2 (29Hofmann R.M. Pickart C.M. J. Biol. Chem. 2001; 276: 27936-27943Abstract Full Text Full Text PDF PubMed Scopus (209) Google Scholar), the UbcH13/Uev1a dimer by itself formed anchorless polyUb chains (supplemental Fig. 1A). With CHIP present, these anchorless Ub chains were formed more rapidly, but in contrast to prior reports, we found that Ub-protein conjugates were also synthesized. These conjugates corresponded to ubiquitinated luciferase, as shown by Western blot analysis (supplemental Fig. 1B). Thus a single E3 can catalyze ubiquitination of the same substrate (luciferase) with two different types of E2s (UbcH5 and UbcH13/U