RGS4 Is Arginylated and Degraded by the N-end Rule Pathway in Vitro
2000; Elsevier BV; Volume: 275; Issue: 30 Linguagem: Inglês
10.1074/jbc.m001605200
ISSN1083-351X
AutoresIlia V. Davydov, Alexander Varshavsky,
Tópico(s)RNA modifications and cancer
ResumoThe N-end rule relates the in vivohalf-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells. The N-end rule relates the in vivohalf-life of a protein to the identity of its N-terminal residue. We used an expression-cloning screen to search for mouse proteins that are degraded by the ubiquitin/proteasome-dependent N-end rule pathway in a reticulocyte lysate. One substrate thus identified was RGS4, a member of the RGS family of GTPase-activating proteins that down-regulate specific G proteins. A determinant of the RGS4 degradation signal (degron) was located at the N terminus of RGS4, because converting cysteine 2 to either glycine, alanine, or valine completely stabilized RGS4. Radiochemical sequencing indicated that the N-terminal methionine of the lysate-produced RGS4 was replaced with arginine. Since N-terminal arginine is a destabilizing residue not encoded by RGS4 mRNA, we conclude that the degron of RGS4 is generated through the removal of N-terminal methionine and enzymatic arginylation of the resulting N-terminal cysteine. RGS16, another member of the RGS family, was also found to be an N-end rule substrate. RGS4 that was transiently expressed in mouse L cells was short-lived in these cells. However, the targeting of RGS4 for degradation in this in vivo setting involved primarily another degron, because N-terminal variants of RGS4 that were stable in reticulocyte lysate remained unstable in L cells. ubiquitin N-terminal amidase Arg-tRNA-protein transferase Ub-X-nsP41–254 glutathione transferase regulator of G-proteinsignaling methionine aminopeptidase GTPase-activating protein deubiquitylating enzyme 5′-adenylylimidodiphosphate 3-(cyclohexylamino)propanesulfonic acid polyacrylamide gel electrophoresis polymerase chain reaction open reading frame A multitude of regulatory circuits, including those that control the cell cycle, cell differentiation, and responses to stress, involve metabolically unstable proteins (1Maniatis T. Genes Dev. 1999; 13: 505-510Crossref PubMed Scopus (368) Google Scholar, 2Nasmyth K. Trends Biochem. Sci. 1999; 24: 98-104Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar, 3Peters J.-M. King R.W. Deshaies R.J. Peters J.-M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Publishing Corp., New York1998: 345-387Crossref Google Scholar, 4Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 76: 425-479Crossref Scopus (6793) Google Scholar, 5Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar). A short in vivohalf-life of a regulator provides a way to generate its spatial gradients and allows for rapid adjustments of its concentration, or subunit composition, through changes in the rate of its synthesis or degradation. Damaged or otherwise abnormal proteins tend to be short-lived as well (6Wickner S. Maurizi M.R. Gottesman S. Science. 1999; 286: 1888-1893Crossref PubMed Scopus (907) Google Scholar). Features of proteins that confer metabolic instability are called degradation signals or degrons (7Varshavsky A. Cell. 1991; 64: 13-15Abstract Full Text PDF PubMed Scopus (118) Google Scholar, 8Laney J.D. Hochstrasser M. Cell. 1999; 97: 427-430Abstract Full Text Full Text PDF PubMed Scopus (389) Google Scholar). The essential component of one degradation signal, called the N-degron, is a destabilizing N-terminal residue of a protein (9Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1359) Google Scholar). A set of amino acid residues that are destabilizing in a given cell 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. The N-end rule pathway is present in all organisms examined, from mammals and plants to fungi and prokaryotes (10Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 11Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (146) Google Scholar).In eukaryotes, an N-degron consists of two determinants, a destabilizing N-terminal residue and an internal lysine or lysines (12Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 13Johnson E.S. Gonda D.K. Varshavsky A. Nature. 1990; 346: 287-291Crossref PubMed Scopus (108) Google Scholar, 14Pickart C.M. FASEB J. 1997; 11: 1055-1066Crossref PubMed Scopus (305) Google Scholar, 15Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar). The Lys residue is the site of formation of a multiubiquitin chain (16Chau V. Tobias J.W. Bachmair A. Marriott D. Ecker D.J. Gonda D.K. Varshavsky A. Science. 1989; 243: 1576-1583Crossref PubMed Scopus (1106) Google Scholar). The N-end rule pathway is thus one of the pathways of the ubiquitin (Ub)1 system. Ub is a 76-residue protein whose covalent conjugation to other proteins plays a role in a vast range of biological processes (4Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 76: 425-479Crossref Scopus (6793) Google Scholar, 5Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar, 17Hochstrasser M. Annu. Rev. Genet. 1996; 30: 405-439Crossref PubMed Scopus (1452) Google Scholar). In most of them, Ub acts through routes that involve the degradation of ubiquitylated 2Ubiquitin whose C-terminal (Gly-76) carboxyl group is covalently linked to another compound is called the ubiquityl moiety, the derivative terms beingubiquitylation and ubiquitylated. The term Ub refers to both free ubiquitin and the ubiquityl moiety. This nomenclature (5Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar), which is also recommended by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (74Webb, E. C. (ed) (1992) Enzyme Nomenclature 1992, p.527, Academic Press, New YorkGoogle Scholar), brings ubiquitin-related terms in line with the standard chemical terminology. 2Ubiquitin whose C-terminal (Gly-76) carboxyl group is covalently linked to another compound is called the ubiquityl moiety, the derivative terms beingubiquitylation and ubiquitylated. The term Ub refers to both free ubiquitin and the ubiquityl moiety. This nomenclature (5Varshavsky A. Trends Biochem. Sci. 1997; 22: 383-387Abstract Full Text PDF PubMed Scopus (513) Google Scholar), which is also recommended by the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (74Webb, E. C. (ed) (1992) Enzyme Nomenclature 1992, p.527, Academic Press, New YorkGoogle Scholar), brings ubiquitin-related terms in line with the standard chemical terminology. proteins by the 26 S proteasome, an ATP-dependent multisubunit protease (18Baumeister W. Walz J. Zühl F. Seemüller E. Cell. 1998; 92: 367-380Abstract Full Text Full Text PDF PubMed Scopus (1299) Google Scholar, 19DeMartino G.N. Slaughter C.A. J. Biol. Chem. 1999; 274: 22123-22126Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar).The N-end rule has a hierarchic structure. In the yeastSaccharomyces cerevisiae, Asn and Gln are tertiary destabilizing N-terminal residues in that they function through their deamidation, by the NTA1-encoded 3Throughout the text, the names of genes are italicized and all uppercase. The names of proteins are roman and all uppercase. This usage, a modification of the existing conventions (73Stewart A. Trends in Genetics Nomenclature Guide. Elsevier Science, Ltd., Cambridge, U.K.1995Google Scholar), provides a uniform nomenclature in a text that refers to both fungal and metazoan genes and proteins. 3Throughout the text, the names of genes are italicized and all uppercase. The names of proteins are roman and all uppercase. This usage, a modification of the existing conventions (73Stewart A. Trends in Genetics Nomenclature Guide. Elsevier Science, Ltd., Cambridge, U.K.1995Google Scholar), provides a uniform nomenclature in a text that refers to both fungal and metazoan genes and proteins.N-terminal amidase (Nt-amidase), to yield the secondary destabilizing residues Asp and Glu (20Baker R.T. Varshavsky A. J. Biol. Chem. 1995; 270: 12065-12074Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). The destabilizing activity of N-terminal Asp and Glu requires their conjugation, by the ATE1-encoded Arg-tRNA-protein transferase (R-transferase), to Arg, one of the primary destabilizing residues (21Balzi E. Choder M. Chen W. Varshavsky A. Goffeau A. J. Biol. Chem. 1990; 265: 7464-7471Abstract Full Text PDF PubMed Google Scholar, 22Li J. Pickart C.M. Biochemistry. 1995; 34: 15829-15837Crossref PubMed Scopus (34) Google Scholar). The primary destabilizing N-terminal residues are bound directly by UBR1, also called N-recognin, the E3 (recognition) component of the N-end rule pathway (10Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 11Kwon Y.T. Reiss Y. Fried V.A. Hershko A. Yoon J.K. Gonda D.K. Sangan P. Copeland N.G. Jenkins N.A. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 7898-7903Crossref PubMed Scopus (146) Google Scholar).In mammals, the deamidation step is mediated by two Nt-amidases, NtN-amidase and NtQ-amidase, which are specific, respectively, for N-terminal Asn and Gln (23Stewart A.E. Arfin S.M. Bradshaw R.A. J. Biol. Chem. 1995; 270: 25-28Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar, 24Grigoryev S. Stewart A.E. Kwon Y.T. Arfin S.M. Bradshaw R.A. Jenkins N.A. Copeland N.G. Varshavsky A. J. Biol. Chem. 1996; 271: 28521-28532Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). In vertebrates, the set of secondary destabilizing residues contains not only Asp and Glu but also Cys, which is a stabilizing residue in yeast (25Gonda 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, 26Davydov I.V. Patra D. Varshavsky A. Arch. Biochem. Biophys. 1998; 357: 317-325Crossref PubMed Scopus (11) Google Scholar). The mammalian counterpart of the yeast R-transferase Ate1p exists as two distinct species, ATE1–1 and ATE1–2, that are produced through alternative splicing of Ate1 pre-mRNA (27Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar). Both ATE1–1 and ATE1–2 are similar in specificity to the ATE1-encoded yeast R-transferase, in that these R-transferases can arginylate N-terminal Asp and Glu, but cannot arginylate N-terminal Cys (27Kwon Y.T. Kashina A.S. Varshavsky A. Mol. Cell. Biol. 1999; 19: 182-193Crossref PubMed Scopus (107) Google Scholar), suggesting the existence of a distinct R-transferase specific for N-terminal Cys.UBR1 (N-recognin) of yeast and mammals has two binding sites for the primary destabilizing N-terminal residues of either proteins or short peptides. The type 1 site is specific for the basic N-terminal residues Arg, Lys, and His. The type 2 site is specific for the bulky hydrophobic N-terminal residues Phe, Leu, Trp, Tyr, and Ile (25Gonda 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, 28Reiss Y. Kaim D. Hershko A. J. Biol. 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Chem. 1999; 274: 9871-9880Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar).The known functions of the N-end rule pathway include the control of peptide import in S. cerevisiae, through the degradation of CUP9, a transcriptional repressor of the peptide transporter PTR2 (30Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google Scholar) (this control includes a positive feedback mediated by the type 1 and type 2 sites of UBR1 (31Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (164) Google Scholar)); the degradation of GPA1, one of two Gα proteins in S. cerevisiae (32Schauber C. Chen L. Tongaonkar P. Vega I. Madura K. Genes Cells. 1998; 3: 307-319Crossref PubMed Scopus (19) Google Scholar); and the degradation of alphaviral RNA polymerases and other viral proteins in infected metazoan cells (33Lawson T.G. Gronros D.L. Evans P.E. Bastien M.C. Michalewich K.M. Clark J.K. Edmonds J.H. Graber K.H. Werner J.A. Lurvey B.A. Cate J.M. J. Biol. Chem. 1999; 274: 9871-9880Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar, 34deGroot R.J. Rümenapf T. Kuhn R.J. Strauss J.H. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8967-8971Crossref PubMed Scopus (122) Google Scholar). Physiological N-end rule substrates were also identified among the proteins secreted into the cytosol of the mammalian cell by intracellular parasites such as the bacteriumListeria monocytogenes (35Sijts A.J. Pilip I. Pamer E.G. J. Biol. Chem. 1997; 272: 19261-19268Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Selective perturbation of the N-end rule pathway was reported to interfere with mammalian cell differentiation (36Obin M. Mesco E. Gong X. Haas A.L. Joseph J. Taylor A. J. Biol. Chem. 1999; 274: 11789-11795Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 37Hondermarck H. Sy J. Bradshaw R.A. Arfin S.M. Biochem. Biophys. Res. Commun. 1992; 30: 280-288Crossref Scopus (31) Google Scholar) and with limb regeneration in amphibians (38Taban C.H. Hondermarck H. Bradshaw R.A. Boilly B. Experientia (Basel). 1996; 52: 865-870Crossref PubMed Scopus (15) Google Scholar). Studies of the Ub-dependent proteolysis of endogenous proteins in muscle extracts suggested that the N-end rule pathway plays a role in catabolic states that result in muscle atrophy (39Solomon V. Baracos V. Sarraf P. Goldberg A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12602-12607Crossref PubMed Scopus (123) Google Scholar).Until the present work, physiological substrates of Nt-amidases and R-transferases were unknown in either yeast or larger eukaryotes. Engineered N-end rule substrates, including the substrates of Nt-amidases and R-transferases, can be produced in vivothrough the Ub fusion technique, in which a Ub-X-reporter fusion is cleaved, cotranslationally, after the last residue of Ub by deubiquitylating enzymes (DUBs) (40Wilkinson K. Hochstrasser M. Peters J.-M. Harris J.R. Finley D. Ubiquitin and the Biology of the Cell. Plenum Publishing Corp., New York1998: 99-125Crossref Google Scholar), yielding a reporter protein bearing the predetermined N-terminal residue X (9Bachmair A. Finley D. Varshavsky A. Science. 1986; 234: 179-186Crossref PubMed Scopus (1359) Google Scholar, 10Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar, 41Baker R.T. Curr. Opin. Biotechnol. 1996; 7: 541-546Crossref PubMed Scopus (77) Google Scholar).In the present work, we employed a modification of the cDNA-based sib-selection strategy in a transcription-translation lysate from rabbit reticulocytes (42Lustig K.D. Stukenberg P.T. McGarry T.J. King R.W. Cryns V.L. Mead P.E. Zon L.I. Yuan J. Kirschner M.W. Methods Enzymol. 1997; 283: 83-99Crossref PubMed Scopus (66) Google Scholar, 43Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) to identify putative physiological substrates of the N-end rule pathway. Specifically, we used dipeptides bearing destabilizing N-terminal residues as selective inhibitors of the N-end rule pathway, and we screened for mouse cDNAs that expressed proteins whose relative abundance in the lysate was altered in the presence of relevant dipeptides.Among the putative N-end rule substrates identified through the use of this approach was mouse RGS4, a GTPase-activating protein (GAP) for specific Gα subunits of heterotrimeric G proteins, and a member of the family of RGS (regulator of G proteinsignaling) proteins (44De Vries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 45Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 46Heximer S.P. Srinivasa S.P. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 47Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 48DiBello P.R. Garrison T.R. Apanovitch D.M. Hoffman G. Shuey D.J. Mason K. Cockett M.I. Dohlman H.G. J. Biol. Chem. 1998; 273: 5780-5784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 49Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 50Wieland T. Chen C.K. Simon M.I. J. Biol. Chem. 1997; 272: 8853-8856Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). We discovered that in addition to the expected removal of N-terminal Met from the newly formed RGS4, the resulting N-terminal Cys of RGS4 was arginylated, presumably by a distinct R-transferase whose Cys specificity is different from that of the known R-transferases. Thus modified RGS4 bore N-terminal Arg, a primary destabilizing residue, and was degraded by the N-end rule pathway in reticulocyte lysate.DISCUSSIONWe searched for substrates of the mammalian N-end rule pathway by testing some of the previously known candidates and also by employing a modification of the sib selection-based in vitro screen (42Lustig K.D. Stukenberg P.T. McGarry T.J. King R.W. Cryns V.L. Mead P.E. Zon L.I. Yuan J. Kirschner M.W. Methods Enzymol. 1997; 283: 83-99Crossref PubMed Scopus (66) Google Scholar,43Stukenberg P.T. Lustig K.D. McGarry T.J. King R.W. Kuang J. Kirschner M.W. Curr. Biol. 1997; 7: 338-348Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar) in a transcription-translation reticulocyte lysate. Two N-end rule substrates thus identified were mouse RGS4 and RGS16. These proteins are members of the RGS family of GTPase-activating proteins (GAPs) that down-regulate specific G proteins (44De Vries L. Farquhar M.G. Trends Cell Biol. 1999; 9: 138-144Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 45Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 46Heximer S.P. Srinivasa S.P. Bernstein L.S. Bernard J.L. Linder M.E. Hepler J.R. Blumer K.J. J. Biol. Chem. 1999; 274: 34253-34259Abstract Full Text Full Text PDF PubMed Scopus (149) Google Scholar, 47Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 48DiBello P.R. Garrison T.R. Apanovitch D.M. Hoffman G. Shuey D.J. Mason K. Cockett M.I. Dohlman H.G. J. Biol. Chem. 1998; 273: 5780-5784Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 49Yan Y. Chi P.P. Bourne H.R. J. Biol. Chem. 1997; 272: 11924-11927Abstract Full Text Full Text PDF PubMed Scopus (126) Google Scholar, 50Wieland T. Chen C.K. Simon M.I. J. Biol. Chem. 1997; 272: 8853-8856Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 67Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (127) Google Scholar). We report the following results.1) The in vitro activated, proteolytically processed larger (80 kDa) subunit of the cysteine protease m-calpain was previously shown to bear N-terminal Lys (59Brown N. Crawford C. FEBS Lett. 1993; 322: 65-68Crossref PubMed Scopus (58) Google Scholar), a type 1-destabilizing residue in the N-end rule (10Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (715) Google Scholar). We expressed this subunit, termed Lys-mCL, in the reticulocyte lysate as a Ub-Lys-mCL fusion. The resulting Lys-mCL was not a substrate of the N-end rule pathway, despite the presence of a destabilizing N-terminal residue. Since an N-degron is a bipartite signal (12Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (317) Google Scholar, 13Johnson E.S. Gonda D.K. Varshavsky A. Nature. 1990; 346: 287-291Crossref PubMed Scopus (108) Google Scholar, 15Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar), the absence of active N-degron from Lys-mCL could be due to the absence of a targetable internal Lys residue, the second determinant of an N-degron. Another possibility is a steric hindrance in the binding of UBR1 to the N-terminal lysine of Lys-mCL. Thus, a destabilizing N-terminal residue is not the sole essential determinant of an active N-degron, as demonstrated previously with engineered N-end rule substrates (12Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (317) Google Scholar).2) A major fraction (75–85%) of mouse RGS4 synthesized in reticulocyte lysate was rapidly degraded by a pathway that was proteasome-dependent and apparently also Ub-dependent. The other 15–25% of the newly made RGS4 was found to be stable in the lysate.3) The degradation of RGS4 was inhibited by dipeptides bearing type 1-destabilizing N-terminal residues (Arg-β-Ala or Lys-Ala) but was unaffected by dipeptides bearing a type 2-destabilizing N-terminal residue (Trp-Ala) or a type 3 residue (Ala-Lys and Ala-Arg).4) Radiochemical sequencing of RGS4 produced in reticulocyte lysate and labeled with either [3H]arginine, [3H]lysine, or [3H]leucine revealed the presence of posttranslationally conjugated Arg at the N terminus of RGS4. The deduced N-terminal sequence of RGS4 was Met-Cys-Lys-Gly-. The observed/inferred sequence was Arg-Cys-Lys-Gly-. This result and, independently, the fact that degradation of RGS4 was inhibited by type 1 but not by type 2 dipeptides strongly suggested the following model: the N-terminal Met of newly formed RGS4 is removed by MetAPs; the resulting N-terminal Cys is arginylated by R-transferase; and the arginylated RGS4 is targeted for processive degradation by the UBR1-encoded E3α and the rest of the N-end rule pathway.5) In agreement with a prediction of this model, the degradation of RGS4 in reticulocyte lysate was found to require Cys-2 residue, which becomes N-terminal after the MetAP-mediated cotranslational removal of Met. Specifically, the RGS4 mutants C2G, C2V, and C2A, in which Cys-2 was replaced with Gly, Val or Ala, were completely stable in the lysate. The former two residues are stabilizing in the N-end rule, and neither of the 3 residues is expected to interfere with the removal of N-terminal Met by MetAPs (64Walker K.W. Bradshaw R.A. J. Biol. Chem. 1999; 274: 13403-13409Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar).6) A Lys → Ser replacement at the encoded position 3 of RGS4 also completely stabilized the resulting RGS4K3S protein. However, a Lys → Arg replacement at this position had no effect on the degradation of RGS4K3R. Thus, Lys-3 is not required for ubiquitylation of RGS4. However, the presence of a basic residue (either Lys or Arg) immediately after Cys is essential for the RGS4 degradation by the N-end rule pathway. One possibility is that the requirement for a basic residue at this position reflects the substrate specificity of an uncharacterized R-transferase that arginylates the N-terminal Cys.7) The mouse protein Gβ5L, a member of the family of β subunits of heterotrimeric G proteins (66Watson A.J. Agaray A.M. Slepak V.Z. Simon M.I. J. Biol. Chem. 1996; 271: 28154-28160Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar) that bore the (initial) N-terminal Met-Cys, was tested and found not to be an N-end rule substrate, similarly to the RGS4K3S mutant, which also bore the (initial) N-terminal sequence Met-Cys but was not degraded by the N-end rule pathway. Thus, the N-terminal Cys of wild type RGS4 is an essential determinant of its N-degron, but other N terminus-proximal residues are relevant as well.8) Similarities among the N-terminal sequences of RGS4, RGS5, and RGS16 (67Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (127) Google Scholar) suggested that RGS5 and RGS16 may also be N-end rule substrates. This prediction was tested, thus far, with RGS16, and was confirmed.9) RGS4 was transiently expressed in mouse L cells and found to be an unstable protein, with the half-life of 40–50 min. The in vivo degradation of RGS4 was proteasome-dependent, as indicated by the nearly complete stabilization of RGS4 in the presence of proteasome inhibitor MG132. However, the targeting of RGS4 in thisin vivo setting involved primarily a degron distinct from the Cys-based N-degron, because N-terminal variants of RGS4 such as RGS4C2V and RGS4C2G, which were completely stable in reticulocyte lysate, remained unstable in L cells.We consider the latter result first. 15–25% of the reticulocyte lysate-produced RGS4 was resistant to degradation by the N-end rule pathway (paragraph 2 above). An analogous protection of RGS4 against targeting by the N-end rule pathway in L cells might involve a much higher fraction of the newly formed RGS4. The mechanism of protection may be a modification of the N-terminal Cys, for example, its palmitoylation (45Berman D.M. Gilman A.G. J. Biol. Chem. 1998; 273: 1269-1272Abstract Full Text Full Text PDF PubMed Scopus (444) Google Scholar, 47Tu Y. Popov S. Slaughter C. Ross E.M. J. Biol. Chem. 1999; 274: 38260-38267Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar, 62Druey K.M. Blumer K.J. Kang V.H. Kehrl J.H. Nature. 1996; 379: 742-746Crossref PubMed Scopus (404) Google Scholar, 67Srinivasa S.P. Bernstein L.S. Blumer K.J. Linder M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5584-5589Crossref PubMed Scopus (127) Google Scholar) or acetylation (68Cook R.K. Sheff D.R. Rubenstein P.A. J. Biol. Chem. 1991; 266: 16825-16833Abstract Full Text PDF PubMed Google Scholar) that would be expected to preclude the arginylation and degradation of RGS4 by the N-end rule pathway. Thus it is possible, indeed likely, that there is a kinetic competition among these reactions at the N-terminal Cys of a nascent RGS4 protein. In experiments to address this model, we added varying amounts of acetyl-CoA or palmitoyl-CoA (the substrates of N-terminal acetylases and palmitoyltransferases) to reticulocyte lysate; no effect of the added compounds on the degradation of newly formed RGS4 in the lysate was observed. 5I. Davydov and A. Varshavsky, unpublished data. A second possibility is a spatial localization of RGS4 in L cells that protects it from degradation by the N-end rule pathway but leaves RGS4 still targetable by another proteasome-dependent pathway(s) that is inactive in reticulocytes. Yet another possible reason for the difference between the results with reticulocyte lysateversus L cells is that the cysteine branch of the N-end rule pathway may be cell type-specific; for example, a Cys-specific R-transferase is present in reticulocytes but might be expressed at a lower level in L cells. In recent experiments, RGS4 was expressed in Xenopus laevis oocytes, through microinjection of RGS4 mRNA. It was found that, similarly to the results with reticulocyte extracts, RGS4 was degraded in oocytes by the cysteine branch of the N-end rule pathway. 6J. Sheng, I. Davydov, and A. Varshavsky,
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