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• W2041774555 abstract "Gln3p is a GATA-type transcription activator of nitrogen catabolite repressible (NCR) genes. Gln3p was recently found to be hyperphosphorylated in a TOR-dependent manner and resides in the cytoplasm in high quality nitrogen. In contrast, during nitrogen starvation or rapamycin treatment, Gln3p becomes rapidly dephosphorylated and accumulates in the nucleus, thereby activating nitrogen catabolite repression genes. However, a detailed mechanistic understanding is lacking for the regulation of Gln3p nucleocytoplasmic distribution. In this study, we applied a functional genomics approach to identify the nuclear transport factors for Gln3p. We found that yeast karyopherin α/Srp1p and Crm1p are required for the nuclear import and export of Gln3p, respectively. Similarly, the Ran GTPase pathway is also involved in the nuclear translocation of Gln3p. Finally, we show that Srp1p preferentially interacts with the hypophosphorylated versusthe hyperphosphorylated Gln3p. These findings define a possible mechanism for regulated nucleocytoplasmic transport of Gln3p by phosphorylation in vivo. Gln3p is a GATA-type transcription activator of nitrogen catabolite repressible (NCR) genes. Gln3p was recently found to be hyperphosphorylated in a TOR-dependent manner and resides in the cytoplasm in high quality nitrogen. In contrast, during nitrogen starvation or rapamycin treatment, Gln3p becomes rapidly dephosphorylated and accumulates in the nucleus, thereby activating nitrogen catabolite repression genes. However, a detailed mechanistic understanding is lacking for the regulation of Gln3p nucleocytoplasmic distribution. In this study, we applied a functional genomics approach to identify the nuclear transport factors for Gln3p. We found that yeast karyopherin α/Srp1p and Crm1p are required for the nuclear import and export of Gln3p, respectively. Similarly, the Ran GTPase pathway is also involved in the nuclear translocation of Gln3p. Finally, we show that Srp1p preferentially interacts with the hypophosphorylated versusthe hyperphosphorylated Gln3p. These findings define a possible mechanism for regulated nucleocytoplasmic transport of Gln3p by phosphorylation in vivo. nitrogen catabolite repression nuclear localization signal glutathione S-transferase immunofluorescence monoclonal antibody calf intestine alkaline phosphatase human immunodeficiency virus, type 1 4′,6-diamidino-2-phenylindole, dialactate In response to nitrogen levels and sources, eukaryotic cells selectively regulate expression of genes involved in utilization and transport of the available nutrients. In the presence of rich nitrogen sources, the expression of genes necessary for utilization and transport of poor nitrogen sources is repressed. These genes become derepressed in a poor nitrogen source such as glutamate. This phenomenon is called nitrogen catabolite repression (NCR)1 (reviewed in Ref. 1Magasanik B. The Molecular and Cellular Biology of Yeast Saccharomyces. 2. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1992: 283-318Google Scholar). Genetic analysis in budding yeast led to the identification of regulatory pathways that detect the quality of nutrients and regulate gene expression. A number of GATA-type transcription factors have now been identified, including Gln3p, Nil1p/Gat1p, and Nil2p/Gzf3p (2Courchesne W.E. Magasanik B. J. Bacteriol. 1988; 170: 708-713Crossref PubMed Scopus (129) Google Scholar, 3Coffman J.A. Rai R. Cunningham T. Svetlov V. Cooper T.G. Mol. Cell. Biol. 1996; 16: 847-858Crossref PubMed Scopus (121) Google Scholar, 4Stanbrough M. Rowen D.W. Magasanik B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 9450-9454Crossref PubMed Scopus (133) Google Scholar). In particular, Gln3p has been the primary focus for most subsequent studies. It is a transcriptional activator of NCR genes when only poor nitrogen sources are available or during nitrogen starvation. Ure2p is a yeast pre-prion protein genetically antagonistic to Gln3p (5Coschigano P.W. Magasanik B. Mol. Cell. Biol. 1991; 11: 822-832Crossref PubMed Scopus (222) Google Scholar,6Wickner R.B. Science. 1994; 264: 566-569Crossref PubMed Scopus (1086) Google Scholar). Recent studies have shed light into the regulatory events leading to activation of Gln3p. TOR is the yeast target of rapamycin (7Heitman J. Movva N.R. Hall M.N. Science. 1991; 253: 905-909Crossref PubMed Scopus (1550) Google Scholar, 8Kunz J. Henriquez R. Schneider U. Deuter-Reinhard M. Movva N.R. Hall M.N. Cell. 1993; 73: 585-596Abstract Full Text PDF PubMed Scopus (728) Google Scholar, 9Cafferkey R. McLaughlin M.M. Young P.R. Johnson R.K. Livi G.P. Gene (Amst.). 1994; 141: 133-136Crossref PubMed Scopus (41) Google Scholar, 10Stan R. McLaughlin M.M. Cafferkey R. Johnson R.K. Rosenberg M. Livi G.P. J. Biol. Chem. 1994; 269: 32027-32030Abstract Full Text PDF PubMed Google Scholar, 11Zheng X.F. Florentino D. Chen J. Crabtree G.R. Schreiber S.L. Cell. 1995; 82: 121-130Abstract Full Text PDF PubMed Scopus (246) Google Scholar, 12Lorenz M.C. Heitman J. J. Biol. Chem. 1995; 270: 27531-27537Abstract Full Text Full Text PDF PubMed Scopus (203) Google Scholar) and a key player of nutrient-mediated signal transduction (recently reviewed in Refs. 13Dennis P.B. Fumagalli S. Thomas G. Curr. Opin. Genet. Dev. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 14Kuruvilla F. Schreiber S.L. Chem. Biol. 1999; 6: R129-R136Abstract Full Text PDF PubMed Scopus (61) Google Scholar, 15Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1729) Google Scholar, 16Rohde J. Heitman J. Cardenas M. J. Biol. Chem. 2001; 276: 7027-7036Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar). Major NCR transcription factors, including Gln3p, Nil1p/Gat1p, and Nil2p/Gzf3p interact with both Tor1p and Tor2p (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Nitrogen starvation or inhibition of TOR by rapamycin causes rapid dephosphorylation and nuclear accumulation of Gln3p in vivo(17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (799) Google Scholar) and expression of a wide variety of NCR genes (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 19Cardenas M. Cutler N. Lorenz M. Di Como C. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (479) Google Scholar, 20Hardwick J.S. Kuruvilla F. Tong J.K. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (466) Google Scholar, 21Shamji A. Kuruvilla F. Schreiber S. Curr. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). TOR is a protein serine/threonine kinase (22Bertram P.G. Zeng C. Thorson J. Shaw A.S. Zheng X.F. Curr. Biol. 1998; 8: 1259-1267Abstract Full Text Full Text PDF PubMed Google Scholar, 23Jiang Y. Broach J.R. EMBO J. 1999; 18: 2782-2792Crossref PubMed Scopus (275) Google Scholar, 24Alarcon C.M. Heitman J. Cardenas M.E. Mol. Biol. Cell. 1999; 10: 2531-2546Crossref PubMed Scopus (65) Google Scholar) and appears to be responsible for Gln3p phosphorylation (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) and may also regulate Gln3p dephosphorylation (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (799) Google Scholar). In addition to the classic NCR transcription factors, TOR also mediates nitrogen signaling to regulate Rtg1/3p, transcription factors involved in regulation of several genes of the trichloroacetic acid cycle (25Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar). Although hyperphosphorylation of Gln3p correlates well with its cytoplasmic retention and dephosphorylation is consistent with its nuclear accumulation, it remains to be determined whether and how phosphorylation directly influences the nuclear accumulation of Gln3p. Recent progress has defined the basic mechanism of nuclear transport (26Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 27Nakielny S. Dreyfuss G. Cell. 1999; 99: 677-690Abstract Full Text Full Text PDF PubMed Scopus (651) Google Scholar, 28Wente S.R. Science. 2000; 288: 1374-1377Crossref PubMed Scopus (216) Google Scholar). Nuclear import and export factors interact with distinct targeting signals and share common functional and structural domains. These are collectively called karyopherins (also importins/exportins, transportins). Analysis of the Saccharomyces data base revealed a total of 14 proteins belonging to the karyopherin β family, defined partly by a ∼150-amino acid region required for binding to the small GTPase Ran (29Gorlich D. Curr. Opin. Cell Biol. 1997; 9: 412-419Crossref PubMed Scopus (257) Google Scholar, 30Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (212) Google Scholar). Some of these karyopherin family members have been shown to serve as receptors for the import and/or export of diverse cargo molecules such as proteins and tRNA (reviewed in Ref. 31Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar). Each karyopherin is likely specific for a distinct targeting signal. The classical nuclear targeting signal is typified by the SV40 large T antigen nuclear localization signal (NLS) that is rich in basic amino acids (32Kalderon D. Roberts B.L. Richardson W.D. Smith A.E. Cell. 1984; 39: 499-509Abstract Full Text PDF PubMed Scopus (1866) Google Scholar, 33Lanford R.E. Butel J.S. Virology. 1985; 147: 72-80Crossref PubMed Scopus (13) Google Scholar). The import factor recognizing this NLS is a heterodimer of karyopherin α and karyopherin β (also termed importins) (extensively reviewed in Refs.26Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar, 31Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar, and 34Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1009) Google Scholar). In the cytoplasm, karyopherin α serves as an adaptor to directly bind the NLS, whereas karyopherin β binds to karyopherin α. The complex then docks at the nuclear pore, translocates across the pore, and is disassembled in the nucleus. In contrast to the NLS, a major nuclear export signal is characterized by a small leucine-rich sequence that is bound by the export factor Crm1/Xpo1 (also called exportin) and Ran-GTP in the nucleus (35Fornerod M. Ohno M. Yoshida M. Mattaj I.W. Cell. 1997; 90: 1051-1060Abstract Full Text Full Text PDF PubMed Scopus (1741) Google Scholar, 36Fukuda M. Asano S. Nakamura T. Adachi M. Yoshida M. Yanagida M. Nishida E. Nature. 1997; 390: 308-311Crossref PubMed Scopus (1028) Google Scholar, 37Kudo N. Khochbin S. Nishi K. Kitano K. Yanagida M. Yoshida M. Horinouchi S. J. Biol. Chem. 1997; 272: 29742-29751Crossref PubMed Scopus (181) Google Scholar, 38Neville M. Stutz F. Lee L. Davis L.I. Rosbash M. Curr. Biol. 1997; 7: 767-775Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 39Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (623) Google Scholar, 40Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar). This complex translocates across the nuclear pore and is disassembled in the cytoplasm. The small GTPase Ran is thought to impose directionality to transport processes because its regulators are specifically compartmentalized within the cell (reviewed in Refs. 26Corbett A.H. Silver P.A. Microbiol. Mol. Biol. Rev. 1997; 61: 193-211Crossref PubMed Scopus (169) Google Scholar and 34Mattaj I.W. Englmeier L. Annu. Rev. Biochem. 1998; 67: 265-306Crossref PubMed Scopus (1009) Google Scholar). Ran-GTP concentration is presumably high in the nucleus, whereas Ran-GDP predominates in the cytoplasm. Ran-GTP binds to the karyopherin β family members to facilitate release of import factors from their cargo in the nucleus (41Gorlich D. Pante N. Kutay U. Aebi U. Bischoff F.R. EMBO J. 1996; 15: 5584-5594Crossref PubMed Scopus (533) Google Scholar, 42Chi N.C. Adam E.J. Visser G.D. Adam S.A. J. Cell Biol. 1996; 135: 559-569Crossref PubMed Scopus (158) Google Scholar, 43Rexach M. Blobel G. Cell. 1995; 83: 683-692Abstract Full Text PDF PubMed Scopus (665) Google Scholar) or to mediate the formation of export complexes consisting of the export receptor, cargo, and Ran-GTP (recently reviewed in Ref. 31Gorlich D. Kutay U. Annu. Rev. Cell Dev. Biol. 1999; 15: 607-660Crossref PubMed Scopus (1674) Google Scholar). Cells can respond to extracellular signals by modulating gene expression, which requires the transfer of information from the plasma membrane to the nucleus. Protein kinase cascades are commonly involved in transducing these signals, and they typically culminate in the phosphorylation of transcription factors. Phosphorylation of both transcription factors and kinases results in regulation of their nuclear localization, suggesting that control of the subcellular localization of these proteins is important for the response to extracellular signals (reviewed in Refs. 44Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar and 45Komeili A. O'Shea E.K. Curr. Opin. Cell Biol. 2000; 12: 355-360Crossref PubMed Scopus (87) Google Scholar). Phosphorylation can directly regulate the recognition of targeting signals by soluble karyopherins and effectively modulates nucleocytoplasmic translocation (reviewed in Refs. 44Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar and 45Komeili A. O'Shea E.K. Curr. Opin. Cell Biol. 2000; 12: 355-360Crossref PubMed Scopus (87) Google Scholar). For example, phosphorylation of the phosphate-regulated transcription factor Pho4p at Ser-152 disrupts its association with the karyopherin β Pse1p and inhibits its nuclear import (46Kaffman A. Rank N.M. O'Shea E.K. Genes Dev. 1998; 12: 2673-2683Crossref PubMed Scopus (204) Google Scholar, 47Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (249) Google Scholar). In this study, we found that at least two nuclear transport factors, karyopherin α/Srp1p and Crm1p/Xpo1p, are required for the import and export of Gln3p, respectively. Mutation in Srp1p blocked the nuclear import of Gln3p and the expression of the Gln3p-dependent gene GAP1 by rapamycin treatment or nitrogen starvation. In contrast, shifting to the nonpermissive growth temperature in thecrm1-1ts mutant was sufficient to cause nuclear accumulation of Gln3p and expression of GAP1 in high quality nitrogen and in the absence of rapamycin. Furthermore, bacterially produced karyopherin α/Srp1p binds preferentially to the hypophosphorylated form of Gln3p. Our results indicate that Gln3p is dynamically shuttling between the cytoplasm and the nucleus. TOR-dependent phosphorylation appears to inhibit the ability of Gln3p to enter into the nucleus by reducing its affinity for the karyopherin α/Srp1p. All of the yeast strains are shown in Table I. The deletion and temperature-sensitive mutants for karyopherins have been described previously (40Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, 48Belanger K.D. Kenna M.A. Wei S. Davis L.I. J. Cell Biol. 1994; 126: 619-630Crossref PubMed Scopus (107) Google Scholar, 49Traglia H.M. 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The concentration of rapamycin used throughout this study is 200 nm.Table IYeast strains used in this studyYeast strainsGenotypeSourceBY4741Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0Res. Genetics1-aResearch Genetics, Huntsville, ALSZy158Matα ade2∷hisG his3Δ200 leu2Δ0 lys2Δ0 met15Δ0 trp1Δ63 ura3Δ0 GLN3-MYC9 -TRP1Ref. 17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google ScholarSZy432Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δlph2Res. GeneticsSZy481Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δsxm1Res. GeneticsSZy568Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δkap114Res. GeneticsSZy569Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δpdr1Res. GeneticsSZy570Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δlos1Res. GeneticsSZy571Mata hisΔ1 leu2Δ0 met15Δ0 ura3Δ0 Δmsn5Res. GeneticsSZy429Matα ura3–52 kap104∷ura3∷HIS3Ref. 50Seedorf M. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google ScholarEE1bMataura3–52 tyr1 his4 his7 rna1–1tsRef. 49Traglia H.M. Atkinson N.S. Hopper A.K. Mol. Cell. Biol. 1989; 9: 2989-2999Crossref PubMed Google Scholarxpo1–1Matα ade2–1 ura3–1 his3–11,15 trp1–1 leu2–3,112 xpol∷LEU2(xpo1–1 on HIS3)Ref.40Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (937) Google ScholarY1705Matα ura3–52 ade2–101 his3–11,15 trp1Δ901 cse1–1csRef. 53Xiao Z. McGrew J.T. Schroeder A.J. Fitzgerald-Hayes M. Mol. Cell. Biol. 1993; 13: 4691-4702Crossref PubMed Scopus (112) Google ScholarT255aMata ura3–52 lys2–801 mtr10Ref. 54Kadowaki T. Chen S. Hitomi M. Jacobs E. Kumagai C. Liang S. Schneiter R. Singleton D. Wisniewska J. Tartakoff A.M. J. Cell Biol. 1994; 126: 649-659Crossref PubMed Scopus (144) Google ScholarKBY44Mata trp1 lys2 ura3 ade2 ade3 nup1∷LEU2 srp1cs(Nup1 on URA3/ADE3)Ref. 48Belanger K.D. Kenna M.A. Wei S. Davis L.I. J. Cell Biol. 1994; 126: 619-630Crossref PubMed Scopus (107) Google ScholarPSY1199Matα ade2Δ∷hisG ade8Δ100∷KANR ura3Δ leu2Δ1 his3Δ200 nmd5Δ∷HIS3Ref. 51Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (348) Google ScholarPSY967Mata ura3–52 leu2Δ1 his3Δ200 kap123Δ∷HIS3Ref. 50Seedorf M. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google ScholarPSY1201Mata ura3–52 leu2Δ1 trp1Δ63 pse1–1csRef. 50Seedorf M. Silver P.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8590-8595Crossref PubMed Scopus (116) Google Scholar1-a Research Genetics, Huntsville, AL Open table in a new tab For the plasmid expressing GST-Srp1 (pSW347), oligonucleotides were used in the polymerase chain reaction to generate the SRP1 open reading frame flanked by BamHI restriction sites. The fragment was inserted into BamHI-digested pGEX-3x, such that the coding sequence for GST was in frame with the second codon of SRP1. To generate pRS315-GLN3-MYC9 and pRS416-GLN3-MYC9, the chromosomal gene GLN3-MYC9 and its natural promoter were generated by polymerase chain reaction from genomic DNA prepared from the GLN3-MYC9 strain (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) and cloned into pRS315 (LEU CEN) or pRS416 (URA CEN) using theSalI and NotI restriction sites. The resultant plasmids were transformed into yeast and shown to express Gln3p-MYC9 at a level comparable with that of the chromosomal GLN3-MYC9. pRS315-GLN3-MYC9 and pRS416-GLN3-MYC9 were transformed into wild type and mutant strains and used for both phosphorylation and indirect immunofluorescence (IF) studies. Exponential wild type and mutant yeast cultures were treated with 200 nm rapamycin. Aliquots of yeast cultures were withdrawn at different times. Total yeast RNAs were prepared using the phenol freezing extraction method (55Schmitt M.E. Brown T.A. Trumpower B.L. Nucleic Acids Res. 1990; 18: 3091-3092Crossref PubMed Scopus (1152) Google Scholar). 20-μg total yeast RNA samples were separated on denaturing agarose gels, transferred onto nylon filters, hybridized to 32P-labeled DNA probes, and detected by phosphorus imaging. For the temperature- and cold-sensitive mutants, yeast were first grown to the early log phase, shifted to nonpermissive temperatures for 2–3 h, and then treated with 200 nm rapamycin. Log phase yeast cells were harvested and lysed with glass beads in disruption buffer (50 mm Tris-HCl, pH 7.5, 150 mm NaCl, 1 mm EDTA, 1% Nonidet P-40 plus a mixture of protease inhibitors; Roche Molecular Biochemicals) by vortexing. 1 mg of total protein was used for immunoprecipitation with 5 μg of monoclonal antibody (mAb) 9E10 for MYC. For Western blotting analysis, 20-μg protein samples were used for gel electrophoresis and detected by ECL (Amersham Pharmacia Biotech) with mAb 9E10. For the phosphatase treatment, cell extracts containing Gln3p-MYC9were incubated with CIP buffer alone, 20 units of CIP (Roche Molecular Biochemicals), or 20 units CIP plus phosphatase inhibitor 10 mm Na4P2O7 for 10 min at 30 °C. The wild type and mutant yeast strains expressing the Gln3p-MYC9 fusions were used for indirect IF studies. For the cold- and temperature-sensitive mutants, the yeast cells were grown to mid-log phase at their permissive temperatures (30 °C for srp1 cs; 23 °C for all other mutants) and then shifted to the nonpermissive temperatures for 2–3 h (20 °C for srp1 cs, 35 °C for crm1-1 ts, and 37 °C for all other temperature-sensitive mutants). 200 nm rapamycin was then added for the duration indicated in each experiment. The IF studies were carried out as described previously (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar) using anti-MYC monoclonal antibody 9E10, and incubation was continued for an additional time as indicated for each experiment. Bacteria were grown at 37 °C in 250 ml of liquid broth medium containing 100 μg/ml ampicillin to an optical density of 0.4 and were induced with 1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h. The culture was chilled on ice for 10 min, and the cells were pelleted and resuspended in 15 ml of lysis buffer (1× phosphate-buffered saline, 1 mm phenylmethylsulfonyl fluoride, 1 mm EDTA, 1× protease inhibitor mixture, and 5 mm β-mercaptoethanol). The suspension was subjected to two freeze-thaw cycles, and the cells were finally disrupted by sonication. The cell lysate was clarified by centrifugation for 20 min at 20,000 × g at 4 °C. The recombinant proteins were purified by glutathione-Sepharose. The in vitro binding assays for the recombinant proteins were carried out as described previously (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Rapamycin treatment or nitrogen starvation leads to nuclear accumulation of Gln3p and activation of Gln3p-dependent genes such as GAP1 (general aminoacid permease 1) (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (799) Google Scholar). We reasoned that mutations in the Gln3p import factor would prevent Gln3p from accumulating in the nucleus in the presence of rapamycin. Under such conditions, there would be no expression of GAP1. In contrast, dysfunction of the export pathway would cause constitutive nuclear accumulation of Gln3p and a high level of GAP1expression in the absence of rapamycin. Therefore, GAP1 can be conveniently used as a reporter to monitor the import/export of Gln3p by examining the effect of their mutations on GAP1expression (Fig. 1a). There are a total of 14 karyopherin βs and one karyopherin α in theSaccharomyces cerevisiae genome as revealed by genomic sequence analysis (30Pemberton L.F. Blobel G. Rosenblum J.S. Curr. Opin. Cell Biol. 1998; 10: 392-399Crossref PubMed Scopus (212) Google Scholar, 56Gorlich D. Dabrowski M. Bischoff F.R. Kutay U. Bork P. Hartmann E. Prehn S. Izaurralde E. J. Cell Biol. 1997; 138: 65-80Crossref PubMed Scopus (350) Google Scholar). Therefore, we devised a functional genomics approach to identify the factors that affect Gln3p nuclear transport by monitoring the expression of GAP1 in individual karyopherin mutants in the presence or the absence of rapamycin. We have screened all the yeast karyopherin mutants using Northern blot analysis for GAP1. As expected, GAP1 expression patterns in a majority of the karyopherin mutants were similar to the wild type strain; GAP1 expression was essentially undetectable in the absence of rapamycin but increased significantly in the presence of rapamycin (Fig. 1b). However,GAP1 expression remained very low or undetectable both in the presence and in the absence of rapamycin in two mutant strains,srp1 cs and cse1-1 cs (Fig.1b). Both srp1 cs andcse1-1 cs are cold-sensitive mutants. The induction of GAP1 by rapamycin was normal at the permissive temperature (30 °C) (data not shown) but not at the nonpermissive temperature (20 °C) (Fig. 1b). Cse1p is a karyopherin required for the export of Srp1p from the nucleus to the cytoplasm (57Kutay U. Bischoff F.R. Kostka S. Kraft R. Gorlich D. Cell. 1997; 90: 1061-1071Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar,58Hood J.K. Silver P.A. J. Biol. Chem. 1998; 273: 35142-35146Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar). The cse1-1 cs mutation may affectGAP1 expression indirectly by preventing the return of Srp1p to the cytoplasm. Kap95p is a co-factor for karyopherin-α/Srp1p. Therefore, a loss-of-function mutation in KAP95 was also expected to inhibit GAP1 expression in the presence of rapamycin. Unfortunately, the two kap95 mutants we analyzed grew extremely poorly, which precluded us from obtaining good quality RNA from these strains to address this point. Taken together, thesrp1 and cse1 phenotypes suggest that Srp1p mediates Gln3p import. In contrast, crm1-1 ts, a temperature-sensitive mutation of CRM1, a known exporting factor, led to a high level of GAP1 expression even in the absence of rapamycin in high quality nitrogen at the restrictive temperature (35 °C) (Fig. 1b) but not at the permissive temperature (25 °C) (data not shown). Thus, Crm1p appears to be the karyopherin responsible for Gln3p nuclear export. To confirm the above findings, we directly examined the effects ofsrp1cs and crm1-1 tsmutations on the localization of Gln3p by indirect immunofluorescence. In agreement, we found that Gln3p remained in the cytoplasm in the absence and the presence of rapamycin in thesrp1cs mutant at the nonpermissive temperature (20 °C) in high quality nitrogen (Fig.2a). In contrast, Gln3p localization was normal at the permissive temperature (30 °C) (Fig.2a). We also showed that Gln3p was exclusively localized to the nucleus both in the absence and the presence of rapamycin in thecrm1-1 ts mutant at 35 °C (Fig.2b). Taken together, our data indicate that Srp1p and Crm1p are the karyopherins that mediate Gln3p nuclear entry and exit. Under physiological conditions, nitrogen sources and levels control Gln3p localization. To further establish the role of Srp1p in nitrogen signal transduction, we examined Gln3p localization in thesrp1cs mutant strain under different nitrogen conditions (Fig. 3). We found that Gln3p was primarily located in the cytoplasm in rich nitrogen but rapidly accumulated in the nucleus during nitrogen starvation in the wild type strain at both 30 °C and 20 °C and in thesrp1cs mutant strain at 30 °C (permissive temperature). In contrast, Gln3p remained in the cytoplasm under nitrogen starvation in the srp1cs mutant strain at the nonpermissive temperature (20 °C). Therefore, Srp1p is required for the regulated Gln3p localization by nitrogen conditions.Figure 3Nuclear accumulation of Gln3p during nitrogen starvation requires a functional karyopherin α/Srp1p.Exponentially growing wild type (WT) andsrp1 cs mutant cells expressing Gln3p-MYC9 were shifted to nonpermissive temperature (20 °C) for 2 h and then switched to synthetic complete (+N) medium or synthetic complete medium without nitrogen sources (−N) for 30 min. The localization of Gln3p-MYC9 was examined by indirect immunofluorescence staining with mAb 9E10. Yeast nuclei were stained with DAPI.View Large Image Figure ViewerDownload (PPT) The small GTPase Ran/Gsp1p plays an essential role in nucleocytoplasmic transport (56Gorlich D. Dabrowski M. Bischoff F.R. Kutay U. Bork P. Hartmann E. Prehn S. Izaurralde E. J. Cell Biol. 1997; 138: 65-80Crossref PubMed Scopus (350) Google Scholar, 59Izaurralde E. Kutay U. von Kobbe C. Mattaj I.W. Gorlich D. EMBO J. 1997; 16: 6535-6547Crossref PubMed Scopus (494) Google Scholar). The RanGTP gradient is maintained by two proteins: RanGAP/Rna1p, which is located in the cytoplasm (60Bischoff F. Klebe C. Kretschmer J. Wittinghofer A. Ponstingl H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 2587-2591Crossref PubMed Scopus (417) Google Scholar, 61Hopper A. Traglia H. Dunst R. J. Cell Biol. 1990; 111: 309-321Crossref PubMed Scopus (167) Google Scholar, 62Mahajan R. Delphin C. Guan T. Gerace L. Melchior F. Cell. 1997; 88: 97-107Abstract Full Text Full Text PDF PubMed Scopus (1006) Google Scholar, 63Matunis M. Coutavas E. Blobel G. J. Cell Biol. 1996; 135: 1457-1470Crossref PubMed Scopus (957) Google Scholar, 64Melchior F. Weber K. Gerke V. Mol. Biol. Cell. 1993; 4: 569-581Crossref PubMed Scopus (78) Google Scholar), and RanGEF/Prp20p, which is exclusively found in the nucleus (65Bischoff F.R. Ponstingl H. Nature. 1991; 354: 80-82Crossref PubMed Scopus (539) Google Scholar). Disruption of this gradient results in failure of nucleocytoplasmic transport. To determine whether Gln3p transport involves the RanGTP gradient, we investigated the effect of a temperature-sensitive mutation in RanGAP (rna1-1 ts) on the expression of GAP1 and the localization of Gln3p in the absence and the presence of rapamycin. We found that this mutation completely abolished the expression of GAP1 (Fig.4a) and the nuclear import of Gln3p in the presence of rapamycin at the restrictive temperature (37 °C) (Fig. 4b). Therefore, the RanGTP gradient is critical for Gln3p import. This is consistent with the requirement for Srp1, an adaptor that binds to a Ran-GTP binding karyopherin-β (Kap95). Karyopherin βs are known to directly bind either to their cargo proteins or to an adaptor. To establish that Srp1p is an adaptor for Gln3p import and to understand the mechanism of regulated nuclear import of Gln3p, we investigated possible protein-protein interactions between Srp1p and Gln3p. We prepared extracts from log phase yeast cells in high quality nitrogen-containing medium. Under such yeast growth condition, Gln3p was present predominantly as hyperphosphorylated forms (Fig.5a, lane 1,arrow, and Ref. 17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). However, there was still a residual amount of Gln3p in the dephosphorylated form (Fig. 5a,lane 1, double arrow, and Ref. 17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). This is consistent with the observation that a small amount of Gln3p is present in the nucleus even when yeast are grown in the nutrient-rich medium, presumably because of the need of limited Gln3p molecules for the basal expression of Gln3p-dependent genes (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). We incubated the yeast extracts with bacterially produced, affinity-purified GST on glutathione-Sepharose beads or GST-Srp1p on glutathione-Sepharose beads. We found that Gln3p specifically bound to GST-Srp1p but not to GST alone (Fig. 5a). More interestingly, GST-Srp1p only bound to the dephosphorylated but not the hyperphosphorylated Gln3p (Fig. 5a). In this experiment, the same amount of GST-Srp1p was used in each sample as indicated by Coomassie Blue staining (Fig.5b). To directly demonstrate that phosphorylation regulates the interaction between Gln3p and Srp1p, we treated the cell extracts with CIP. CIP resulted in dephosphorylation of Gln3p as indicated by the increase of its gel mobility (Fig. 6a,lane 2, and Ref. 17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). This dephosphorylation was completely blocked by Na4P2O7, a potent phosphatase inhibitor. We found that GST-Srp1p indeed preferentially bound to the dephosphorylated Gln3p (Fig. 6a, lanes 4–6). The small amounts of hypophosphorylated and dephosphorylated Gln3p in the CIP-untreated or CIP/Na4P2O7 sample were barely detectable (because of the short exposure of the Western blot). The same amount of GST-Srp1p was used for each different sample as indicated by Coomassie Blue staining (Fig. 6b). Taken together, these results show that Gln3p binds to karyopherin α/Srp1p and that phosphorylation of Gln3p inhibits this interaction. Cellular responses to nitrogen availability and the quality of nitrogen nutrients require regulated nuclear transport of the GATA-type transcription factor Gln3p. Gln3p is phosphorylated in a TOR-dependent manner and localizes predominantly to the cytoplasm when yeast are grown in high quality nitrogen-containing medium. In contrast, Gln3 becomes dephosphorylated and accumulates in the nucleus when yeast are grown in poor nitrogen, grown under nitrogen starvation conditions, or treated with rapamycin (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (799) Google Scholar). In this study, we sought to understand how phosphorylation of Gln3p regulates its nucleocytoplasmic transport and the nuclear transport machinery that moves Gln3p into and out of the nucleus. We found that thesrp1cs mutation specifically blocked the nuclear import of Gln3p at nonpermissive temperatures in the presence of rapamycin. In addition, Gln3p binds specifically to bacterially expressed GST-Srp1p in vitro. Taken together, these results indicate that Srp1p is the nuclear import factor for Gln3p. Crm1p is required for the nuclear export of many proteins, including Hog1p (51Ferrigno P. Posas F. Koepp D. Saito H. Silver P.A. EMBO J. 1998; 17: 5606-5614Crossref PubMed Scopus (348) Google Scholar), the HIV1 Rev protein (38Neville M. Stutz F. Lee L. Davis L.I. Rosbash M. Curr. Biol. 1997; 7: 767-775Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar), Dbp5p (66Hodge C.A. Colot H.V. Stafford P. Cole C.N. EMBO J. 1999; 18: 5778-5788Crossref PubMed Scopus (162) Google Scholar), and Yap1p (67Yan C. Lee L.H. Davis L.I. EMBO J. 1998; 17: 7416-7429Crossref PubMed Scopus (205) Google Scholar, 68Kuge S. Toda T. Iizuka N. Nomoto A. Genes Cells. 1998; 3: 521-532Crossref PubMed Scopus (136) Google Scholar). We found that the crm1-1ts mutation caused constitutive nuclear retention of Gln3p at the restrictive temperature in high quality nitrogen in the absence of rapamycin (Fig.2b). Consequently, there was a high expression level ofGAP1 under such conditions (Fig. 1). Many Crm1p substrates contain a leucine-rich nuclear export sequence (l-X2–3-(F/I/L/V/M)-X2–3-L-X-(L/I)) (36Fukuda M. Asano S. Nakamura T. Adachi M. Yoshida M. Yanagida M. Nishida E. Nature. 1997; 390: 308-311Crossref PubMed Scopus (1028) Google Scholar, 37Kudo N. Khochbin S. Nishi K. Kitano K. Yanagida M. Yoshida M. Horinouchi S. J. Biol. Chem. 1997; 272: 29742-29751Crossref PubMed Scopus (181) Google Scholar, 38Neville M. Stutz F. Lee L. Davis L.I. Rosbash M. Curr. Biol. 1997; 7: 767-775Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar, 39Ossareh-Nazari B. Bachelerie F. Dargemont C. Science. 1997; 278: 141-144Crossref PubMed Scopus (623) Google Scholar, 40Stade K. Ford C.S. Guthrie C. Weis K. Cell. 1997; 90: 1041-1050Abstract Full Text Full Text PDF PubMed Scopus (937) Google Scholar, 69Fornerod M. van Deursen J. van Baal S. Reynolds A. Davis D. Murti K.G. Fransen J. Grosveld G. EMBO J. 1997; 16: 807-816Crossref PubMed Scopus (398) Google Scholar). Interestingly, Gln3p also contains a motif (335LHGTMRPLSL) characteristic of a leucine-rich nuclear export signal. Deletion of this leucine-rich sequence causes nuclear accumulation of Gln3p in nitrogen-rich medium and constitutive expression ofGAP1. 2J. Carvalho and X. F. S. Zheng, unpublished data. Taken together, our results indicate that Crm1p is the nuclear export factor for Gln3p. A defect in nuclear export as a result of thecrm1-1ts mutation is sufficient to cause nuclear accumulation of Gln3p in nitrogen-rich medium, suggesting that a small amount of Gln3p shuttles between the cytoplasm and the nucleus when yeast are grown in nitrogen-rich medium. This observation is also consistent with the presence of a small amount of dephosphorylated Gln3p in the same conditions (Fig. 5a). Phosphorylation is broadly involved in the regulated transport of many important proteins between the cytoplasm and nucleus (reviewed in Refs.44Hood J.K. Silver P.A. Curr. Opin. Cell Biol. 1999; 11: 241-247Crossref PubMed Scopus (114) Google Scholar and 45Komeili A. O'Shea E.K. Curr. Opin. Cell Biol. 2000; 12: 355-360Crossref PubMed Scopus (87) Google Scholar). Phosphorylation within or adjacent to the NLS sequence can dramatically affect recognition of cargo proteins by the karyopherins. One such example is Pho4p, a phosphate-regulated transcription factor. Pho4p is phosphorylated and localized in the cytoplasm when yeast is grown in phosphate-rich medium, whereas in low phosphate medium, Pho4p is dephosphorylated and localized to the nucleus. Pho4p nuclear translocation requires the karyopherin Pse1. Phosphorylation decreases the affinity of Pho4p for Pse1p and results in the cytoplasmic retention of Pho4p (46Kaffman A. Rank N.M. O'Shea E.K. Genes Dev. 1998; 12: 2673-2683Crossref PubMed Scopus (204) Google Scholar, 47Komeili A. O'Shea E.K. Science. 1999; 284: 977-980Crossref PubMed Scopus (249) Google Scholar). A similar scenario may also be true for Gln3p and possibly other NCR-sensitive transcription activators and repressors (a model is shown in Fig. 7). During nitrogen starvation or rapamycin treatment, TOR-dependent phosphorylation decreases, which is possibly accompanied by an increase in the phosphatase activity toward Gln3p (17Bertram P.G. Choi J. Carvalho J. Ai W.D. Zeng C.B. Chan T.F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 18Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (799) Google Scholar). As a result, the dephosphorylated form of Gln3p predominates and is imported into the nucleus as a result of its interaction with Srp1p. Our results provide further support for a common mechanism by which nutrient-sensing transcription factors are controlled by phosphorylation-dependent interaction with the nuclear transport machinery. We are grateful to A. Hopper, A. Tartakoff, K. Weis, L. Davis, M. Fitzgerald-Hayes, M. Johnston, and P. Silver for strains, K. Mishra for construction of pSW347, and D. Dean for use of the fluorescence microscopes. We also thank other members of the Zheng laboratory for insightful discussions." @default.
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• W2041774555 title "Phosphorylation Regulates the Interaction between Gln3p and the Nuclear Import Factor Srp1p" @default.
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