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• W2080972425 abstract "The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116·Ufm1 formation was greatly accelerated by Ufl1. The C20orf116·Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms. The ubiquitin fold modifier 1 (Ufm1) is the most recently discovered ubiquitin-like modifier whose conjugation (ufmylation) system is conserved in multicellular organisms. Ufm1 is known to covalently attach with cellular protein(s) via a specific E1-activating enzyme (Uba5) and an E2-conjugating enzyme (Ufc1), but its E3-ligating enzyme(s) as well as the target protein(s) remain unknown. Herein, we report both a novel E3 ligase for Ufm1, designated Ufl1, and an Ufm1-specific substrate ligated by Ufl1, C20orf116. Ufm1 was covalently conjugated with C20orf116. Although Ufl1 has no obvious sequence homology to any other known E3s for ubiquitin and ubiquitin-like modifiers, the C20orf116·Ufm1 formation was greatly accelerated by Ufl1. The C20orf116·Ufm1 conjugate was cleaved by Ufm1-specific proteases, implying the reversibility of ufmylation. The conjugation was abundant in the liver and lungs of Ufm1-transgenic mice, fractionated into membrane fraction, and impaired in Uba5 knock-out cells. Intriguingly, immunological analysis revealed localizations of Ufl1 and C20orf116 mainly to the endoplasmic reticulum. Our results provide novel insights into the Ufm1 system involved in cellular regulation of multicellular organisms. Protein-modifying systems contribute to functional changes in target proteins and/or to amplification of genetic information. Ubiquitylation is an example of the protein modification systems in which ubiquitin, composed of 76 amino acids, is covalently conjugated with target proteins (1.Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6736) Google Scholar). The covalent modification of cellular proteins with ubiquitin regulates a diverse array of biological processes, including protein degradation, cell cycle control, DNA repair, signal/transcriptional regulation, and stress response (1.Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6736) Google Scholar, 2.Mukhopadhyay D. Riezman H. Science. 2007; 315: 201-205Crossref PubMed Scopus (934) Google Scholar, 3.Pickart C.M. Eddins M.J. Biochim. Biophys. Acta. 2004; 1695: 55-72Crossref PubMed Scopus (995) Google Scholar). Ubiquitylation is carried out by an elaborate enzymatic reaction consisting of three sequential steps. In the initial step, ubiquitin is activated by a ubiquitin-activating enzyme, E1, which forms a high energy thioester bond with ubiquitin via adenylation in an ATP-dependent manner. In the second step, the E1-activated ubiquitin is transferred to a ubiquitin-conjugating enzyme, E2, in a thioester linkage. In some cases, E2 can directly transfer the ubiquitin to substrate proteins in an isopeptide linkage; however, E2 mostly requires the participation of a ubiquitin-ligating enzyme, E3, to achieve substrate-specific ubiquitylation reaction in the cells. Although only one E1 had been thought to activate ubiquitin, another novel E1, such as Uba6/E1-L2, was discovered recently in higher eukaryotes (4.Groettrup M. Pelzer C. Schmidtke G. Hofmann K. Trends Biochem. Sci. 2008; 33: 230-237Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 5.Jin J. Li X. Gygi S.P. Harper J.W. Nature. 2007; 447: 1135-1138Crossref PubMed Scopus (254) Google Scholar). Compared with the two E1s, E2 forms relatively large families. Whereas there are 13 E2s in Saccharomyces cerevisiae, the human genome encodes ∼30 E2s containing a core Ubc domain composed of about 150 amino acids in addition to several E2 variants (6.Scheffner M. Nuber U. Huibregtse J.M. Nature. 1995; 373: 81-83Crossref PubMed Scopus (721) Google Scholar), indicating evolutional variability. E3 has unexpectedly enormous diversity for strict attachment of ubiquitin to the target proteins, ensuring a variety of roles in ubiquitylation. Indeed, the number of E3s, which contain a core domain such as RING, HECT, and U-box domain, is estimated to exceed 1,000 in humans (7.Rotin D. Kumar S. Nat. Rev. Mol. Cell Biol. 2009; 10: 398-409Crossref PubMed Scopus (744) Google Scholar). Similar to E3s, deubiquitylating enzymes, in which about 100 genes are encoded in the human genome, also regulate the ubiquitylation because deubiquitylating enzymes remove ubiquitin from the target protein (8.Amerik A.Y. Hochstrasser M. Biochim. Biophys. Acta. 2004; 1695: 189-207Crossref PubMed Scopus (720) Google Scholar, 9.D'Andrea A. Pellman D. Crit. Rev. Biochem. Mol. Biol. 1998; 33: 337-352Crossref PubMed Scopus (226) Google Scholar, 10.Nijman S.M. Luna-Vargas M.P. Velds A. Brummelkamp T.R. Dirac A.M. Sixma T.K. Bernards R. Cell. 2005; 123: 773-786Abstract Full Text Full Text PDF PubMed Scopus (1370) Google Scholar), ensuring the reversibility of ubiquitylation. A set of novel molecules called ubiquitin-like proteins (UBLs) 4The abbreviations used are: UBLubiquitin-like proteinUfSPUfm1-specific proteasetgtransgenicGSTglutathione S-transferaseGFPgreen fluorescent proteinaaamino acidsMEFmouse embryonic fibroblastICSIintracytoplasmic sperm injectionLCliquid chromatographyMS/MStandem mass spectrometryPCIproteasome-COP9-initiation factorMBPmaltose-binding protein. with structural similarities to ubiquitin were identified recently (11.Schulman B.A. Harper J.W. Nat. Rev. Mol. Cell Biol. 2009; 10: 319-331Crossref PubMed Scopus (553) Google Scholar). In addition to ubiquitylation for protein degradation, it is generally considered that protein modification by UBLs serves many proteolysis-independent events, such as molecule assembly and functional conversion of proteins. The UBLs include SUMO, NEDD8, UCRP/ISG15, FAT10, Urm1, and Ufm1 as well as Atg8 and Atg12 proteins, which are involved in autophagy. These UBLs possess the C-terminal conserved glycine residue and are covalently attached to the target proteins or phospholipids through reaction cascades in a manner analogous to the ubiquitylation pathway. The E1 and E2 involved in UBL reactions have been identified. Although the E3s for small ubiquitin-related modifier, such as protein inhibitor of activated STATs, were also discovered recently (12.Chung C.D. Liao J. Liu B. Rao X. Jay P. Berta P. Shuai K. Science. 1997; 278: 1803-1805Crossref PubMed Scopus (795) Google Scholar, 13.Liu B. Liao J. Rao X. Kushner S.A. Chung C.D. Chang D.D. Shuai K. Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 10626-10631Crossref PubMed Scopus (626) Google Scholar, 14.Schmidt D. Müller S. Cell Mol. Life Sci. 2003; 60: 2561-2574Crossref PubMed Scopus (220) Google Scholar, 15.Shuai K. Liu B. Nat. Rev. Immunol. 2005; 5: 593-605Crossref PubMed Scopus (338) Google Scholar), it remains elusive whether or not E3s for other UBLs exist. ubiquitin-like protein Ufm1-specific protease transgenic glutathione S-transferase green fluorescent protein amino acids mouse embryonic fibroblast intracytoplasmic sperm injection liquid chromatography tandem mass spectrometry proteasome-COP9-initiation factor maltose-binding protein. Among the UBLs, the most recently identified, Ufm1 (ubiquitin fold modifier 1), conjugates to target protein(s) via unique E1- and E2-like enzymes (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar, 17.Mizushima T. Tatsumi K. Ozaki Y. Kawakami T. Suzuki A. Ogasahara K. Komatsu M. Kominami E. Tanaka K. Yamane T. Biochem. Biophys. Res. Commun. 2007; 362: 1079-1084Crossref PubMed Scopus (29) Google Scholar, 18.Sasakawa H. Sakata E. Yamaguchi Y. Komatsu M. Tatsumi K. Kominami E. Tanaka K. Kato K. Biochem. Biophys. Res. Commun. 2006; 343: 21-26Crossref PubMed Scopus (42) Google Scholar). Ufm1 is synthesized as a pro-form and cleaved at the C terminus by the specific cysteine proteases, UfSP1 and UfSP2, to expose the conserved glycine residue (19.Kang S.H. Kim G.R. Seong M. Baek S.H. Seol J.H. Bang O.S. Ovaa H. Tatsumi K. Komatsu M. Tanaka K. Chung C.H. J. Biol. Chem. 2007; 282: 5256-5262Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). Thereafter, the mature Ufm1 is activated by a specific E1-like enzyme, Uba5, forming a high energy thioester bond. The activated Ufm1 is then transferred to an E2-like enzyme, Ufc1, in a similar thioester linkage. Although Ufm1 forms covalent complexes with cellular proteins in HEK293 cells and mouse tissues (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar), to date, it is still puzzling whether a specific E3 is required for the Ufm1-conjugated reaction (ufmylation). In addition, the biological roles of the Ufm1-modifying system are largely unknown. To unravel these two issues, identification of the target protein(s) for ufmylation is essential. In the present study, we identified and characterized C20orf116 as the substrate for Ufm1. Subsequently, we found an E3-ligating enzyme for Ufm1, Ufl1, by proteomics using C20orf116 as bait and finally revealed the reversibility of the C20orf116·Ufm1 conjugate via UfSPs. Furthermore, we generated Ufm1-transgenic (tg) mice and examined the dynamic nature of Ufm1 conjugation profiles in different cell compartments and tissues. The cDNA encoding human C20orf116 was obtained by PCR from human liver cDNA with the C20orf116-s5′ primer (5′-TGGGATCCATGTGGCGCCTGTGTGGTA-3′) and the C20orf116-r3′ primer (5′-GTGCGGCCGCTCAGGCTGGGGCTTGGGCAG-3′). It was then subcloned into pcDNA3 vector (Invitrogen). The FLAG, Myc, and GFP tags were introduced at the C terminus of C20orf116. C20orf116ΔN50 (amino acids 51–314) was generated by PCR and then subcloned into pGEX-6p-1 vector (GE Healthcare) to express GST-fused C20orf116ΔN50 in Escherichia coli. The cDNA encoding human Ufl1 (KIAA0776) was purchased from the Kazusa DNA Research Institute and subcloned into pIRESpuro3 vector (Clontech). The 3× FLAG and GFP tags were introduced at the N terminus of Ufl1. The deletion mutants of Ufl1, named Ufl1 M1 (aa 1–212), Ufl1 M2 (aa 213–794), Ufl1 M3 (aa 453–794), Ufl1 M4 (aa 1–452), and Ufl1 M5 (aa 1–654), were generated by PCR. These cDNAs were subcloned into pIRESpuro3. To express maltose-binding protein (MBP)-fused Ufl1 and Ufl1 mutants in E. coli, these cDNAs were subcloned into pMAL vector (New England BioLabs, Ipswich, MA). All mutations mentioned above were confirmed by DNA sequencing. The cDNAs encoding Ufm1 and UfSPs have been described previously (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar, 19.Kang S.H. Kim G.R. Seong M. Baek S.H. Seol J.H. Bang O.S. Ovaa H. Tatsumi K. Komatsu M. Tanaka K. Chung C.H. J. Biol. Chem. 2007; 282: 5256-5262Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar). The media and reagents for cell culture were purchased from Invitrogen. HEK293 and MEFs were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum, 5 units/ml penicillin, and 50 μg/ml streptomycin. HEK293 cells at subconfluence were transfected with the indicated plasmids using the Neon™ transfection system (Invitrogen). Cells were analyzed at 48 h after transfection. Uba5+/+ and Uba5−/− MEFs prepared from day 10.5 embryos were transfected with pMESVts vector, a SV40 large T antigen expression vector (20.Lee G.H. Ogawa K. Drinkwater N.R. Am. J. Pathol. 1995; 147: 1811-1822PubMed Google Scholar), to establish immortalized MEFs. The immortalized cells were further transfected with C20orf116-FLAG and/or GFP-Ufm1 using the retrovirus vector system (21.Kitamura T. Koshino Y. Shibata F. Oki T. Nakajima H. Nosaka T. Kumagai H. Exp. Hematol. 2003; 31: 1007-1014Abstract Full Text Full Text PDF PubMed Scopus (492) Google Scholar) and then cultured in medium containing 10 μg/ml puromycin and/or 5 μg/ml blasticidin to select stable transformants. For plasmid-based RNA interference against HsUfl1, two oligonucleotides, siTop (5′-GATCCGAAAGTGGTCAGGTCACCATTCAAGAGATGGTGACCTGACCACTTTCTTTTTTGGAAA-3′) and siBottom (5′-AGCTTTTCCAAAAAAGAAAGTGGTCAGGTCACCATCTCTTGAATGGTGACCTGACCACTTTCG-3′), were synthesized. A fragment made by annealing these oligonucleotides was inserted into pSilencer H1 3.0 (Ambion, Austin, TX) via BamHI and HindIII restriction sites, and the resulting vector was transfected to knock down endogenous Ufl1. Immunoblot and immunoprecipitation analyses were performed as described previously (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar). Immunoprecipitation analyses for Ufm1-conjugated proteins were carried out as described below. The cell lysates were denatured by boiling in 1% SDS-containing TNE buffer (10 mm Tris-Cl, pH 7.5, 1% Nonidet P-40, 150 mm NaCl, 1 mm EDTA, and protease inhibitors) and then diluted by the addition of 10-fold volume TNE buffer without SDS followed by immunoprecipitation with anti-GFP or anti-FLAG antibody. 5% of total lysates used for immunoprecipitation and 20% of the immunoprecipitants were applied on each lane. The anti-C20orf116, anti-Ufl1, and anti-Uba5 polyclonal antibodies were raised in rabbits using the purified recombinant C20orf116ΔN50, MBP-Ufl1, and Uba5, respectively, as an antigen. The antibodies for Ufm1 and Ufc1 were described previously (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar). The antibodies for lamin B (M-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), LDH (ab2101; Abcam, Inc., Cambridge, MA), cytochrome c (7H8.2C12; BD Biosciences), Bip (Affinity BioReagents, Inc., Golden, CO), actin (MAB1501R; Chemicon International, Inc., Temecula, CA), FLAG (M2; Sigma), and GFP (Invitrogen) were purchased. Recombinant GST-Ufm1ΔC2, GST-Ufm1ΔC3, GST-Uba5, GST-Ufc1, GST-C20orf116ΔN50, GST-UfSP1, GST-UfSP2, and GST-LC3 were produced in E. coli, and recombinant proteins were purified using glutathione-Sepharose 4B and PreScission protease according to the protocol provided by the manufacturer (Amersham Biosciences). Recombinant MBP-Ufl1 and the deletion mutants were expressed in E. coli, and these recombinant proteins were purified using amylose resin according to the protocol supplied by the manufacturer (New England BioLabs). Recombinant purified MBP-Ufl1 or MBP-Ufl1 mutants and Ufc1 were mixed in TN buffer (10 mm Tris-HCl (pH 7.5) and 150 mm NaCl) for 3 h at 4 °C and subsequently precipitated with amylose resin. The mixtures were washed three times with ice-cold TNE buffer. The bound proteins were eluted using 10 mm maltose and analyzed by SDS-PAGE followed by immunoblot with anti-Ufc1 antibody and Coomassie Brilliant Blue staining. Purified recombinant proteins were dialyzed in 50 mm Tris (pH 8.5), 150 mm NaCl, and 1 mm dithiothreitol (reaction buffer). The purified Ufm1ΔC2 (0.1 μm), Uba5 (0.1 μm), Ufc1 (0.1 μm), C20orf116ΔN50 (0.1 μm), and MBP-Ufl1 (0.1 μm) were mixed in a reaction buffer containing 5 mm ATP and 10 mm MgCl2. The mixtures were incubated at 30 °C for 90 min, and the reaction was stopped by the addition of SDS-sample buffer containing 5% β-mercaptoethanol. For the deconjugation assay, the reconstitution reaction was stopped by the addition of 5 units apyrase (Sigma), followed by incubation at 30 °C for 30 min. UfSP1 (0.1–10 nm) or UfSP2 (0.1–10 nm) was added to the reaction mixture and then incubated at 30 °C for 15 min. FLAG- and His-tagged Ufm1 (FLAGHis-Ufm1) cDNA was introduced into the expression vector, pBsCAG2, which contains cytomegalovirus enhancer and chicken β-actin promoter, β-actin intron, and rabbit β-globin poly(A) signal (CAG) (22.Niwa H. Yamamura K. Miyazaki J. Gene. 1991; 108: 193-199Crossref PubMed Scopus (4524) Google Scholar). The resulting construct (designated CAG-FLAGHis-Ufm1) was dissolved in 10 mm Tris-HCl (pH 7.6), 0.1 mm EDTA and then stored at 4 °C until use. Sperm suspension (1 × 106/29 μl) was mixed with 1 μl of the DNA solution (30 ng/μl) and incubated at ambient temperature for 2 min. Intracytoplasmic sperm injection (ICSI) was carried out according to the method of Kimura and Yanagimachi (23.Kimura Y. Yanagimachi R. Biol. Reprod. 1995; 52: 709-720Crossref PubMed Scopus (881) Google Scholar) with minor modifications. The oocytes were examined ∼6 h after ICSI for survival and activation. Oocytes with two well developed pronuclei were recorded at 7 h after ICSI to assess the fertilization ability. Two-cell stage embryos, cultured for ∼24 h and developed from oocytes fertilized by ICSI, were transferred to the oviducts of recipient females. 193 oocytes were microinjected, 157 (81.3%) survived injection, and 144 (91.7%) cleaved to the two-cell stage. Transfer of 144 embryos produced 49 offspring (34.0%). Of the 49 offspring generated by ICSI, four (8.2%) carried transgene DNA. To assess germ line transmission, four founder mice were mated with C57BL/6J mice. The transmission rate of the transgene to the F1 progeny, determined by PCR, was 46.7% (7 of 15), 47.4% (9 of 19), 54.5% (6 of 11), and 47.1% (8 of 17) in four founder mice (208, 503, 603, and 609 lines), respectively. These rates were within the variation range of the expected value, 50%, based on the Mendelian pattern. One of the tg lines, FLAGHis-Ufm1#503, was used in this study. Genotyping was carried out by PCR analysis. Mice were fed ad libitum a standard diet and maintained in an air-conditioned and light-controlled room (23 ± 1 °C, 55 ± 5%, 12/12 h light/dark). All animal experiments were performed in accordance with guidelines for the laboratory animal experimentation at Juntendo University School of Medicine. Fresh mouse livers were homogenized in the homogenate buffer (0.25 m sucrose, 10 mm HEPES (pH 7.4), and 1 mm dithiothreitol) using a Potter-Elvehjem homogenizer. The homogenate was centrifuged at 500 × g for 10 min. The supernatant was centrifuged at 10,000 × g for 30 min. The resultant precipitate was used as the mitochondria-lysosome fraction. The supernatant was further centrifuged at 100,000 × g for 60 min. The resulting supernatant and precipitate were used as the cytosol and microsome fractions, respectively. The nuclear fraction was prepared as described previously (24.Blobel G. Potter V.R. Science. 1966; 154: 1662-1665Crossref PubMed Scopus (981) Google Scholar, 25.Komatsu M. Waguri S. Koike M. Sou Y.S. Ueno T. Hara T. Mizushima N. Iwata J. Ezaki J. Murata S. Hamazaki J. Nishito Y. Iemura S. Natsume T. Yanagawa T. Uwayama J. Warabi E. Yoshida H. Ishii T. Kobayashi A. Yamamoto M. Yue Z. Uchiyama Y. Kominami E. Tanaka K. Cell. 2007; 131: 1149-1163Abstract Full Text Full Text PDF PubMed Scopus (1669) Google Scholar). HeLa cells were transfected with C20orf116-GFP or GFP-Ufl1. At 24 h after transfection, the cells were fixed with ice-cold methanol and immunostained with anti-calreticulin (Affinity BioReagents) and anti-β-COP antibodies (Affinity BioReagents). All fluorescence images were obtained using a fluorescence microscope (Q550FV; Leica) equipped with a cooled charge-coupled device camera (CTR MIC; Leica). Pictures were taken using Leica Qfluoro software (Leica). When the C-terminal conserved glycine residue in ubiquitin and Ufm1, essential for conjugation, is substituted with alanine residue, ubiquitin and Ufm1 form stable conjugates with the target proteins, which acquire resistance against attacks of deconjugating enzymes (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar). Taking advantage of this property, the FLAG-tagged Ufm1G82A mutant was constructed. The mutant, in which the C-terminal glycine residue was substituted with an alanine residue, was expressed in HEK293 cells. Thereafter, the lysates were immunoprecipitated with anti-FLAG antibody, and the immunoprecipitants were then subjected to direct nanoflow LC-MS/MS analysis (26.Natsume T. Yamauchi Y. Nakayama H. Shinkawa T. Yanagida M. Takahashi N. Isobe T. Anal. Chem. 2002; 74: 4725-4733Crossref PubMed Scopus (179) Google Scholar) in order to isolate the interacting protein(s), including target protein(s) (supplemental Table 1). A new uncharacterized protein, C20orf116, was identified as a Ufm1-interacting protein. C20orf116 is composed of 314 amino acids with a predicted molecular mass of ∼38 kDa and has a C-terminal proteasome-COP9-initiation factor (PCI) domain (Fig. 1A), which is found in proteins known to be involved in the formation of very large protein complexes like the proteasome and signalosome (27.Aravind L. Ponting C.P. Protein Sci. 1998; 7: 1250-1254Crossref PubMed Scopus (112) Google Scholar, 28.Hofmann K. Bucher P. Trends Biochem. Sci. 1998; 23: 204-205Abstract Full Text Full Text PDF PubMed Scopus (236) Google Scholar, 29.Wei N. Deng X.W. Annu. Rev. Cell Dev. Biol. 2003; 19: 261-286Crossref PubMed Scopus (380) Google Scholar). This protein was also identified as a Ufc1-interacting protein by LC-MS/MS analysis using Ufc1 as bait (data not shown). This protein is conserved in multicellular organisms but not in yeasts, like Ufm1, Uba5, Ufc1, and UfSPs (16.Komatsu M. Chiba T. Tatsumi K. Iemura S. Tanida I. Okazaki N. Ueno T. Kominami E. Natsume T. Tanaka K. EMBO J. 2004; 23: 1977-1986Crossref PubMed Scopus (219) Google Scholar, 19.Kang S.H. Kim G.R. Seong M. Baek S.H. Seol J.H. Bang O.S. Ovaa H. Tatsumi K. Komatsu M. Tanaka K. Chung C.H. J. Biol. Chem. 2007; 282: 5256-5262Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar) (supplemental Fig. 1). Immunoblot analysis with anti-C20orf116 antibody showed ubiquitous expression although with tissue-specific differences (e.g. specifically highly expressed in the liver) (Fig. 1B). Because C20orf116 was identified as both a Ufm1- and Ufc1-interacting protein, we first examined whether C20orf116 is a substrate for Ufm1 in vivo. To do this, GFP-tagged Ufm1 (GFP-Ufm1) was expressed alone and co-expressed with C-terminal FLAG-tagged C20orf116 (C20orf116-FLAG) in HEK293 cells. Each cell lysate was prepared and subjected to immunoprecipitation analysis with anti-GFP antibody followed by immunoblotting with anti-GFP and anti-C20of116 antibodies. Expression of GFP-Ufm1 resulted in the appearance of several bands that shifted to higher molecular weight (see the band shown by asterisks in Fig. 1C, top, lane 8). Because we did not detect any higher bands when Ufm1ΔC3 lacking the C-terminal glycine 83 of mature Ufm1 (GFP-Ufm1ΔC3) was expressed (Fig. 1C, lane 10), these higher size bands were thought to be conjugates between GFP-Ufm1 and endogenous proteins. By co-expression of GFP-Ufm1 with C20orf116-FLAG, an additional band with a molecular mass of 80 kDa (see the band shown by an arrowhead) was detected (Fig. 1C, top, lane 9). The band was also clearly recognized by anti-C20orf116 antibody (Fig. 1C, bottom, lane 9), suggesting the presence of C20orf116-FLAG·GFP-Ufm1 conjugate. To further confirm this result, C20orf116-FLAG was immunoprecipitated by anti-FLAG antibody and subsequently immunoblotted with anti-GFP and anti-C20orf116 antibodies. Consistent with the above data, a higher sized band (see the band shown by an arrowhead) was observed when C20orf116-FLAG was co-expressed with GFP-Ufm1 but not alone or with GFP-Ufm1ΔC3 (Fig. 1C, bottom, lanes 12, 14, and 15). The band was also recognized by anti-GFP antibody (Fig. 1C, top, lane 14). These results indicate the existence of the C20orf116-FLAG·GFP-Ufm1 complex. Because the conjugate had an apparent molecular mass of 80 kDa, it could be composed of one GFP-Ufm1 (40 kDa) and one C20orf116-FLAG (40 kDa). Next, to determine the lysine residue of C20orf116 for Ufm1 conjugation, we prepared several point mutants in which each lysine residue of C20orf116 conserved across species was substituted with an arginine residue (K116R, K121R, K124R, K128R, K193R, K224R, K227R, and K267R) and a lysineless mutant in which all conserved lysine residues were substituted with arginine residues (K-less in Fig. 1D). Then we examined the effect of each mutant on the conjugate formation. As shown in Fig. 1D, immunoprecipitation experiments revealed that the K267R mutant was less likely to form a conjugate with GFP-Ufm1 than the wild-type C20orf116 and other mutants (Fig. 1D, lane 12), although some conjugate formation was still observed. In contrast, the lysineless mutant completely abolished the conjugation formation (Fig. 1D, lane 13). These results suggest that Lys-267 is the main lysine residue for Ufm1 conjugation, although other lysine residue(s) could become the site when Lys-267 was mutated. There is also a possibility that K267R mutation causes a conformational change of the PCI domain and subsequently disrupts the protein-protein interaction, leading to loss of the ufmylation. In the next step, we tested whether Ufm1 can conjugate with C20orf116 in vitro. The in vitro reconstitution assay was performed by using recombinant proteins expressed in E. coli. Due to insolubilization of the full-length C20orf116, we used C20orf116 with the N-terminal 50 amino acids deleted, whose region is rich in hydrophobic residues (C20orf116ΔN50). Recombinant Uba5, Ufc1, mature Ufm1 (i.e. Ufm1ΔC2) with an exposed C-terminal glycine 83 residue, and C20orf116ΔN50 were purified, mixed, and incubated in the presence or absence of ATP, and then they were subjected to SDS-PAGE under reducing conditions. Ufm1ΔC3 was used as a negative control. A ∼40 kDa band, corresponding to the C20orf116ΔN50·Ufm1ΔC2 complex, was observed, which was dependent on Uba5 and Ufc1 (Fig. 1E, lanes 2, 4, and 5). This complex was not observed when the reaction mixture was ATP-free (Fig. 1E, lane 1). Moreover, Ufm1ΔC3 could not form the complex in this reaction (Fig. 1E, lane 3). Furthermore, Ufm1 did not form a conjugate with the unrelated protein LC3 in these assay conditions (Fig. 1E, lane 6), implying substrate specificity of Ufm1. Taken together, these results indicate that C20orf116 is a real substrate for Ufm1. To identify the proteins that regulate C20orf116 conjugation with Ufm1, we carried out direct nanoflow LC-MS/MS analysis using C20orf116-FLAG as bait. The analysis identified an uncharacterized protein, KIAA0776, that was named Ufl1 (Ufm1-ligase 1) (supplemental Table 1; for explanation, see below). Ufl1 is composed of 794 amino acids with a predicted molecular mass of ∼90 kDa (Fig. 2A) and has no typical functional domain or homology with other known proteins. This protein was also identified as an Ufc1-interacting protein by the LC-MS/MS analysis using Ufc1 as bait (data not shown; see Fig. 2E). Like C20orf116, this protein is conserved in multicellular organisms but not in yeasts (supplemental Fig. 1). Immunoblot analysis with anti-Ufl1 antibody showed ubiquitous expression in tissues, with exceptionally high expression in the liver (Fig. 2B). Next, to determine the C20orf116-interacting domain of Ufl1, we constructed a series of FLAG-tagged deletion mutants of Ufl1 (Fig. 2C), and they were expressed in HEK293 cells. Each cell lysate was prepared and then immunoprecipitated with anti-FLAG antibody followed by immunoblot analysis with anti- FLAG and anti-C20orf116 antibodies. As predicted, the wild-type Ufl1 (aa 1–794) efficiently interacted with endogenous C20orf116 (Fig. 2D, lane 9). Similarly, the C-terminal deleted mutants of Ufl1, such as M4 (aa 1–452) and M5 (aa 1–654), also interacted with C20orf116 (Fig. 2D, lanes 13 and 14). The interaction with C20orf116 was still noted in the N-terminal region of Ufl1 named M1 (aa 1–212), although the immunoblot signal was very weak (Fig. 2D, lane 10). In contrast, the N-terminal deleted mutants of Ufl1, including M2 (aa 213–794) and M3 (aa 453–794), did not interact with C20orf116 (Fig. 2D, lanes 11 and 12). We also confirmed the interaction between recombinant C20orf116 and Ufl1 M1 (supplemental Fig. 2), indicating direct interaction via the N-terminal region of Ufl1. In contrast to C20orf116 interaction with Ufl1, there was hardly any in vivo interaction between Ufl1 and Ufc1 (data not shown). However, an in vitro pull-down assay with recombinant proteins expressed in E. coli revealed a direct interaction between Ufl1 and Ufc1 (Fig. 2E, lane 9). Furthermore, the pull-down assay with mutant recombinant Ufl1 proteins showed clearly that the N-terminal region (aa 1–212) is sufficient for the interaction with Ufc1 (Fig. 2E, lane 10). Taken together, these results suggest that in addition to the presence of the intracellular stable com" @default.
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• W2080972425 title "A Novel Type of E3 Ligase for the Ufm1 Conjugation System" @default.
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