Amino Acids Induce Peptide Uptake via Accelerated Degradation of CUP9, the Transcriptional Repressor of the PTR2 Peptide Transporter
2008; Elsevier BV; Volume: 283; Issue: 43 Linguagem: Inglês
10.1074/jbc.m803980200
ISSN1083-351X
AutoresZanxian Xia, Glenn Turner, Cheol‐Sang Hwang, Christopher Byrd, Alexander Varshavsky,
Tópico(s)Polyamine Metabolism and Applications
ResumoMultiple pathways link expression of PTR2, the transporter of di- and tripeptides in the yeast Saccharomyces cerevisiae, to the availability and quality of nitrogen sources. Previous work has shown that induction of PTR2 by extracellular amino acids requires, in particular, SSY1 and PTR3. SSY1 is structurally similar to amino acid transporters but functions as a sensor of amino acids. PTR3 acts downstream of SSY1. Expression of the PTR2 peptide transporter is induced not only by amino acids but also by dipeptides with destabilizing N-terminal residues. These dipeptides bind to UBR1, the ubiquitin ligase of the N-end rule pathway, and allosterically accelerate the UBR1-dependent degradation of CUP9, a transcriptional repressor of PTR2. UBR1 targets CUP9 through its internal degron. Here we demonstrate that the repression of PTR2 by CUP9 requires TUP1 and SSN6, the corepressor proteins that form a complex with CUP9. We also show that the induction of PTR2 by amino acids is mediated by the UBR1-dependent acceleration of CUP9 degradation that requires both SSY1 and PTR3. The acceleration of CUP9 degradation is shown to be attained without increasing the activity of the N-end rule pathway toward substrates with destabilizing N-terminal residues. We also found that GAP1, a general amino acid transporter, strongly contributes to the induction of PTR2 by Trp. Although several aspects of this complex circuit remain to be understood, our findings establish new functional links between the amino acids-sensing SPS system, the CUP9-TUP1-SSN6 repressor complex, the PTR2 peptide transporter, and the UBR1-dependent N-end rule pathway. Multiple pathways link expression of PTR2, the transporter of di- and tripeptides in the yeast Saccharomyces cerevisiae, to the availability and quality of nitrogen sources. Previous work has shown that induction of PTR2 by extracellular amino acids requires, in particular, SSY1 and PTR3. SSY1 is structurally similar to amino acid transporters but functions as a sensor of amino acids. PTR3 acts downstream of SSY1. Expression of the PTR2 peptide transporter is induced not only by amino acids but also by dipeptides with destabilizing N-terminal residues. These dipeptides bind to UBR1, the ubiquitin ligase of the N-end rule pathway, and allosterically accelerate the UBR1-dependent degradation of CUP9, a transcriptional repressor of PTR2. UBR1 targets CUP9 through its internal degron. Here we demonstrate that the repression of PTR2 by CUP9 requires TUP1 and SSN6, the corepressor proteins that form a complex with CUP9. We also show that the induction of PTR2 by amino acids is mediated by the UBR1-dependent acceleration of CUP9 degradation that requires both SSY1 and PTR3. The acceleration of CUP9 degradation is shown to be attained without increasing the activity of the N-end rule pathway toward substrates with destabilizing N-terminal residues. We also found that GAP1, a general amino acid transporter, strongly contributes to the induction of PTR2 by Trp. Although several aspects of this complex circuit remain to be understood, our findings establish new functional links between the amino acids-sensing SPS system, the CUP9-TUP1-SSN6 repressor complex, the PTR2 peptide transporter, and the UBR1-dependent N-end rule pathway. Biological processes addressed by this study include the mechanisms and regulation of peptide import. Peptides can serve as a source of amino acids and nitrogen in all organisms. The import of di- and tripeptides (di/tripeptides) in the yeast Saccharomyces cerevisiae has been shown to be regulated by the N-end rule pathway, one proteolytic pathway of the ubiquitin (Ub) 4The abbreviations used are: Ub, ubiquitin; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; βgal, β-galactosidase; UPR, Ub-protein-reference. -proteasome system (1Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google Scholar, 2Du 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, 3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar, 4Homann O.R. Cai H. Becker J.M. Lindquist S.L. PLoS Genet. 2005; 1: e80Crossref PubMed Scopus (56) Google Scholar). The N-end rule relates the in vivo half-life of a protein to the identity of its N-terminal residue (reviewed in Refs. 5Varshavsky A. Proc. 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In eukaryotes, this pathway recognizes several kinds of degradation signals (degrons), including a set called N-degrons (5Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 12142-12149Crossref PubMed Scopus (721) Google Scholar, 6Mogk A. Schmidt R. Bukau B. Trends Cell Biol. 2007; 17: 165-172Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar, 7Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Xia Z. Webster A. Du F. Piatkov K. Ghislain M. Varshavsky A. J. Biol. Chem. 2008; 283: 24011-24028Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). Specific N-degrons that are active (recognized) in a cell give rise to that cell's N-end rule. The determinants of an N-degron in a substrate protein are a destabilizing N-terminal residue that bears the unmodified N-terminal amino group, a substrate's internal Lys residue (the site of formation of a poly-Ub chain), and a nearby conformationally disordered region (11Bachmair A. Varshavsky A. Cell. 1989; 56: 1019-1032Abstract Full Text PDF PubMed Scopus (321) Google Scholar, 12Suzuki T. Varshavsky A. EMBO J. 1999; 18: 6017-6026Crossref PubMed Scopus (94) Google Scholar, 13Inobe T. Matouschek A. Curr. Opin. Struct. Biol. 2008; 18: 43-51Crossref PubMed Scopus (33) Google Scholar). The N-end rule has a hierarchic structure. In eukaryotes, N-terminal Asn and Gln are tertiary destabilizing residues in that they function through their enzymatic deamidation, to yield the secondary destabilizing N-terminal residues Asp and Glu (Ref. 7Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, and references therein). Destabilizing activity of N-terminal Asp and Glu requires their conjugation to Arg, one of the primary destabilizing residues, by the ATE1-encoded Arg-tRNA-protein transferase (arginyl-transferase or R-transferase) (14Kwon 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 (262) Google Scholar, 15Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 16Hu R.-G. Brower C.S. Wang H. Davydov I.V. Sheng J. Zhou J. Kwon Y.T. Varshavsky A. J. Biol. Chem. 2006; 281: 32559-32573Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 17Hu R.-G. Wang H. Xia Z. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar, 18Lee 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 (191) 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, which is arginylated after its oxidation to Cys-sulfinate or Cys-sulfonate (14Kwon 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 (262) Google Scholar). The in vivo oxidation of N-terminal Cys requires NO, as well as oxygen (O2) or its derivatives (15Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 18Lee 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 (191) Google Scholar). The N-end rule pathway is thus a sensor of NO, through the ability of this pathway to destroy proteins with N-terminal Cys, at rates controlled by NO, O2, and their derivatives. E3 Ub ligases of the N-end rule pathway are called N-recognins (7Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 10Xia Z. Webster A. Du F. Piatkov K. Ghislain M. Varshavsky A. J. Biol. Chem. 2008; 283: 24011-24028Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 19Kwon 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 (123) Google Scholar). They recognize (bind to) primary destabilizing N-terminal residues. (The term “Ub ligase” denotes either an E2–E3 holoenzyme or its E3 component.) At least four N-recognins, including UBR1, mediate the N-end rule pathway in mammals and other multicellular eukaryotes (7Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The known N-recognins share a ∼70-residue motif called the UBR box. Mouse UBR1 and UBR2 are sequelogous (similar in sequence) 200-kDa RING-type E3 Ub ligases that are 47% identical. Several other mammalian N-recognins, either confirmed or putative ones, are HECT-type or SCF-type Ub ligases that share the UBR motif with the RING-type UBR1 and UBR2 but are largely nonsequelogous to them otherwise (20Tasaki 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 (245) Google Scholar, 21Tasaki T. Sohr R. Xia Z. Hellweg R. Hörtnagl H. Varshavsky A. Kwon Y.T. J. Biol. Chem. 2007; 282: 18510-18520Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). 5A note on 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 (22Varshavsky A. Curr. 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The functions of the N-end rule pathway include: (i) the sensing of heme, owing to inhibition, in both yeast and mammals, of the ATE1 R-transferase by hemin (Fe3+-heme), which also inhibits N-recognins, the latter at least in yeast; (ii) the sensing of NO and O2, and the resulting control of signaling by transmembrane receptors, through the conditional, NO/O2-mediated degradation of G-protein regulators RGS4, RGS5, and RGS16; (iii) regulation of import of short peptides, through the degradation, modulated by peptides, of CUP9, the repressor of import; (iv) fidelity of chromosome segregation, through degradation of a separase-produced cohesin fragment; (v) regulation of apoptosis, through degradation of a caspase-processed inhibitor of apoptosis; (vi) a multitude of processes mediated by the transcription factor c-FOS, a conditional substrate of the N-end rule pathway; (vii) regulation of the human immunodeficiency virus replication cycle, through degradation of human immunodeficiency virus integrase; and (viii) regulation of meiosis, spermatogenesis, neurogenesis, and cardiovascular development in mammals, and leaf senescence in plants (Refs. 2Du 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, 3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar, 4Homann O.R. Cai H. Becker J.M. Lindquist S.L. PLoS Genet. 2005; 1: e80Crossref PubMed Scopus (56) Google Scholar, 7Tasaki T. Kwon Y.T. Trends Biochem. Sci. 2007; 32: 520-528Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar, 14Kwon 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 (262) Google Scholar, 15Hu R.-G. Sheng J. Xin Q. Xu Z. Takahashi T.T. Varshavsky A. Nature. 2005; 437: 981-986Crossref PubMed Scopus (239) Google Scholar, 17Hu R.-G. Wang H. Xia Z. Varshavsky A. Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 76-81Crossref PubMed Scopus (93) Google Scholar, 18Lee M.J. Tasaki T. Moroi K. An J.Y. Kimura S. Davydov I.V. Kwon Y.T. Proc. Natl. 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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 (193) Google Scholar). The N-end rule pathway of S. cerevisiae is mediated by a single N-recognin, UBR1, a 225-kDa sequelog of mammalian UBR1 and UBR2 (2Du 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, 10Xia Z. Webster A. Du F. Piatkov K. Ghislain M. Varshavsky A. J. Biol. Chem. 2008; 283: 24011-24028Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar, 27Xie Y. Varshavsky A. EMBO J. 1999; 18: 6832-6844Crossref PubMed Scopus (143) Google Scholar). S. cerevisiae UBR1 contains at least three substrate-binding sites. The type-1 site is specific for basic N-terminal residues of polypeptides (Arg, Lys, and His). The type-2 site is specific for bulky hydrophobic N-terminal residues (Trp, Phe, Tyr, Leu, and Ile). The third binding site of UBR1 recognizes an internal (non-N-terminal) degron in target proteins. The third binding site of UBR1 is autoinhibited but can be allosterically activated through a conformational change that is caused by the binding of short peptides, such as dipeptides, to the other, type-1 and type-2, binding sites of UBR1. The known substrate of the third binding site of UBR1 is CUP9 (1Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google Scholar, 2Du 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, 3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar), a homeodomain protein and transcriptional regulator, largely a repressor, of more than 30 genes in S. cerevisiae. 6C. S. Hwang and A. Varshavsky, unpublished data. The regulon of CUP9 includes PTR2, a gene encoding the main transporter of di/tripeptides (28Cai H. Hauser M. Naider F. Becker J.M. Eukaryot. Cell. 2007; 6: 1805-1813Crossref PubMed Scopus (36) Google Scholar, 29Cai H. Kauffman S. Naider F. Becker J.M. Genetics. 2006; 172: 1459-1476Crossref PubMed Scopus (35) Google Scholar). The reversal of UBR1 autoinhibition by imported di/tripeptides with destabilizing N-terminal residues accelerates the UBR1-dependent ubiquitylation of CUP9, leads to its faster degradation, and thereby causes a derepression of PTR2. The resulting positive-feedback circuit allows S. cerevisiae to detect the presence of extracellular peptides and to react by increasing their uptake (2Du 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, 3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar). The evolution of peptide-import circuits is under conflicting selective pressures, because the ability of a cell to import peptides confers both a benefit (utilization of peptides as food) and a vulnerability to toxins that resemble short peptides. Although PTR2 is the major transporter of di/tripeptides in S. cerevisiae, some di/tripeptides can also be imported (with a low efficacy) by DAL5, whose major function is the import of other nitrogen sources, such as allantoate and ureidosuccinate (4Homann O.R. Cai H. Becker J.M. Lindquist S.L. PLoS Genet. 2005; 1: e80Crossref PubMed Scopus (56) Google Scholar). Another set of S. cerevisiae peptide transporters comprises OPT1 (HGT1) and OPT2, which have partially overlapping functions, do not import di/tripeptides, but can import peptides of 4–5 residues. In addition, OPT1 (HGT1) is a high affinity importer of glutathione, a “noncanonical” tripeptide (Ref 30Wiles A.M. Cai H. Naider F. Becker J.M. Microbiology. 2006; 152: 3133-3145Crossref PubMed Scopus (32) Google Scholar, and references therein). Similarly to the PTR2 transporter of di/tripeptides, the expression of OTP2 is down-regulated by CUP9, whereas the expression of OPT1 (HGT1) is independent of CUP9 (30Wiles A.M. Cai H. Naider F. Becker J.M. Microbiology. 2006; 152: 3133-3145Crossref PubMed Scopus (32) Google Scholar). In addition to PTR2 and OPT2, the N-end rule pathway also controls the expression of DAL5, but in a manner opposite to that of the other two transporters: whereas CUP9 is a transcriptional repressor of PTR2 and OPT2, CUP9 apparently up-regulates the expression of DAL5 (28Cai H. Hauser M. Naider F. Becker J.M. Eukaryot. Cell. 2007; 6: 1805-1813Crossref PubMed Scopus (36) Google Scholar, 30Wiles A.M. Cai H. Naider F. Becker J.M. Microbiology. 2006; 152: 3133-3145Crossref PubMed Scopus (32) Google Scholar). It is unknown whether CUP9 down-regulates a repressor of DAL5 or whether CUP9 acts, in the context of DAL5, as a transcriptional activator. The induction of the PTR2 peptide transporter by di/tripeptides, a process controlled by the UBR1-CUP9 circuit (3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar), is just one of regulatory inputs that couple PTR2 expression to the availability and quality of nutrients. For example, PTR2 expression is down-regulated by certain nitrogen sources, including ammonia, but not by other nitrogen sources, such as urea and allantoin (31Godard P. Urrestarazu A. Vissers S. Kontos K. Bontempi G. van Helden J. André B. Mol. Cell. Biol. 2007; 27: 3065-3086Crossref PubMed Scopus (184) Google Scholar). The underlying systems, including the N-end rule pathway, ensure that a cell does not waste resources synthesizing large amounts of the PTR2 transporter in the absence of extracellular peptides, or when a more efficacious nitrogen source, such as ammonia, is present. PTR2 is also induced by extracellular amino acids, particularly leucine or tryptophan (32Island M.D. Naider F. Becker J.M. J. Bacteriol. 1987; 169: 2132-2136Crossref PubMed Google Scholar). 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Genet. 2004; 9: 121-153Crossref Google Scholar, 41Klasson H. Fink G.R. Ljungdahl P.O. Mol. Cell. Biol. 1999; 19: 5405-5416Crossref PubMed Scopus (154) Google Scholar), a disposition that recurs with other nutrient sensors as well (37Boles E. André B. Top. Curr. Genet. 2004; 9: 121-153Crossref Google Scholar, 42Holsbeeks I. Lagatie O. Nuland A.V. Van de Velde S. Thevelein J.M. Trends Biochem. Sci. 2004; 29: 556-564Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). Both the inferred design of SSY1 and experimental evidence suggest that it is the concentration ratio of an amino acid across the plasma membrane, rather than the absolute concentration of extracellular amino acid that determines the signaling output by SSY1 (36Wu B. Ottow K. Poulsen P. Gaber R.F. Albers E. Kielland-Brandt M.C. J. Cell Biol. 2006; 173: 327-331Crossref PubMed Scopus (58) Google Scholar). Activated SSY1 induces expression of a regulon that includes the PTR2 peptide transporter and amino acid transporters such as AGP1, BAP2, BAP3, TAT1, TAT2, and GNP1 (38Fosberg H. Ljungdahl P.O. Curr. Genet. 2001; 40: 91-109Crossref PubMed Scopus (162) Google Scholar). PTR3 and SSY5 are peripheral membrane proteins associated with SSY1 (41Klasson H. Fink G.R. Ljungdahl P.O. Mol. Cell. Biol. 1999; 19: 5405-5416Crossref PubMed Scopus (154) Google Scholar). SSY5 is a protease regulated, in particular, by PTR3. SSY5 can cleave, and thereby activate, the latent (conditionally cytosolic) transcriptional activators STP1 and STP2, leading to their import into the nucleus and the induction of genes that encode, in particular, amino acid transporters (34Andréasson C. Heessen S. Ljungdahl P.O. Genes Dev. 2006; 20: 1563-1568Crossref PubMed Scopus (52) Google Scholar, 35Abdel-Sater F. Bakkoury E.I. Urrestarazu A. Vissers S. Andre B. Mol. Cell. Biol. 2004; 24: 9771-9785Crossref PubMed Scopus (65) Google Scholar, 36Wu B. Ottow K. Poulsen P. Gaber R.F. Albers E. Kielland-Brandt M.C. J. Cell Biol. 2006; 173: 327-331Crossref PubMed Scopus (58) Google Scholar, 37Boles E. André B. Top. Curr. Genet. 2004; 9: 121-153Crossref Google Scholar, 39Boban M. Ljungdahl P.O. Genetics. 2007; 176: 2087-2097Crossref PubMed Scopus (28) Google Scholar, 40Liu Z. Thornton J. Spirek M. Butow R.A. Mol. Cell. Biol. 2008; 28: 551-563Crossref PubMed Scopus (51) Google Scholar, 43Andréasson C. Ljungdahl P.O. Genes Dev. 2002; 16: 3158-3172Crossref PubMed Scopus (97) Google Scholar). In the present work, we show that an extracellular amino acid such as Trp acts via the SPS system to induce the PTR2-mediated import of di/tripeptides through the acceleration of degradation of CUP9 (the repressor of import) by the UBR1-dependent N-end rule pathway. The bulk of this effect of Trp on the rate of CUP9 degradation requires both SSY1 and PTR3. At the same time, no significant activation of the N-end rule pathway toward substrates with destabilizing N-terminal residues (i.e. toward substrates with N-degrons) was observed under these conditions, suggesting a differential regulation of three substrate-binding sites of the UBR1 Ub ligase. We also show that the repression of PTR2 by CUP9 requires the global corepressors TUP1 and SSN6, and that GAP1, a general amino acid transporter, strongly contributes to the induction of PTR2 by Trp. Although several aspects of this complex circuit remain to be understood, our findings establish new functional links between the amino acids-sensing SPS system, the CUP9-TUP1-SSN6 repressor complex, the PTR2 peptide transporter, and the UBR1-dependent N-end rule pathway. Yeast Strains, Plasmids, and Genetic Techniques—The S. cerevisiae strains used in this study are described in Table 1. AVY24 and AVY25 were constructed in the background of strain RJD347 (MATα ura3–52; a gift from Dr. R. 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Chem. 1995; 270: 17442-17456Abstract Full Text Full Text PDF PubMed Scopus (680) Google Scholar)AVY24MATα ura3-52 ssy1Δ::myc3-URA3-myc3This studyAVY25MATα ura3-52 ptr3Δ::myc3-URA3-myc3This studyAVY26MATα ura3-52 ubr1Δ::HisGThis studyAVY27MATα ura3-52 ssy1Δ::myc3This studyAVY28MATα ura3-52ptr3Δ::myc3This studyAVY30MATα leu2-3,112 ubr1Δ::LEU2(3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar)AVY31MATα leu2-3,112 cup9Δ::LEU2(3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar)AVY32MATα LEU2(3Turner G.C. Du F. Varshavsky A. Nature. 2000; 405: 579-583Crossref PubMed Scopus (165) Google Scholar)AVY50MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 cup9Δ::LEU2CBY19 in Ref. 1Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google ScholarAVY51MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 ubr1Δ::HIS3 cup9Δ::LEU2CBY17 in Ref. 1Byrd C. Turner G.C. Varshavsky A. EMBO J. 1998; 17: 269-277Crossref PubMed Scopus (104) Google ScholarAVY60MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 ssn6Δ::HisGThis studyAVY61MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 tup1Δ::HisGThis studyAVY62MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 ubr1Δ::HIS3 ssn6Δ::HisGThis studyAVY63MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 ubr1Δ::HIS3 tup1Δ::HisGThis studyAVY64MATa ura3-52 his3-Δ200 leu2-3,112 trp1-Δ63 lys2-801 cup9Δ::LEU2 ssn6Δ:::HisGThis studyAVY65MATa ura3-5
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