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• W2043893670 abstract "Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin relatedmodifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Δurm1 and Δuba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells. Protein conjugation, such as ubiquitination, is the process by which the C-terminal glycine of a small modifier protein is covalently attached to target protein(s) through sequential reactions with an activating enzyme and conjugating enzymes. Here we report on a novel protein conjugation system in yeast. A newly identified ubiquitin relatedmodifier, Urm1 is a 99-amino acid protein terminated with glycine-glycine. Urm1 is conjugated to target proteins, which requires the C-terminal glycine of Urm1. At the first step of this reaction, Urm1 forms a thioester with a novel E1-like protein, Uba4. Δurm1 and Δuba4 cells showed a temperature-sensitive growth phenotype. Urm1 and Uba4 show similarity to prokaryotic proteins essential for molybdopterin and thiamin biosynthesis, although the Urm1 system is not involved in these pathways. This is the fifth conjugation system in yeast, following ubiquitin, Smt3, Rub1, and Apg12, but it is unique in respect to relation to prokaryotic enzyme systems. This fact may provide an important clue regarding evolution of protein conjugation systems in eukaryotic cells. ubiquitin-activating enzyme ubiquitin carrier protein ubiquitin-protein isopeptide ligase polymerase chain reaction hemagglutinin molybdopterin open reading frame dithiothreitol HA-tagged Urm1 Myc-tagged Uba4 wild-type In recent years, it has been unveiled that protein-protein conjugation plays an indispensable role in several cellular processes in eukaryotes. The best example is the ubiquitin system (1.Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (514) Google Scholar, 2.Bonifacino J.S. Weissman A.M. Annu. Rev. Cell Dev. Biol. 1998; 14: 19-57Crossref PubMed Scopus (533) Google Scholar, 3.Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1180) Google Scholar, 4.Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6822) Google Scholar). Ubiquitin is activated by the ubiquitin-activating enzyme (E1)1 with consumption of ATP and then forms a thioester with E1 between the C-terminal glycine of ubiquitin and the active site cysteine of E1. Ubiquitin is subsequently transferred to one of the ubiquitin carrier proteins (E2). It is finally conjugated to substrate proteins via an isopeptide bond between the C-terminal carboxy group of glycine in ubiquitin and an ε-amino group of lysine in substrates. E3 enzymes or complexes often catalyze this final step. Ubiquitination is the tag for selective degradation by the 26 S proteasome and for endocytosis of many cell surface proteins. In the yeast, Saccharomyces cerevisiae, three other conjugation systems have been reported. Smt3, which was originally isolated as a high-copy suppressor of mif2 mutations, is covalently attached to several targets (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar). Some of the targets were recently identified to be septins (6.Takahashi Y. Iwase M. Konishi M. Tanaka M. Toh-e A. Kikuchi Y. Biochem. Biophys. Res. Commun. 1999; 259: 582-587Crossref PubMed Scopus (85) Google Scholar, 7.Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-993Crossref PubMed Scopus (326) Google Scholar). This conjugation reaction requires Uba2 and Ubc9 as E1- and E2-like enzymes, respectively (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 8.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar). Rub1 is also a modifier protein that is conjugated to Cdc53 in a Uba3- and Ubc12-dependent manner (9.Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 10.Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (278) Google Scholar). In the course of studies on Apg proteins essential for autophagy, we recently discovered the fourth protein conjugation system in yeast. The Apg12 modifier protein is covalently attached to Apg5 through a series of covalent intermediates with Apg7 and Apg10 (11.Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1267) Google Scholar, 12.Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar, 13.Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar). Amino acid sequences of Smt3 and Rub1 are similar to that of ubiquitin, whereas Apg12 is not homologous to ubiquitin. This finding led us to speculate that more protein conjugation systems exist than ever expected. Here we describe the fifth protein conjugation system in yeast, which has affinity to the molybdopterin and thiamin biosynthetic pathways in various species, including prokaryotes. An S. cerevisiae strain used for gene disruption was KA31 (MATα ura3 leu2 his3 trp1) (14.Irie K. Takase M. Lee K.S. Levin D.E. Araki H. Matsumoto K. Oshima Y. Mol. Cell. Biol. 1993; 13: 3076-3083Crossref PubMed Scopus (259) Google Scholar). Characterization of phenotype and immunochemical analysis were performed with Δurm1 (MATαura3 leu2 his3 trp1 Δurm1::HIS3), Δuba4 (MATα ura3 leu2 his3 trp1Δuba4::LEU2), and Δurm1Δuba4 (MATα ura3 leu2 his3 trp1 Δurm1::HIS3Δuba4::LEU2) strains. For two-hybrid screening or analysis, PJ69–4A strain (MAT a ura3 leu2 his3 trp1 gal4Δ gal80ΔLYS2::GAL1-HIS3 GAL2-ADE2 met2::GAL7-lacZ) was used (15.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Cells were grown either in YPD medium (1% yeast extract, 2% peptone, and 2% glucose) or in SD medium containing nutritional supplements. The 1.6-kbAccI–AccI genomic DNA fragment containingURM1 was amplified by PCR and was cloned into theSmaI site of pRS426. A BglII site was created immediately after the initiation codon (ATG), where 3× hemagglutinin (HA) epitopes were introduced to generate pHA-URM1. Similarly, theUBA4 gene was PCR-amplified, subcloned into pRS424, and tagged with 3× Myc epitopes at its N terminus. For gene disruption, pUC18Δurm1, containing the URM1 gene fragment in which theSacI–SacI fragment was replaced withHIS3, and pUC18Δuba4, containing the UBA4 gene in which the whole UBA4 gene was replaced withLEU2, were used. A bait plasmid (pGBD-URM1) encoding Urm1 fused in-frame to the C-terminal end of the Gal4 DNA binding domain was constructed (15.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Two-hybrid screen was performed using the system described by James et al. (15.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). The strain PJ69–4A was sequentially transformed with pGBD-URM1 and a mixture of yeast genomic two-hybrid libraries fused to the Gal4 activation domain (Y2HLA-C1, -C2, and -C3) (15.James P. Halladay J. Craig E.A. Genetics. 1996; 144: 1425-1436Crossref PubMed Google Scholar). Transformants were selected for growth on Ade− Trp− Leu− plates. An insert in pGAD plasmid of each positive clone was isolated by colony PCR and identified by DNA sequencing and Southern blot hybridization. Positive interaction was verified by co-transformation of pGBD-URM1 and each of the recovered prey plasmids. Whole cell extracts were prepared by suspending cells in 0.2 m NaOH, 0.5% 2-mercaptoethanol and precipitated with acetone. The extracts were separated by SDS-polyacrylamide gel electrophoresis followed by immunoblotting using anti-HA monoclonal antibody (16B12; BAbCO). For immunoprecipitation, cell lysates were prepared by homogenizing with glass beads and were precipitated with 16B12 or anti-Myc monoclonal antibody (9E10; BAbCO) as described previously (13.Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar, 16.Kamada Y. Qadota H. Python C.P. Anraku Y. Ohya Y. Levin D.E. J. Biol. Chem. 1996; 271: 9193-9196Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). Mutation and deletion constructs were generated by PCR-based site-directed mutagenesis and confirmed by automated DNA sequencing. In the yeastS. cerevisiae, whose whole genome sequence has been uncovered, a simple BLAST search could not identify any more ubiquitin-related modifier proteins. We then took notice of putative prototypes of ubiquitination-like protein activation systems. InEscherichia coli, the C-terminal glycine residues of small proteins, MoaD (a small subunit of molybdopterin (MPT) synthase) and ThiS, are activated in an ATP-dependent manner by E1-like enzymes, MoeB (MPT synthase sulfurylase) and ThiF, respectively (17.Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar,18.Taylor S.V. Kelleher N.L. Kinsland C. Chiu H.-J. Costello C.A. Backstrom A.D. McLafferty F.W. Begley T.P. J. Biol. Chem. 1998; 273: 16555-16560Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). These reactions are essential for MPT and thiamin biosynthesis, and the MoaD system is also conserved in eukaryotes except for S. cerevisiae (19.Appleyard M.V.C.L. Sloan J. Kana'n G.J.M. Heck I.S. Kinghorn J.R. Unkles S.E. J. Biol. Chem. 1998; 273: 14869-14876Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar, 20.Sloan J. Kinghorn J.R. Unkles S.E. Nucleic Acids Res. 1999; 27: 854-858Crossref PubMed Scopus (23) Google Scholar, 21.Unkles S.E. Heck I.S. Appleyard M.V.C.L. Kinghorn J.R. J. Biol. Chem. 1999; 274: 19286-19293Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). We performed a PSI BLAST search using the sequences of MoaD and ThiS and identified an uncharacterized open reading frame (ORF), YIL008w, in yeast. It encodes a 99-amino acid protein with a predicted relative molecular mass of 11.0 kDa. As it would function in a pathway other than those of the MPT and thiamin biosynthesis (discussed below), we named it Urm1 (ubiquitinrelated modifier 1). Urm1 shows 23 and 20% identity to MoaD and ThiS, respectively. Significant homology is observed in the C-terminal region of these proteins (Fig.1 A). Urm1, ThiS, and MoaD possess the C-terminal glycine-glycine motif, which is a common feature of ubiquitin and ubiquitin-related modifiers. Unlike other ubiquitin-related modifiers, Urm1 does not have any C-terminal extension after the glycine-glycine residues. Although Urm1 does not show apparent overall homology to ubiquitin and other modifiers, it shows limited homology to Smt3 at its C-terminal region. Possible counterparts of Urm1 exist in higher eukaryotes, including human. A human cDNA (AI816106) encodes a protein that is 42% identical to amino acids 10–99 of Urm1 (Fig. 1 B). Thus, Urm1 fulfills a basic function in eukaryotic cells. Because Urm1 has the characteristic C-terminal domain, we postulated that Urm1 would be activated by E1-like enzymes. Using Urm1 as a bait, we performed a two-hybrid screen and obtained several positive clones containing fragments of ORF YHR111w. Strong two-hybrid interaction was actually observed between Urm1 and the full sequence of YHR111w protein. YHR111w encodes a 440-amino acid protein with a predicted molecular mass of 49.4 kDa. We named it Uba4, because the region containing residues 46–196 shows high similarity to the corresponding regions in Uba1 (ubiquitin-activating enzyme, E1) (22.McGrath J.P. Jentsch S. Varshavsky A. EMBO J. 1991; 10: 227-236Crossref PubMed Scopus (188) Google Scholar) (Fig.2 A) and other E1-like enzymes (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 9.Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 12.Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar, 23.Dohmen R.J. Stappen R. McGrath J.P. Forrová H. Kolarov J. Goffeau A. Varshavsky A. J. Biol. Chem. 1995; 270: 18099-18109Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar) (not shown) including a conserved ATP binding motif (GXGXXG). Intriguingly, the overall sequence of Uba4 is closely similar to MPT synthase sulfurylase of various species including E. coli MoeB and E. coli ThiF (Fig.2 B). If Uba4 is a Urm1-activating enzyme, Urm1 and Uba4 would form a thioester on the analogy of the ubiquitin-related systems. The lysates of Δurm1Δuba4 strains expressing a 3× HA-tagged Urm1 (HAUrm1) and/or 3× Myc-tagged Uba4 (MycUba4) were immunoprecipitated with anti-Myc antibody, and the resulting precipitates were analyzed by Western blotting. A conjugate linked by a thioester bond can be detected as a reducing reagent-sensitive band on a Western blot. In the case of the cells expressing both HAUrm1 and MycUba4, a 76-kDa band was detected with anti-Myc antibody, in addition to a 58-kDa band corresponding to free MycUba4 under non-reduced condition (Fig. 3, lanes 7 and8). The 76-kDa band was also precipitated with anti-HA antibody (not shown). The intensity of this band was decreased with increasing concentrations of a reducing reagent, DTT (Fig. 3,lanes 8–12). In contrast, an 18-kDa band corresponding to free HAUrm1 appeared in the precipitate after the DTT treatment (Fig. 3, lanes 8–12). These results suggest thatHAUrm1 interacts with MycUba4 through a thioester bond, and Uba4 functions as a Urm1-activating enzyme. We next determined whether Urm1 is actually conjugated to some target proteins. The extract of the Δurm1 cells expressing HAUrm1 by a 2μ-based plasmid was treated with 2-mercaptoethanol and subjected to Western blotting. A major band at 37 kDa was detected in addition to the 18-kDa band that corresponds to HAUrm1 (Fig. 4 A, WT). The 37-kDa band was not observed in the cells expressingHAUrm1ΔG in which the C-terminal glycine was deleted (Fig. 4 A, ΔG). This indicates that Urm1 is covalently attached to at least one target protein, most probably through an isopeptide bond between the C-terminal glycine of Urm1 and a lysine residue in the target. As shown in Fig. 4 A, there were many other faint bands in the WT, most of which were weakly observed in the ΔG cells. However, a band at 33 kDa (arrow head) disappeared in the ΔG cells, suggesting that the 33-kDa molecule is another conjugate or a degradation product of the 37-kDa conjugate. These conjugate(s) were not found in the Δuba4 cells (Fig.4 B, Δuba4), confirming that Urm1 activation by Uba4 is a prerequisite for the conjugate formation. As shown in Fig.2 B, a metal binding motif (CXXC) of Uba4 is conserved in MPT synthase sulfurylase and ThiF and also in other E1-like enzymes (Uba2, Uba3, and Apg7) (not shown). In the case of MoeB, it was shown that MoeB contains stoichiometric zinc (17.Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar). As the active-site cysteines of these E1-like enzymes in eukaryotes are 10–20 amino acid residues downstream from the metal binding motif (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 9.Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 12.Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar), a Cys225 of Uba4 is the most possible active-site cysteine. Indeed, the mutants in which the Cys225 was replaced by serine or alanine were unable to catalyze the conjugate formation (Fig.4 B, C225S and C225A). URM1 was not essential because the Δurm1mutant was able to grow on YPD or non-fermentable carbon source media (glycerol, ethanol, and acetic acid). However, the growth was slightly retarded at 23 or 30 °C and severely impaired at 37 °C (Fig.5 A). This growth defect was suppressed by addition of an osmotic stabilizer (1 msorbitol) in the medium (Fig. 5 B). As shown in TableI, the phenotype of the Δuba4 strain was the same as the Δurm1strain, and the Δurm1Δuba4 strain also showed the same temperature sensitivity without additive effect. Furthermore, Urm1ΔG, Uba4C225S, and Uba4C225A mutants, which are defective in the conjugate formation (Fig. 4), showed the same phenotype. These results imply that Urm1 and Uba4 act in the same pathway, and conjugate formation is essential to fulfill a role of the system.Table ITemperature sensitivity caused by mutations in Urm1 and Uba4StrainGrowth at 37 °CHostExpressed protein(s)YPD1 M sorbitol/YPDWild-type (KA31)None++Δurm1None−+Δurm1Urm1++Δurm1Urm1ΔG−+Δuba4None−+Δurm1 Δuba4None−+Δurm1 Δuba4Urm1, Uba4++Δurm1 Δuba4Urm1−+Δurm1 Δuba4Urm1, Uba4C225S−+Δurm1 Δuba4Urm1, Uba4C225A−+This test was carried out in the same way as described in Fig. 5. Open table in a new tab This test was carried out in the same way as described in Fig. 5. Here we have shown the novel Urm1 conjugation system in yeast. This is the fifth protein conjugation system in yeast, successive to the ubiquitin, Smt3 (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 8.Johnson E.S. Blobel G. J. Biol. Chem. 1997; 272: 26799-26802Abstract Full Text Full Text PDF PubMed Scopus (406) Google Scholar, 24.Li S.J. Hochstrasser M. Nature. 1999; 398: 246-251Crossref PubMed Scopus (604) Google Scholar), Rub1 (9.Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 10.Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (278) Google Scholar), and Apg12 (11.Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1267) Google Scholar, 12.Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar, 13.Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar, 25.Mizushima N. Noda T. Ohsumi Y. EMBO J. 1999; 18: 3888-3896Crossref PubMed Scopus (338) Google Scholar) systems. Although the ubiquitin system is well known, the other systems are rather new. Smt3 and Rub1 have been considered to be modifier proteins (5.Johnson E.S. Schwienhorst I. Dohmen R.J. Blobel G. EMBO J. 1997; 16: 5509-5519Crossref PubMed Scopus (441) Google Scholar, 26.Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1452) Google Scholar), and some of their substrates were recently identified (6.Takahashi Y. Iwase M. Konishi M. Tanaka M. Toh-e A. Kikuchi Y. Biochem. Biophys. Res. Commun. 1999; 259: 582-587Crossref PubMed Scopus (85) Google Scholar, 7.Johnson E.S. Blobel G. J. Cell Biol. 1999; 147: 981-993Crossref PubMed Scopus (326) Google Scholar, 9.Liakopoulos D. Doenges G. Matuschewski K. Jentsch S. EMBO J. 1998; 17: 2208-2214Crossref PubMed Scopus (306) Google Scholar, 10.Lammer D. Mathias N. Laplaza J.M. Jiang W. Liu Y. Callis J. Goebl M. Estelle M. Genes Dev. 1998; 12: 914-926Crossref PubMed Scopus (278) Google Scholar). As the fourth yeast system, we previously found the Apg12 system in our apg mutant collection (11.Mizushima N. Noda T. Yoshimori T. Tanaka Y. Ishii T. George M.D. Klionsky D.J. Ohsumi M. Ohsumi Y. Nature. 1998; 395: 395-398Crossref PubMed Scopus (1267) Google Scholar, 12.Tanida I. Mizushima N. Kiyooka M. Ohsumi M. Ueno T. Ohsumi Y. Kominami E. Mol. Biol. Cell. 1999; 10: 1367-1379Crossref PubMed Scopus (324) Google Scholar, 13.Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar). These discoveries increasingly revealed the importance of protein conjugation systems. However, this field is still in the early stage of development. Among the five conjugation systems in yeast, only the Urm1 system has high affinity to the prokaryotic enzyme systems. So far, the ubiquitination-like protein conjugation systems have been discovered and focused almost exclusively in eukaryotic cells. However, at least the first step, i.e. ATP-dependent protein activation by E1-like proteins, apparently has ancient prototypes such as the MoaD and ThiS activation systems in E. coli (17.Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar). Primary function of the MoaD and ThiS systems has been assigned to sulfur transfer. After activation by MoeB, MoaD receives sulfur to its C-terminal glycine and donates it to MPT precursor Z. Pitterle and Rajagopalan (27.Pitterle D.M. Rajagopalan K.V. J. Biol. Chem. 1993; 268: 13499-13505Abstract Full Text PDF PubMed Google Scholar) suggested that MoaD might be covalently attached to MoaE (a large subunit of MPT synthase). However, the relationship between generation of the MoaD-MoaE complex and the ATP-dependent enzyme reaction remains unknown. The Urm1 conjugation system that has similarity to these systems may provide a missing link between ATP-dependent cofactor sulfuration and ATP-dependent protein conjugation. Furthermore, our results could suggest that MoaD may first form an adenylate and then a thioester with MoeB prior to sulfuration as previously suggested (17.Rajagopalan K.V. Biochem. Soc. Trans. 1997; 25: 757-761Crossref PubMed Scopus (64) Google Scholar), and eukaryotes have probably utilized such a kind of enzyme reaction systems to create a world of the ubiquitination-type protein conjugation systems. The exact function of the Urm1 system is still to be determined, and future identification of substrate(s) will disclose its physiological roles. We assume that the Urm1 system must function in a distinctive pathway from the MPT and thiamin biosynthetic systems by the following reasons: it was suggested that the yeast S. cerevisiae lost MPT and its precursor, although the MPT biosynthetic system is well conserved in eukaryotes (20.Sloan J. Kinghorn J.R. Unkles S.E. Nucleic Acids Res. 1999; 27: 854-858Crossref PubMed Scopus (23) Google Scholar, 21.Unkles S.E. Heck I.S. Appleyard M.V.C.L. Kinghorn J.R. J. Biol. Chem. 1999; 274: 19286-19293Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In higher eukaryotes, MoaD homologues are distinct from Urm1 homologues (20.Sloan J. Kinghorn J.R. Unkles S.E. Nucleic Acids Res. 1999; 27: 854-858Crossref PubMed Scopus (23) Google Scholar) (Fig. 1 B and data not shown). As for thiamin biosynthesis, a yeast counterpart of ThiF is assigned to Thi4 (28.Praekelt U.M. Byrne K.L. Meacock P.A. Yeast. 1994; 10: 481-490Crossref PubMed Scopus (79) Google Scholar, 29.Hohmann S. Meacock P.A. Biochim. Biophys. Acta. 1998; 1385: 201-219Crossref PubMed Scopus (148) Google Scholar) that is different from Uba4. Furthermore, we observed that the temperature sensitivity of the Δurm1 and Δuba4 cells was not supressed by thiamin addition (data not shown). Finally, although humans do not synthesize thiamin, Urm1 is highly conserved in human. Our successful discovery of the new conjugation system implies that protein conjugation is more prevailing in the eukaryotic cells than ever predicted from simple sequence similarity. Actually, we have recently reported that Apg10 acts as a protein-conjugating enzyme (E2) for the Apg12 modifier, although it shows no homology to ubiquitin E2 enzymes (13.Shintani T. Mizushima N. Ogawa Y. Matsuura A. Noda T. Ohsumi Y. EMBO J. 1999; 18: 5234-5241Crossref PubMed Scopus (234) Google Scholar). Now it is reasonable to speculate that protein conjugation provides more generalized apparatus to modify the fate and function of target proteins. We thank Philip James (University of Wisconsin) for providing the yeast strain, vectors, and genomic libraries for the two-hybrid screening and analysis." @default.
• W2043893670 created "2016-06-24" @default.
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• W2043893670 date "2000-03-01" @default.
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• W2043893670 title "A Protein Conjugation System in Yeast with Homology to Biosynthetic Enzyme Reaction of Prokaryotes" @default.
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• W2043893670 doi "https://doi.org/10.1074/jbc.275.11.7462" @default.
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