Arginyltransferase, Its Specificity, Putative Substrates, Bidirectional Promoter, and Splicing-derived Isoforms
2006; Elsevier BV; Volume: 281; Issue: 43 Linguagem: Inglês
10.1074/jbc.m604355200
ISSN1083-351X
AutoresRonggui Hu, Christopher S. Brower, Haiqing Wang, Ilia V. Davydov, Jun Sheng, Jian‐Min Zhou, Yong Tae Kwon, Alexander Varshavsky,
Tópico(s)Endoplasmic Reticulum Stress and Disease
ResumoSubstrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu, and (oxidized) Cys, function through their conjugation to Arg, one of destabilizing N-terminal residues that are recognized directly by the pathway's ubiquitin ligases. The conjugation of Arg is mediated by arginyltransferase, encoded by ATE1. Through its regulated degradation of specific proteins, the arginylation branch of the N-end rule pathway mediates, in particular, the cardiovascular development, the fidelity of chromosome segregation, and the control of signaling by nitric oxide. We show that mouse ATE1 specifies at least six mRNA isoforms, which are produced through alternative splicing, encode enzymatically active arginyltransferases, and are expressed at varying levels in mouse tissues. We also show that the ATE1 promoter is bidirectional, mediating the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (glucose-regulated protein 78) and protein-disulfide isomerase as putative physiological substrates of arginyltransferase. Purified isoforms of arginyltransferase that contain the alternative first exons differentially arginylate these proteins in extract from ATE1-/- embryos, suggesting that specific isoforms may have distinct functions. Although the N-end rule pathway is apparently confined to the cytosol and the nucleus, and although GRP78 and protein-disulfide isomerase are located largely in the endoplasmic reticulum, recent evidence suggests that these proteins are also present in the cytosol and other compartments in vivo, where they may become N-end rule substrates. Substrates of the N-end rule pathway include proteins with destabilizing N-terminal residues. Three of them, Asp, Glu, and (oxidized) Cys, function through their conjugation to Arg, one of destabilizing N-terminal residues that are recognized directly by the pathway's ubiquitin ligases. The conjugation of Arg is mediated by arginyltransferase, encoded by ATE1. Through its regulated degradation of specific proteins, the arginylation branch of the N-end rule pathway mediates, in particular, the cardiovascular development, the fidelity of chromosome segregation, and the control of signaling by nitric oxide. We show that mouse ATE1 specifies at least six mRNA isoforms, which are produced through alternative splicing, encode enzymatically active arginyltransferases, and are expressed at varying levels in mouse tissues. We also show that the ATE1 promoter is bidirectional, mediating the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (glucose-regulated protein 78) and protein-disulfide isomerase as putative physiological substrates of arginyltransferase. Purified isoforms of arginyltransferase that contain the alternative first exons differentially arginylate these proteins in extract from ATE1-/- embryos, suggesting that specific isoforms may have distinct functions. Although the N-end rule pathway is apparently confined to the cytosol and the nucleus, and although GRP78 and protein-disulfide isomerase are located largely in the endoplasmic reticulum, recent evidence suggests that these proteins are also present in the cytosol and other compartments in vivo, where they may become N-end rule substrates. A protein substrate of the ubiquitin (Ub) 2The abbreviations used are: Ub, ubiquitin; R-transferase, Arg-tRNA-protein transferase or arginyltransferase; Ndp, primary destabilizing N-terminal residue; Nds, secondary destabilizing N-terminal residue; E3, ubiquitinprotein ligase; PDI, protein-disulfide isomerase; ER, endoplasmic reticulum; RT, reverse transcription; EF, embryonic fibroblast; MS/MS, tandem mass spectrometry; EST, expressed sequence tag; TR-PDI, translocon-resident protein-disulfide isomerase complex; βgal, β-galactosidase. 2The abbreviations used are: Ub, ubiquitin; R-transferase, Arg-tRNA-protein transferase or arginyltransferase; Ndp, primary destabilizing N-terminal residue; Nds, secondary destabilizing N-terminal residue; E3, ubiquitinprotein ligase; PDI, protein-disulfide isomerase; ER, endoplasmic reticulum; RT, reverse transcription; EF, embryonic fibroblast; MS/MS, tandem mass spectrometry; EST, expressed sequence tag; TR-PDI, translocon-resident protein-disulfide isomerase complex; βgal, β-galactosidase.-proteasome system, which controls the levels of many intracellular proteins, is conjugated to Ub through the action of ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin-protein ligase (E3) (1Varshavsky A. Protein Sci. 2006; 15: 647-654Crossref PubMed Scopus (58) Google Scholar, 2Hershko A. Ciechanover A. Varshavsky A. Nat. Med. 2000; 10: 1073-1081Crossref Scopus (567) Google Scholar, 3Pickart C. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 4Elsasser S. Finley D. Nat. Cell Biol. 2005; 7: 16-23Crossref PubMed Scopus (301) Google Scholar, 5Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1662) Google Scholar, 6Fang S. Weissman A.M. Cell. Mol. Life Sci. 2004; 61: 1546-1561Crossref PubMed Google Scholar, 7Hochstrasser M. Cell. 2006; 124: 27-34Abstract Full Text Full Text PDF PubMed Scopus (214) Google Scholar). The term “Ub ligase” denotes either an E2-E3 holoenzyme or its E3 component. The selectivity of ubiquitylation is mediated largely by E3, which recognizes a degradation signal (degron) of a substrate (1Varshavsky A. Protein Sci. 2006; 15: 647-654Crossref PubMed Scopus (58) Google Scholar, 3Pickart C. Cell. 2004; 116: 181-190Abstract Full Text Full Text PDF PubMed Scopus (585) Google Scholar, 8Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 9Glotzer M. Murray A.W. Kirschner M. Nature. 1991; 349: 132-138Crossref PubMed Scopus (1891) Google Scholar, 10Johnson E.S. Bartel B., W. Varshavsky A. EMBO J. 1992; 11: 497-505Crossref PubMed Scopus (211) Google Scholar, 11Sheng J. Kumagai A. Dunphy W.G. Varshavsky A. EMBO J. 2002; 21: 6061-6071Crossref PubMed Scopus (39) Google Scholar, 12Ardley H.C. Robinson P.A. Essays Biochem. 2005; 41: 15-30Crossref PubMed Google Scholar). The E3 Ub ligases are an exceptionally large family, with more than 500 distinct E3s in a mammal (5Petroski M.D. Deshaies R.J. Nat. Rev. Mol. Cell Biol. 2005; 6: 9-20Crossref PubMed Scopus (1662) Google Scholar, 12Ardley H.C. Robinson P.A. Essays Biochem. 2005; 41: 15-30Crossref PubMed Google Scholar, 13Cardozo T. Pagano M. Nat. Rev. Mol. Cell Biol. 2004; 5: 739-751Crossref PubMed Scopus (875) Google Scholar, 14Peters J.-M. Mol. Cell. 2002; 9: 931-943Abstract Full Text Full Text PDF PubMed Scopus (770) Google Scholar). A ubiquitylated protein bears a covalently linked poly-Ub chain and is targeted for processive degradation by the 26 S proteasome (15Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1301) Google Scholar, 16Rape M. Jentsch S. Nat. Cell Biol. 2002; 4: E113-E116Crossref PubMed Scopus (96) Google Scholar, 17Wolf D.H. Hilt W. Biochim. Biophys. Acta. 2004; 1695: 19-31Crossref PubMed Scopus (217) Google Scholar, 18Rechsteiner M. Hill C.P. Trends Cell Biol. 2005; 15: 27-33Abstract Full Text Full Text PDF PubMed Scopus (308) Google Scholar, 19Collins G.A. Tansey W.P. Curr. Opin. Genet. Dev. 2006; 16: 197-202Crossref PubMed Scopus (213) Google Scholar). Ub has other functions as well, including nonproteolytic ones (20Finley D. Bartel B. Varshavsky A. Nature. 1989; 338: 394-401Crossref PubMed Scopus (554) Google Scholar, 21Schnell J.D. Hicke L. J. Biol. Chem. 2003; 278: 35857-35860Abstract Full Text Full Text PDF PubMed Scopus (345) Google Scholar). An essential determinant of one class of degrons, called N-degrons, is a destabilizing N-terminal residue of a substrate. The set of destabilizing residues in a given cell type yields a rule, called the N-end rule, which relates the in vivo half-life of a protein to the identity of its N-terminal residue (1Varshavsky A. Protein Sci. 2006; 15: 647-654Crossref PubMed Scopus (58) Google Scholar, 22Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1367) Google Scholar, 23Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 24Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (120) Google Scholar, 25Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (238) Google Scholar, 26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar). In eukaryotes, the N-degron consists of three determinants: a destabilizing N-terminal residue of a protein substrate, its internal Lys residue(s) (the site of formation of a poly-Ub chain), and a conformationally flexible region(s) in the vicinity of these determinants that is required for the substrate's ubiquitylation and/or degradation (8Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (318) Google Scholar, 23Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 27Johnson E.S. Gonda D.K. Varshavsky A. Nature. 1990; 346: 287-291Crossref PubMed Scopus (108) Google Scholar, 28Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar, 29Prakash S. Tian L. Ratliff K.S. Lehotzky R.E. Matouschek A. Nat. Struct. Mol. Biol. 2004; 11: 830-837Crossref PubMed Scopus (356) Google Scholar). The N-end rule has a hierarchic structure (Fig. 1A). In eukaryotes, N-terminal Asn and Gln are tertiary destabilizing residues (denoted as Ndt) in that they function through their enzymatic deamidation (30Baker R.T. Varshavsky A. J. Biol. Chem. 1995; 270: 12065-12074Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 31Kwon Y.T. Balogh S.A. Davydov I.V. Kashina A.S. Yoon J.K. Xie Y. Gaur A. Hyde L. Denenberg V.H. Varshavsky A. Mol. Cell. Biol. 2000; 20: 4135-4148Crossref PubMed Scopus (87) Google Scholar), to yield the secondary destabilizing N-terminal residues Asp and Glu (denoted as Nds). The destabilizing activity of the N-terminal Asp and Glu requires their conjugation, by ATE1-encoded isoforms of Arg-tRNA-protein transferase (arginyltransferase or R-transferase), to Arg, one of the primary destabilizing residues (denoted as Ndp) (26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar, 32Balzi E. Choder M. Chen W. Varshavsky A. Goffeau A. J. Biol. Chem. 1990; 265: 7464-7471Abstract Full Text PDF PubMed Google Scholar, 33Li J. Pickart C.M. Biochemistry. 1995; 34: 15829-15837Crossref PubMed Scopus (34) Google Scholar, 34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar, 35Davydov I.V. Varshavsky A. J. Biol. Chem. 2000; 275: 22931-22941Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 36Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (257) Google Scholar, 37Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (187) Google Scholar). The latter N-terminal residues are recognized by E3 Ub ligases of the N-end rule pathway, called N-recognins (23Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 24Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (120) Google Scholar, 25Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (238) Google Scholar, 38Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (113) Google Scholar, 39An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (70) Google Scholar) (Fig. 1A). The N-end rule pathway of the yeast Saccharomyces cerevisiae is mediated by a single N-recognin, UBR1 (40Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (142) Google Scholar, 41Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar), while at least four N-recognins, including UBR1, mediate this pathway in mammals (24Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (120) Google Scholar, 25Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (238) Google Scholar, 38Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (113) Google Scholar, 39An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (70) Google Scholar, 42Siepmann T.J. Bohnsack R.N. Tokgoz Z. Baboshina O.V. Haas A.L. J. Biol. Chem. 2003; 278: 9448-9457Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar). In mammals and other eukaryotes that produce nitric oxide (NO), the set of arginylated residues contains not only Asp and Glu but also N-terminal Cys (C) (43Gonda D.K. Bachmair A. Wünning I. Tobias J.W. Lane W.S. Varshavsky A. J. Biol. Chem. 1989; 264: 16700-16712Abstract Full Text PDF PubMed Google Scholar), which is arginylated after its oxidation (36Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (257) Google Scholar). The in vivo oxidation of N-terminal Cys requires NO, as well as oxygen (O2) or its derivatives (Fig. 1A) (26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar, 37Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (187) Google Scholar). Although prokaryotes lack Ub conjugation and Ub itself, they contain (Ub-independent) N-end rule pathways (44Tobias J.W. Shrader T.E. Rocap G. Varshavsky A. Science. 1991; 254: 1374-1377Crossref PubMed Scopus (420) Google Scholar, 45Shrader T.E. Tobias J.W. Varshavsky A. J. Bacteriol. 1993; 175: 4364-4374Crossref PubMed Scopus (71) Google Scholar, 46Erbse A. Schmidt R. Bornemann T. Schneider-Mergener J. Mogk A. Zahn R. Dougan D.A. Bukau B. Nature. 2005; 439: 753-756Crossref Scopus (178) Google Scholar, 47Graciet E. Hu R.G. Piatkov K. Rhee J.H. Schwarz E.M. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3078-3083Crossref PubMed Scopus (70) Google Scholar). The sets of destabilizing residues in prokaryotic N-end rules contain secondary destabilizing (Nds) residues as well. Their identities (Arg and Lys in Escherichia coli; Arg, Lys, Asp, and Glu in some other prokaryotes) are either overlapping with, or distinct from, the eukaryotic Nds residues (47Graciet E. Hu R.G. Piatkov K. Rhee J.H. Schwarz E.M. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3078-3083Crossref PubMed Scopus (70) Google Scholar). The activity of Nds residues in prokaryotes requires their conjugation to Leu (an Ndp residue) by Leu-tRNA-protein transferases (L-transferases), in contrast to the conjugation of Arg (an Ndp residue in eukaryotes) to N-terminal Asp, Glu, or (oxidized) Cys (Fig. 1A) (44Tobias J.W. Shrader T.E. Rocap G. Varshavsky A. Science. 1991; 254: 1374-1377Crossref PubMed Scopus (420) Google Scholar, 47Graciet E. Hu R.G. Piatkov K. Rhee J.H. Schwarz E.M. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3078-3083Crossref PubMed Scopus (70) Google Scholar). Prokaryotic L-transferases are of two distinct types, differing in both amino acid sequence and substrate specificity. Remarkably, L-transferases of one class, encoded by bpt genes, are sequelogs of ATE1-encoded eukaryotic R-transferases, despite the fact that Bpt (prokaryotic) aminoacyltransferases conjugate Leu, rather than Arg, to the N termini of cognate substrates (47Graciet E. Hu R.G. Piatkov K. Rhee J.H. Schwarz E.M. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 3078-3083Crossref PubMed Scopus (70) Google Scholar). (In this terminology, “sequelog” and “spalog” denote, respectively, a sequence that is similar, to a specified extent, to another sequence and a three-dimensional structure that is similar, to a specified extent, to another three-dimensional structure (48Varshavsky A. Curr. Biol. 2004; 14: R181-R183Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar). Sequelog and spalog are mnemonically helpful, single word terms whose rigor-conferring advantage is their evolutionary neutrality. The sequelog terminology conveys the fact of sequence similarity (sequelogy) without evolutionary or functional connotations, in contrast to interpretation-laden terms such as homolog, ortholog, and paralog. The latter terms are compatible with the sequelog/spalog terminology and can be employed to convey understanding about functions and common descent, if this information is actually available (48Varshavsky A. Curr. Biol. 2004; 14: R181-R183Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar).) The functions of the N-end rule pathway include the regulation of import of short peptides (through the degradation, modulated by peptides, of the import's repressor) (41Du F. Navarro-Garcia F. Xia Z. Tasaki T. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 14110-14115Crossref PubMed Scopus (84) Google Scholar, 49Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (164) Google Scholar), the fidelity of chromosome segregation (through the degradation of a conditionally produced cohesin fragment) (50Rao H. Uhlmann F. Nasmyth K. Varshavsky A. Nature. 2001; 410: 955-960Crossref PubMed Scopus (228) Google Scholar), the regulation of apoptosis (through the degradation of a caspase-processed inhibitor of apoptosis) (51Ditzel M. Wilson R. Tenev T. Zachariou A. Paul A. Deas E. Meier P. Nat. Cell Biol. 2003; 5: 467-473Crossref PubMed Scopus (205) Google Scholar, 52Varshavsky A. Nat. Cell Biol. 2003; 5: 373-376Crossref PubMed Scopus (92) Google Scholar), the regulation of meiosis (24Kwon Y.T. Xia Z.X. An J.Y. Tasaki T. Davydov I.V. Seo J.W. Xie Y. Varshavsky A. Mol. Cell. Biol. 2003; 23: 8255-8271Crossref PubMed Scopus (120) Google Scholar), the leaf senescence in plants (53Yoshida S. Ito M. Gallis J. Nishida I. Watanabe A. Plant J. 2002; 32: 129-137Crossref PubMed Scopus (117) Google Scholar), as well as neurogenesis and cardiovascular development in mammals (26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar, 36Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (257) Google Scholar, 37Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (187) Google Scholar, 39An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (70) Google Scholar). Mutations in human UBR1, one of several functionally overlapping N-recognins (Fig. 1A) (25Tasaki T. Mulder L.C.F. Iwamatsu A. Lee M.J. Davydov I.V. Varshavsky A. Muesing M. Kwon Y.T. Mol. Cell. Biol. 2005; 25: 7120-7136Crossref PubMed Scopus (238) Google Scholar, 39An J.Y. Seo J.W. Tasaki T. Lee M.J. Varshavsky A. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2006; 103: 6212-6217Crossref PubMed Scopus (70) Google Scholar), are the cause of Johanson-Blizzard syndrome, which includes mental retardation, physical malformations, and severe pancreatitis (54Zenker M. Mayerle J. Lerch M.M. Tagariello A. Zerres K. Durie P.R. Beier M. Hülskamp G. Guzman C. Rehder H. Beemer F.A. Hamel B. Vanlieferinghen P. Gershoni-Baruch R. Vieira M.W. Dumic M. Auslender R. Gil-da-Silva-Lopes V.L. Steinlicht S. Rauh R. Shalev S.A. Thiel C. Winterpacht A. Kwon Y.T. Varshavsky A. Reis A. Nat. Genet. 2005; 37: 1345-1350Crossref PubMed Scopus (190) Google Scholar). The abnormalities of UBR1-/- mice (38Kwon Y.T. Xia Z. Davydov I.V. Lecker S.H. Varshavsky A. Mol. Cell. Biol. 2001; 21: 8007-8021Crossref PubMed Scopus (113) Google Scholar) include pancreatic insufficiency (54Zenker M. Mayerle J. Lerch M.M. Tagariello A. Zerres K. Durie P.R. Beier M. Hülskamp G. Guzman C. Rehder H. Beemer F.A. Hamel B. Vanlieferinghen P. Gershoni-Baruch R. Vieira M.W. Dumic M. Auslender R. Gil-da-Silva-Lopes V.L. Steinlicht S. Rauh R. Shalev S.A. Thiel C. Winterpacht A. Kwon Y.T. Varshavsky A. Reis A. Nat. Genet. 2005; 37: 1345-1350Crossref PubMed Scopus (190) Google Scholar), a less severe version of the defect in human Johanson-Blizzard syndrome (UBR1-/-) patients. The cardiovascular and (probably) other functions of the N-end rule pathway involve the arginylation-mediated degradation of RGS4, RGS5, and RGS16. These “GTPase-activating” proteins function by inhibiting the signaling by specific G proteins, and are themselves down-regulated through the NO/O2-dependent degradation by the N-end rule pathway. The N-terminal Cys residues of RGS4, RGS5, and RGS16 are oxidized in vivo at rates controlled by NO and oxygen, followed by the arginylation of oxidized Cys and processive proteolysis by the rest of the N-end rule pathway (Fig. 1A) (26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar, 35Davydov I.V. Varshavsky A. J. Biol. Chem. 2000; 275: 22931-22941Abstract Full Text Full Text PDF PubMed Scopus (123) Google Scholar, 37Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15030-15035Crossref PubMed Scopus (187) Google Scholar). The arginylation branch of this pathway is also required for degradation of the in vivo-produced fragment of the mammalian RAD21/SCC1 subunit of cohesin. 3J. Zhou, J. Sheng, R.-G. Hu, Y. T. Kwon, and A. Varshavsky, unpublished data. 3J. Zhou, J. Sheng, R.-G. Hu, Y. T. Kwon, and A. Varshavsky, unpublished data. This fragment, a part of circuits that control mitosis and meiosis, is produced through a cleavage by separase (55Uhlmann F. Lottspeich F. Nasmyth K. Nature. 1999; 400: 37-42Crossref PubMed Scopus (750) Google Scholar) and bears N-terminal Glu (an Nds residue) in mammals (56Hauf S. Waizenegger I.C. Peters J.-M. Science. 2001; 293: 1320-1323Crossref PubMed Scopus (376) Google Scholar) but N-terminal Arg (an Ndp residue) in S. cerevisiae (50Rao H. Uhlmann F. Nasmyth K. Varshavsky A. Nature. 2001; 410: 955-960Crossref PubMed Scopus (228) Google Scholar). Given the tripartite structure of N-degrons, it is possible that some physiological substrates of R-transferase would be found to lack a complete N-degron, i.e. that their arginylation would not be followed by their degradation. All eukaryotes examined, from fungi to plants and animals, contain both R-transferases and the N-end rule pathway. The former are sequelogous (similar in sequence) (48Varshavsky A. Curr. Biol. 2004; 14: R181-R183Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) throughout most of their ∼60-kDa spans, even between highly divergent eukaryotes such as fungi and mammals (34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar). A single gene, ATE1, encodes R-transferase in both S. cerevisiae (32Balzi E. Choder M. Chen W. Varshavsky A. Goffeau A. J. Biol. Chem. 1990; 265: 7464-7471Abstract Full Text PDF PubMed Google Scholar) and the mouse or human genomes (34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar, 36Kwon Y.T. Kashina A.S. Davydov I.V. Hu R.-G. An J.Y. Seo J.W. Du F. Varshavsky A. Science. 2002; 297: 96-99Crossref PubMed Scopus (257) Google Scholar), whereas plants such as Arabidopsis thaliana contain two genes, ATE1 and ATE2, which encode sequelogous R-transferases (53Yoshida S. Ito M. Gallis J. Nishida I. Watanabe A. Plant J. 2002; 32: 129-137Crossref PubMed Scopus (117) Google Scholar). Our previous studies described the cloning of yeast and mouse ATE1, and also the finding that mammalian (but not S. cerevisiae) R-transferase occurs as two isoforms, produced through alternative splicing of ATE1 pre-mRNA (32Balzi E. Choder M. Chen W. Varshavsky A. Goffeau A. J. Biol. Chem. 1990; 265: 7464-7471Abstract Full Text PDF PubMed Google Scholar, 34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar). The two mouse R-transferases were shown to be of identical sizes (516 residues), differing by a stretch of 43 residues, encoded by two alternative, adjacent and sequelogous 129-bp exons (34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar). In this work, we continued the analysis of mouse ATE1, identifying six splicing-derived isoforms of ATE1 mRNA and also discovering that the ATE1 transcriptional promoter is bidirectional, driving the expression of both ATE1 and an oppositely oriented, previously uncharacterized gene. In addition, we identified GRP78 (BiP) and protein-disulfide isomerase (PDI), two proteins located primarily in the endoplasmic reticulum (ER), as putative physiological substrates of R-transferases. Several lines of evidence (57Stockton J.D. Merkert M.C. Kellaris K.V. Biochemistry. 2003; 42: 12821-12834Crossref PubMed Scopus (15) Google Scholar, 58Turano C. Coppari S. Altieri F. Ferraro A. J. Cell. Physiol. 2002; 193: 154-163Crossref PubMed Scopus (406) Google Scholar, 59Sun F.C. Wei S. Li C.W. Chang Y.S. Chao C.C. Lai Y.K. Biochem. J. 2006; 396: 31-39Crossref PubMed Scopus (45) Google Scholar, 60Grune T. Reinheckel T. Li R. North J.A. Davies K.J.A. Arch. Biochem. Biophys. 2002; 397: 407-413Crossref PubMed Scopus (38) Google Scholar) suggest that both GRP78 and PDI can be present in non-ER compartments as well, including the cytosol and the nucleus, where these proteins may be targeted by the N-end rule pathway. A recent paper by Rai and Kashina (61Rai R. Kashina A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10123-10128Crossref PubMed Scopus (51) Google Scholar) described two new splicing-derived mouse ATE1 isoforms, in addition to the two previously known ones (34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar). The authors claimed that the four R-transferases could arginylate unmodified N-terminal Cys and also that the two new R-transferases were inactive with N-terminal Asp or Glu (61Rai R. Kashina A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10123-10128Crossref PubMed Scopus (51) Google Scholar). We show that both of these conclusions are incorrect: the activity of known R-transferases toward unmodified N-terminal Cys is negligible, and the two isoforms of R-transferase stated to be inactive with the N-terminal Asp and Glu (61Rai R. Kashina A. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 10123-10128Crossref PubMed Scopus (51) Google Scholar) are actually active with these residues. Yeast Strains, Plasmids, and β-Galactosidase Assay—Synthetic yeast media (62Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2002Google Scholar) contained 0.67% yeast nitrogen base without amino acids (Difco) and either 2% glucose (SD medium) or 2% galactose (SG medium). Synthetic media lacking appropriate nutrients were used to select for (and maintain) specific plasmids. Cells were also grown in rich medium (YPD) (62Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2002Google Scholar). The S. cerevisiae strains were YPH500 (MATα ura3-52 lys-801 ade2-101 his3-Δ200 trp1-Δ63 his3-Δ200 leu2-Δ1), SGY3 (MATα ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1 ate1-Δ2::LEU2), and JD55 (MATα ura3-52 lys2-801 trp1-Δ63 his3-Δ200 leu2-3, 112 ubr1-Δ1::HIS3) (63Ghislain M. Dohmen R.J. Levy F. Varshavsky A. EMBO J. 1996; 15: 4884-4899Crossref PubMed Scopus (236) Google Scholar). AVY34B, a ubr1Δ ate1Δ double mutant, was constructed by replacing the entire ATE1 open reading frame in JD55 (ubr1Δ) by the antibiotic resistance gene G418, using homologous recombination with G418 flanked on either side by 40 bp of ATE1-specific sequences (62Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Smith J.A. Seidman J.G. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York2002Google Scholar). Mutants were selected on SD lacking His and containing G418. G418-resistant isolates were checked by PCR (for the absence of ATE1), by functional assays, and by N-terminal sequencing of X-β-galactosidase (X-βgal) reporters (26Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (235) Google Scholar, 34Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cel
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