Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite Repression and the Mechanism of Its Nuclear Localization during Carbon Starvation in Saccharomyces cerevisiae
2002; Elsevier BV; Volume: 277; Issue: 40 Linguagem: Inglês
10.1074/jbc.m204879200
ISSN1083-351X
AutoresKathleen H. Cox, Jennifer J. Tate, Terrance Cooper,
Tópico(s)DNA Repair Mechanisms
ResumoRegulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g.glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus. Regulated intracellular localization of Gln3, the transcriptional activator responsible for nitrogen catabolite repression (NCR)-sensitive transcription, permits Saccharomyces cerevisiae to utilize good nitrogen sources (e.g.glutamine and ammonia) in preference to poor ones (e.g. proline). During nitrogen starvation or growth in medium containing a poor nitrogen source, Gln3 is nuclear and NCR-sensitive transcription is high. However, when cells are grown in excess nitrogen, Gln3 is localized to the cytoplasm with a concomitant decrease in gene expression. Treating cells with the Tor protein inhibitor, rapamycin, mimics nitrogen starvation. Recently, carbon starvation has been reported to cause nuclear localization of Gln3 and increased NCR-sensitive transcription. Here we show that nuclear localization of Gln3 during carbon starvation derives from its indirect effects on nitrogen metabolism, i.e. Gln3 does not move into the nucleus of carbon-starved cells if glutamine rather than ammonia is provided as the nitrogen source. In addition, these studies have clearly shown Gln3 is not uniformly distributed in the cytoplasm, but rather localizes to punctate or tubular structures. Analysis of these images by deconvolution microscopy suggests that Gln3 is concentrated in or associated with a highly structured system in the cytosol, one that is possibly vesicular in nature. This finding may impact significantly on how we view (i) the mechanism by which Tor regulates the intracellular localization of Gln3 and (ii) how proteins move into and out of the nucleus. nitrogen catabolite repression 4′,6-diamidino-2-phenylindole The budding yeast Saccharomyces cerevisiae is often used as a model with which to elucidate the functions of important mammalian proteins. Few such proteins have generated greater excitement than Tor1/2 and their mammalian counterpart mTor, which are inhibited by the immunosuppressant and antineoplastic drug rapamycin (1Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar, 2Dennis P.B. Fumagalli S. Thomas G. Cur. Opin. Genet. Develop. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 3Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1736) Google Scholar, 4Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 5Cooper T.G. FEMS Microbiol. Rev. 2002; 737: 1-16Google Scholar). Tor1 and the essential Tor2 proteins are reported to be situated at the top of the Tor signal transduction cascade and through it to regulate an exceedingly large number of diverse cellular processes, including: translational initiation, G1-phase progression, autophagy, RNA polymerase I/III function, actin cytoskeleton organization, membrane protein stability, nitrogen catabolite repression (NCR)1-sensitive and retrograde gene expression (1Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar, 2Dennis P.B. Fumagalli S. Thomas G. Cur. Opin. Genet. Develop. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 3Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1736) Google Scholar, 4Rohde J. Heitman J. Cardenas M.E. J. Biol. Chem. 2001; 276: 9583-9586Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 5Cooper T.G. FEMS Microbiol. Rev. 2002; 737: 1-16Google Scholar, 6Shamji A.F. Kuruvilla F.G. Schreiber S.L. Cur. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Edskes H.K. Hanover J.A. Wickner R.B. Genetics. 1999; 153: 585-594PubMed Google Scholar, 8Pierce M.M. Maddelein M.L. Roberts B.T. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13213-13218Crossref PubMed Scopus (17) Google Scholar, 9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar). In this work, we focus on the last two of these processes. NCR is the physiological process by which S. cerevisiaeselectively uses good nitrogen sources in its environment in preference to poor ones (5Cooper T.G. FEMS Microbiol. Rev. 2002; 737: 1-16Google Scholar, 10Hoffman-Bang J. Mol. Biotechnol. 1999; 12: 35-73Crossref PubMed Scopus (181) Google Scholar, 11ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (211) Google Scholar, 12Cooper T.G. Marzluf G. Bambrl R. Mycota III. Springer Verlag, Berlin, Heidelberg1996: 139-169Google Scholar). In the presence of good nitrogen sources (glutamine, asparagine, and in some strains, ammonia), expression of genes encoding the permeases and enzymes needed for utilization of poor nitrogen sources is held at low levels, i.e. expression is repressed. When only poor (proline) or limiting amounts of good nitrogen sources are available, these genes are then expressed at the higher levels needed to scavenge whatever is available in the environment (5Cooper T.G. FEMS Microbiol. Rev. 2002; 737: 1-16Google Scholar, 10Hoffman-Bang J. Mol. Biotechnol. 1999; 12: 35-73Crossref PubMed Scopus (181) Google Scholar, 11ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (211) Google Scholar, 12Cooper T.G. Marzluf G. Bambrl R. Mycota III. Springer Verlag, Berlin, Heidelberg1996: 139-169Google Scholar). Two GATA family transcriptional activators (Gln3 and Gat1/Nil1) are responsible for NCR-sensitive transcription and Ure2 (a yeast prion precursor) inhibits their ability to carry out this function (5Cooper T.G. FEMS Microbiol. Rev. 2002; 737: 1-16Google Scholar, 10Hoffman-Bang J. Mol. Biotechnol. 1999; 12: 35-73Crossref PubMed Scopus (181) Google Scholar, 11ter Schure E.G. van Riel N.A. Verrips C.T. FEMS Microbiol. Rev. 2000; 24: 67-83Crossref PubMed Scopus (211) Google Scholar, 12Cooper T.G. Marzluf G. Bambrl R. Mycota III. Springer Verlag, Berlin, Heidelberg1996: 139-169Google Scholar). In glucose-proline medium, Gln3 and Gat1 are bound to the GATA sequences of the NCR-sensitive CAN1 gene, whereas in glucose-glutamine medium or in agln3Δgat1Δ strain, these GATAs are unoccupied and available to serve as surrogate TATA elements (13Cox K.H. Rai R. Distler M. Daugherty J.R. Coffman J.A. Cooper T.G. J. Biol. Chem. 2000; 275: 17611-17618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Correlating with these observations, Gln3 and Gat1 are localized to the nucleus in the former medium and to the cytoplasm in the latter (13Cox K.H. Rai R. Distler M. Daugherty J.R. Coffman J.A. Cooper T.G. J. Biol. Chem. 2000; 275: 17611-17618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar). Work in four laboratories, using the immunosuppressant drug, rapamycin, has provided insights into the mechanism regulating intracellular localization of Gln3 (14Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar, 15Cardenas M.E. Cutler N.S. Lorenz M.C., Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (484) Google Scholar, 16Hardwick J.S. Kuruvilla F.G. Tong J.F. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar, 17Bertram P.G. Choi J.H. Carvalho J., Ai, W. Zeng C. Chan T.-F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). They found that inhibiting Tor1/2 with rapamycin resulted in dephosphorylation of Gln3 and, in some laboratories, Ure2, and its entry into the nucleus (14Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar, 16Hardwick J.S. Kuruvilla F.G. Tong J.F. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar, 17Bertram P.G. Choi J.H. Carvalho J., Ai, W. Zeng C. Chan T.-F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). Loss of NCR-sensitive gene expression in rna1 and srp1mutants (19Bossinger J. Cooper T.G. J. Bacteriol. 1976; 126: 198-204Crossref PubMed Google Scholar, 20Carvalho J. Bertram P.G. Wente S.R. Zheng X.F.S. J. Biol. Chem. 2001; 276: 25359-25365Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar), led Carvalho et al. (20Carvalho J. Bertram P.G. Wente S.R. Zheng X.F.S. J. Biol. Chem. 2001; 276: 25359-25365Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar) to conclude that these proteins are required for Gln3 entry into the nucleus and that Crm1 mediates its exit. Tap42, a kinase that itself is phosphorylated by Tor (21Di Como C.J. Arndt K.T. Genes Dev. 1996; 10: 1904-1916Crossref PubMed Scopus (442) Google Scholar, 22Jiang Y. Broach J.R. EMBO. 1999; 18: 2782-2792Crossref PubMed Scopus (276) Google Scholar), Tap41, type 2 phosphatases Sit4 and Pph3, Mks1 and Ure2 are the remaining members of the Tor signal transduction cascade reported to be situated between Tor and Gln3/Gat1 (1Barbet N.C. Schneider U. Helliwell S.B. Stansfield I. Tuite M.F. Hall M.N. Mol. Biol. Cell. 1996; 7: 25-42Crossref PubMed Scopus (603) Google Scholar, 2Dennis P.B. Fumagalli S. Thomas G. Cur. Opin. Genet. Develop. 1999; 9: 49-54Crossref PubMed Scopus (249) Google Scholar, 3Schmelzle T. Hall M.N. Cell. 2000; 103: 253-262Abstract Full Text Full Text PDF PubMed Scopus (1736) Google Scholar, 6Shamji A.F. Kuruvilla F.G. Schreiber S.L. Cur. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Edskes H.K. Hanover J.A. Wickner R.B. Genetics. 1999; 153: 585-594PubMed Google Scholar, 8Pierce M.M. Maddelein M.L. Roberts B.T. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13213-13218Crossref PubMed Scopus (17) Google Scholar, 9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar, 14Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar, 15Cardenas M.E. Cutler N.S. Lorenz M.C., Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (484) Google Scholar, 16Hardwick J.S. Kuruvilla F.G. Tong J.F. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar, 17Bertram P.G. Choi J.H. Carvalho J., Ai, W. Zeng C. Chan T.-F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar, 18Kunz J. Schneider U. Howald I. Schmidt A. Hall M.N. J. Biol. Chem. 2000; 275: 37011-37020Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar,23Jacinto E. Guo B. Arndt K.T. Schmelzle T. Hall M.N. Mol. Cell. 2001; 8: 1017-1026Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 24Tate J.J. Cox K.H. Rai R. Cooper T.G. J. Biol. Chem. 2002; 277: 20477-20482Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Recent work (24Tate J.J. Cox K.H. Rai R. Cooper T.G. J. Biol. Chem. 2002; 277: 20477-20482Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), however, demonstrated that Mks1 functions only indirectly in the regulation of Gln3/Gat1. A second set of metabolic genes, those associated with the retrograde response have also been reported to be regulated by the Tor signal transduction cascade (6Shamji A.F. Kuruvilla F.G. Schreiber S.L. Cur. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 7Edskes H.K. Hanover J.A. Wickner R.B. 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Like Gln3 and Gat1, nuclear localization of Rtg1/3 occurs when cells are treated with rapamycin (9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar), which also results in changes in Rtg3 phosphorylation levels (9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar). Although consensus remains to be established on whether nuclear localization of Rtg3 occurs with the hyperphosphorylated form, hypophosphorylated form, or both, correlations between differences in transcription factor phosphorylation and retrograde gene expression are convincing (9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar, 26Sekito T. Liu Z. Thornton J. Butow R.A. Mol. Biol. Cell. 2002; 13: 795-804Crossref PubMed Scopus (84) Google Scholar, 28Sekito T. Thornton J. Butow R.A. Mol. Biol. Cell. 2000; 11: 2103-2115Crossref PubMed Scopus (191) Google Scholar, 33Dilova I. Chen C-Y. Powers T. Curr. Biol. 2002; 12: 389-395Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Rtg2 is required for Rtg1/3 to mediate retrograde gene expression, and Mks1, originally thought also to be required for Rtg1/3-mediated transcription (6Shamji A.F. Kuruvilla F.G. Schreiber S.L. Cur. Biol. 2000; 10: 1574-1581Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar, 8Pierce M.M. Maddelein M.L. Roberts B.T. Wickner R.B. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13213-13218Crossref PubMed Scopus (17) Google Scholar, 9Komeili A. Wedaman K.P. O'Shea E.K. Powers T. J. Cell Biol. 2000; 151: 863-878Crossref PubMed Scopus (180) Google Scholar), is now shown to be a strong negative regulator of Rtg1/3-mediated transcription (24Tate J.J. Cox K.H. Rai R. Cooper T.G. J. Biol. Chem. 2002; 277: 20477-20482Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar,26Sekito T. Liu Z. Thornton J. Butow R.A. Mol. Biol. Cell. 2002; 13: 795-804Crossref PubMed Scopus (84) Google Scholar, 33Dilova I. Chen C-Y. Powers T. Curr. Biol. 2002; 12: 389-395Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). The retrograde gene expression experiments just described collectively demonstrate a relationship between nitrogen and carbon metabolism inS. cerevisiae. Bertram et al. (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar) have recently reported a second major bridge linking carbon and nitrogen metabolism. They reported that starving cells for carbon, like starving them for nitrogen, results in Gln3 being localized to the nucleus and mediating transcription of the NCR-sensitive genes, GAP1,GDH2, and PUT1 (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar). Carbon-starved nuclear localization of Gln3 also correlates with its hyperphosphorylation. Finally, Snf1 has been shown to be required for these carbon starvation-induced changes in Gln3 localization, in phosphorylation, and for the transcription it mediates (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar). Our interest was piqued by these carbon starvation experiments, because here Gln3 is nuclear during the nitrogen excess, which exists during carbon starvation. Investigating this phenomenon, we find that whether or not Gln3 is localized to the nucleus during carbon starvation is dictated by the nature of the nitrogen source, i.e. Gln3 nuclear localization occurs with ammonia but not glutamine as nitrogen source. The effects of carbon starvation with ammonia as the nitrogen source are indirect and most likely caused by the inability of ammonia to be assimilated into glutamate during carbon starvation due to the lack of α-ketoglutarate, the carbon skeleton of glutamate. More important, however, we also found that Gln3 is not uniformly distributed in the cytoplasm during growth in glutamine medium. It appears to be sequestered in or associated with globular/tubular structures. The implications of this distribution generate an alternative way of viewing how Tor influences Gln3 intracellular distribution and NCR-sensitive transcription. Strains used in this work were JK9-3da (MATa, leu2–3,112,ura3–52, rme1, trp1, his4,GAL+, HMLa) and TB123 (MATa, leu2–3,112,ura3–52, rme1, trp1, his4,GAL+, HMLa,GLN3-myc[kanMX4]). Cultures were grown overnight at 30 °C in Difco YNB (without ammonium sulfate or amino acids), 2% glucose, and the indicated nitrogen source. Appropriate auxotropic requirements were supplied when media other than synthetic complete (SC) (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar) were used. Experiments in which yeast were transferred from one medium to another were performed as follows. A sample of the exponentially growing cells (A 600 nm = 0.4–0.7) was harvested from the initial medium for immunofluorescence or RNA analysis. The remaining cells in the culture were collected, washed with the new medium, recollected, and resuspended in an equal volume of the new medium. Additional samples were then collected and processed for RNA isolation or immunofluorescence at the indicated times. Precisely the same format was also used for the transfer of cells from rapamycin-free (initial medium) to rapamycin-containing (new medium) media. Rapamycin (Sigma), from a concentrated stock solution in 90% ethanol/10% Tween 20, was used at a final concentration of 200 ng/ml where indicated. Total RNA was prepared from cultures of strain JK9-3da as previously described (24Tate J.J. Cox K.H. Rai R. Cooper T.G. J. Biol. Chem. 2002; 277: 20477-20482Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Total yeast RNA (9 μg) was separated on denaturing gels, transferred to a nylon membrane, and hybridized with 32P-labeled DNA probes, and the blots were washed as previously described (24Tate J.J. Cox K.H. Rai R. Cooper T.G. J. Biol. Chem. 2002; 277: 20477-20482Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Immunofluorescent staining of yeast was carried out using a modification of the methods of Schwartzet al. (32Schwartz K. Richards K. Botstein D. Mol. Biol. Cell. 1997; 8: 2677-2691Crossref PubMed Scopus (168) Google Scholar). Cultures of strain TB123 were fixed by the addition of 1/10 volume of 37% formaldehyde and incubated with shaking at 30 °C for 10 min. They were then collected by centrifugation at room temperature and incubated in 3.7% formaldehyde in potassium phosphate buffer (40 mm, pH 6.5, containing 0.5 mm MgCl2) for 1 h. After washing and Zymolyase digestion as previously described, cells were applied to poly-l-lysine-coated microscope slides. The slides were blocked overnight at 4 °C using 0.5% bovine serum albumin, 0.5% Tween 20 in phosphate-buffered saline (pH 7.4). All further antibody incubations and washes were performed in this buffer. 9E10(c-myc) (Covance MMS-150P) was used at a dilution of 1:1000 as the primary antibody and either Texas Red-conjugated goat anti-mouse IgG (Jackson ImmunoResearch) or Alexa Fluor 488 goat anti-mouse IgG (Molecular Probes) was used at a dilution of 1:200 as the secondary antibody. To visualize nuclei, 4′,6′-diamino-2-phenylindole (DAPI) was added to the mounting media at a final concentration of 50 μg/ml, and images were collected immediately after mounting. Cells in Figs. 1 and 2 were imaged using a Zeiss Axiophot microscope with a 63× Plan-Apochromat 1.40 oil objective. Images were acquired using AxioVision 3.0 (Zeiss) software and a Zeiss Axio camera. Alternatively, cells shown in Figs.Figure 7, Figure 8, Figure 9 (see below) were imaged using a Zeiss Axioplan 2 imaging microscope with a 100× Plan-Apochromat 1.40 oil objective. Images were acquired using a Zeiss Axio camera and deconvolved using AxioVision 3.0 (Zeiss) software using the constrained iterative algorithm. Three-dimensional views (see Figs. 8, E–G, and 9 below) were produced from 15 images in a two-dimensional Z-stack, spanning about 1.5 μm of the center of a yeast cell using AxioVision Inside 4D (Zeiss) software. Images were rendered using either the maximum projection in which only pixels of the highest intensity along the axis are displayed (Fig. 8, E–G) or the surface mode in which non-transparent surfaces are calculated from gray values (Fig. 9).Figure 2Effect of ammonia or glutamine on intracellular localization of Gln3 in carbon-starved cells grown in YNB media. The experiment was performed as in Fig. 1 except for changes in the media. Top panel, micrographs of cells after 180 min of incubation in YNB-glucose-free ammonium sulfate (A) or YNB-glucose-free glutamine (B) media.Bottom panel, as in Fig. 1.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 7Intracellular distribution of Gln3 under various growth conditions, visualized by conventional and deconvolution microscopy. Yeast cultures (TB123) were grown to mid-log phase in YNB-glutamine medium and processed for immunolocalization (row 3). Alternatively, cultures were grown to mid-log phase in YNB-proline and transferred to YNB-glutamine medium. Aliquots were removed just prior to (row 1), and 1 min after (row 2) the transfer from proline to glutamine medium.Images are presented in pairs: Micrographs A,C, E, and G were imaged using a Zeiss Axioplan 2 imaging microscope. 0.1-μm sections were collected as a Z-stack, and one image from the center of the cell is shown. Micrographs B, D, F, and Hshow these same images deconvolved with AxioVision 3.0 (Zeiss) software using the constrained iterative algorithm.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 9The three-dimensional image described in Fig.8 was rendered using the surface mode in which non-transparent surfaces are calculated from gray values.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 8TB123 cells were grown to mid-log phase in YNB-glutamine medium and processed for immunolocalization.Micrographs A–D were imaged using a Zeiss Axioplan 2 imaging microscope. 0.1-μm sections were collected as a Z-stack, and one image from the center of the cell is shown. The images are shown either as raw data (A) or as deconvolved images (B–D) of Gln3p-mediated (A, B, andD) or of DAPI-mediated fluorescence (C andD). Three-dimensional views (E–G) were produced from 15 images in a two-dimensional Z-stack, spanning about 1.5 μm of the center of a yeast cell using AxioVision Inside 4D (Zeiss) software. Images were rendered using the maximum projection in which only pixels of the highest intensity along the axis are displayed. Three-dimensional images have been rotated on all three axes to provide different views of Gln3 intracellular localization.View Large Image Figure ViewerDownload Hi-res image Download (PPT) A recent report indicates that carbon starvation results in Snf1-dependent nuclear localization of Gln3 (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar). These data, coupled with earlier reports that nitrogen starvation or limitation also results in nuclear localization of Gln3 and Gat1p (13Cox K.H. Rai R. Distler M. Daugherty J.R. Coffman J.A. Cooper T.G. J. Biol. Chem. 2000; 275: 17611-17618Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 14Beck T. Hall M.N. Nature. 1999; 402: 689-692Crossref PubMed Scopus (803) Google Scholar, 15Cardenas M.E. Cutler N.S. Lorenz M.C., Di Como C.J. Heitman J. Genes Dev. 1999; 13: 3271-3279Crossref PubMed Scopus (484) Google Scholar, 16Hardwick J.S. Kuruvilla F.G. Tong J.F. Shamji A.F. Schreiber S.L. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 14866-14870Crossref PubMed Scopus (470) Google Scholar, 17Bertram P.G. Choi J.H. Carvalho J., Ai, W. Zeng C. Chan T.-F. Zheng X.F.S. J. Biol. Chem. 2000; 275: 35727-35733Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar), create a paradox. Cells grown in medium devoid of nitrogen are by definition growing in carbon excess. Conversely, cells grown in medium devoid of carbon are by definition growing in nitrogen excess. It was difficult to understand why Gln3 would react similarly to physiologically opposite conditions, i.e.nitrogen starvation and excess. Therefore, we investigated the phenomenon in greater detail, first by performing and then extending the critical published experiment (31Bertram P.G. Choi J.H. Carvalho J. Chan T.F., Ai, W. Zheng X.F. Mol. Cell. Biol. 2002; 22: 1246-1252Crossref PubMed Scopus (98) Google Scholar). To acquire a crude estimate of the time course of Gln3 redistribution, following perturbation, we scored the intracellular localization of Gln3 in ∼100 cells from random f
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