TORC1 is a central regulator of cell growth in response to amino acid availability, yet little is known about how it is regulated. Here, we performed a reverse genetic screen in yeast for genes necessary to inactivate TORC1. The screen consisted of monitoring the expression of a TORC1 sensitive GFP-based transcriptional reporter in all yeast deletion strains using flow cytometry. We find that in response to amino acid starvation, but not to carbon starvation or rapamycin treatment, cells lacking NPR2 and NPR3 fail to fully (1) activate transcription factors Gln3/Gat1, (2) dephosphorylate TORC1 effector Npr1, and (3) repress ribosomal protein gene expression. Both mutants show proliferation defects only in media containing a low quality nitrogen source, such as proline or ammonia, whereas no defects are evident when cells are grown in the presence of glutamine or peptone mixture. Proliferation defects in npr2Δ and npr3Δ cells can be completely rescued by artificially inhibiting TORC1 by rapamycin, demonstrating that overactive TORC1 in both strains prevents their ability to adapt to an environment containing a low quality nitrogen source. A biochemical purification of each demonstrates that Npr2 and Npr3 form a heterodimer, and this interaction is evolutionarily conserved since the human homologs of NPR2 and NPR3 (NPRL2 and NPRL3, respectively) also co-immunoprecipitate. We conclude that, in yeast, the Npr2/3 complex mediates an amino acid starvation signal to TORC1.
Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, et al. (2002) mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell growth machinery. Cell 110: 163–175.
Sarbassov DD, Ali SM, Kim DH, Guertin DA, Latek RR, et al. (2004) Rictor, a novel binding partner of mTOR, defines a rapamycin-insensitive and raptor-independent pathway that regulates the cytoskeleton. Current biology 14: 1296–1302.
Loewith R, Jacinto E, Wullschleger S, Lorberg A, Crespo JL, et al. (2002) Two TOR complexes, only one of which is rapamycin sensitive, have distinct roles in cell growth control. Molecular Cell 10: 457–468.
Li H, Tsang CK, Watkins M, Bertram PG, Zheng XF (2006) Nutrient regulates Tor1 nuclear localization and association with rDNA promoter. Nature 442: 1058–1061.
Barbet NC, Schneider U, Helliwell SB, Stansfield I, Tuite MF, et al. (1996) TOR controls translation initiation and early G1 progression in yeast. Mol Biol Cell 7: 25–42.
Powers T, Walter P (1999) Regulation of ribosome biogenesis by the rapamycin-sensitive TOR-signaling pathway in Saccharomyces cerevisiae. Mol Biol Cell 10: 987–1000.
Cardenas ME, Cutler NS, Lorenz MC, Di Como CJ, Heitman J (1999) The TOR signaling cascade regulates gene expression in response to nutrients. Genes Dev 13: 3271–3279.
Crespo JL, Powers T, Fowler B, Hall MN (2002) The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine. Proc Natl Acad Sci U S A 99: 6784–6789.
Komeili A, Wedaman KP, O'Shea EK, Powers T (2000) Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors. J Cell Biol 151: 863–878.
Martin DE, Soulard A, Hall MN (2004) TOR regulates ribosomal protein gene expression via PKA and the Forkhead transcription factor FHL1. Cell 119: 969–979.
Smith EM, Finn SG, Tee AR, Browne GJ, Proud CG (2005) The tuberous sclerosis protein TSC2 is not required for the regulation of the mammalian target of rapamycin by amino acids and certain cellular stresses. J Biol Chem 280: 18717–18727.
Nobukuni T, Joaquin M, Roccio M, Dann SG, Kim SY, et al. (2005) Amino acids mediate mTOR/raptor signaling through activation of class 3 phosphatidylinositol 3OH-kinase. Proc Natl Acad Sci U S A 102: 14238–14243.
Byfield MP, Murray JT, Backer JM (2005) hVps34 is a nutrient-regulated lipid kinase required for activation of p70 S6 kinase. J Biol Chem 280: 33076–33082.
Herman PK, Emr SD (1990) Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol 10: 6742–6754.
Juhasz G, Hill JH, Yan Y, Sass M, Baehrecke EH, et al. (2008) The class III PI(3)K Vps34 promotes autophagy and endocytosis but not TOR signaling in Drosophila. J Cell Biol 181: 655–666.
Stanbrough M, Rowen DW, Magasanik B (1995) Role of the GATA factors Gln3p and Nil1p of Saccharomyces cerevisiae in the expression of nitrogen-regulated genes. Proc Natl Acad Sci U S A 92: 9450–9454.
Liu Z, Butow RA (1999) A transcriptional switch in the expression of yeast tricarboxylic acid cycle genes in response to a reduction or loss of respiratory function. Mol Cell Biol 19: 6720–6728.
Neklesa TK, Davis RW (2008) Superoxide anions regulate TORC1 and its ability to bind Fpr1:rapamycin complex. Proc Natl Acad Sci U S A 105: 15166–15171.
Schmidt A, Beck T, Koller A, Kunz J, Hall MN (1998) The TOR nutrient signalling pathway phosphorylates NPR1 and inhibits turnover of the tryptophan permease. Embo J 17: 6924–6931.
De Craene JO, Soetens O, Andre B (2001) The Npr1 kinase controls biosynthetic and endocytic sorting of the yeast Gap1 permease. J Biol Chem 276: 43939–43948.
Gander S, Bonenfant D, Altermatt P, Martin DE, Hauri S, et al. (2008) Identification of the rapamycin-sensitive phosphorylation sites within the Ser/Thr-rich domain of the yeast Npr1 protein kinase. Rapid Commun Mass Spectrom 22: 3743–3753.
Tate JJ, Cox KH, Rai R, Cooper TG (2002) Mks1p is required for negative regulation of retrograde gene expression in Saccharomyces cerevisiae but does not affect nitrogen catabolite repression-sensitive gene expression. J Biol Chem 277: 20477–20482.
Zheng XF, Schreiber SL (1997) Target of rapamycin proteins and their kinase activities are required for meiosis. Proc Natl Acad Sci U S A 94: 3070–3075.
Rigaut G, Shevchenko A, Rutz B, Wilm M, Mann M, et al. (1999) A generic protein purification method for protein complex characterization and proteome exploration. Nat Biotechnol 17: 1030–1032.
Lerman MI, Minna JD (2000) The 630-kb lung cancer homozygous deletion region on human chromosome 3p21.3: identification and evaluation of the resident candidate tumor suppressor genes. The International Lung Cancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium. Cancer Res 60: 6116–6133.
Zabarovsky ER, Lerman MI, Minna JD (2002) Tumor suppressor genes on chromosome 3p involved in the pathogenesis of lung and other cancers. Oncogene 21: 6915–6935.
Li J, Wang F, Haraldson K, Protopopov A, Duh FM, et al. (2004) Functional characterization of the candidate tumor suppressor gene NPRL2/G21 located in 3p21.3C. Cancer Res 64: 6438–6443.
Ueda K, Kawashima H, Ohtani S, Deng WG, Ravoori M, et al. (2006) The 3p21.3 Tumor Suppressor NPRL2 Plays an Important Role in Cisplatin-Induced Resistance in Human Non-Small-Cell Lung Cancer Cells. Cancer Res 66: 9682–9690.
Lin F, Zhang PL, Yang XJ, Prichard JW, Lun M, et al. (2006) Morphoproteomic and molecular concomitants of an overexpressed and activated mTOR pathway in renal cell carcinomas. Ann Clin Lab Sci 36: 283–293.
Kenerson HL, Aicher LD, True LD, Yeung RS (2002) Activated mammalian target of rapamycin pathway in the pathogenesis of tuberous sclerosis complex renal tumors. Cancer Res 62: 5645–5650.
Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, et al. (2001) Enhanced sensitivity of PTEN-deficient tumors to inhibition of FRAP/mTOR. Proceedings of the National Academy of Sciences of the United States of America 98: 10314–10319.
Longtine MS, McKenzie A 3rd, Demarini DJ, Shah NG, Wach A, et al. (1998) Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae. Yeast 14: 953–961.
Williams RM, Primig M, Washburn BK, Winzeler EA, Bellis M, et al. (2002) The Ume6 regulon coordinates metabolic and meiotic gene expression in yeast. Proceedings of the National Academy of Sciences of the United States of America 99: 13431–13436.
