Distinct Phosphatase Requirements and GATA Factor Responses to Nitrogen Catabolite Repression and Rapamycin Treatment in Saccharomyces cerevisiae*

In yeast, rapamycin (Rap)-inhibited TorC1, and the phosphatases it regulates (Sit4 and PP2A) are components of a conserved pathway regulating the response of eukaryotic cells to nutrient availability. TorC1 and intracellular nitrogen levels regulate the localization of Gln3 and Gat1, the activators of nitrogen catabolite repression (NCR)-sensitive genes whose products are required to utilize poor nitrogen sources. In nitrogen excess, Gln3 and Gat1 are cytoplasmic, and NCR-sensitive transcription is repressed. During nitrogen limitation or Rap treatment, Gln3 and Gat1 are nuclear, and transcription is derepressed. We previously demonstrated that the Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for nuclear Gln3 localization differ. We now show that Sit4 and Pph21/22-Tpd3-Cdc55/Rts1 requirements for NCR-sensitive and Rap-induced nuclear Gat1 localization markedly differ from those of Gln3. Our data suggest that Gln3 and Gat1 localizations are controlled by two different regulatory pathways. Gln3 localization predominantly responds to intracellular nitrogen levels, as reflected by its stronger NCR-sensitivity, weaker response to Rap treatment, and strong response to methionine sulfoximine (Msx, a glutamine synthetase inhibitor). In contrast, Gat1 localization predominantly responds to TorC1 regulation as reflected by its weaker NCR sensitivity, stronger response to Rap, and immunity to the effects of Msx. Nuclear Gln3 localization in proline-grown (nitrogen limited) cells exhibits no requirement for Pph21/22-Tpd3/Cdc55, whereas nuclear Gat1 localization under these conditions is absolutely dependent on Pph21/22-Tpd3/Cdc55. Furthermore, the extent to which Pph21/22-Tpd3-Cdc55 is required for the TorC1 pathway (Rap) to induce nuclear Gat1 localization is regulated in parallel with Pph21/22-Tpd3-Cdc55-dependent Gln3 dephosphorylation and NCR-sensitive transcription, being highest in limiting nitrogen and lowest when nitrogen is in excess.

[1]  T. Kinzy,et al.  Mutations in a GTP-binding Motif of Eukaryotic Elongation Factor 1A Reduce Both Translational Fidelity and the Requirement for Nucleotide Exchange* , 1999, The Journal of Biological Chemistry.

[2]  T. Cooper,et al.  Tor1/2 Regulation of Retrograde Gene Expression in Saccharomyces cerevisiae Derives Indirectly as a Consequence of Alterations in Ammonia Metabolism* , 2003, Journal of Biological Chemistry.

[3]  T. Cooper,et al.  G1n3p is capable of binding to UAS(NTR) elements and activating transcription in Saccharomyces cerevisiae , 1996, Journal of bacteriology.

[4]  S. Schreiber,et al.  Rapamycin-modulated transcription defines the subset of nutrient-sensitive signaling pathways directly controlled by the Tor proteins. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[5]  X. Zheng,et al.  Domains of Gln3p Interacting with Karyopherins, Ure2p, and the Target of Rapamycin Protein* , 2003, The Journal of Biological Chemistry.

[6]  Stephen Garrett,et al.  Multiple roles of Tap42 in mediating rapamycin-induced transcriptional changes in yeast. , 2003, Molecular cell.

[7]  E. Dubois,et al.  Tor Pathway Control of the Nitrogen-responsive DAL5 Gene Bifurcates at the Level of Gln3 and Gat1 Regulation in Saccharomyces cerevisiae* , 2008, Journal of Biological Chemistry.

[8]  T. Cooper,et al.  Differing responses of Gat1 and Gln3 phosphorylation and localization to rapamycin and methionine sulfoximine treatment in Saccharomyces cerevisiae. , 2006, FEMS yeast research.

[9]  James R Broach,et al.  How Saccharomyces responds to nutrients. , 2008, Annual review of genetics.

[10]  B. Birren,et al.  Proof and evolutionary analysis of ancient genome duplication in the yeast Saccharomyces cerevisiae , 2004, Nature.

[11]  J. Thornton,et al.  Retrograde signaling is regulated by the dynamic interaction between Rtg2p and Mks1p. , 2003, Molecular cell.

[12]  B. Magasanik,et al.  Interaction of the GATA factor Gln3p with the nitrogen regulator Ure2p in Saccharomyces cerevisiae , 1996, Journal of bacteriology.

[13]  Yu Jiang,et al.  Interaction with Tap42 is required for the essential function of Sit4 and type 2A phosphatases. , 2003, Molecular biology of the cell.

[14]  Janet Thornton,et al.  Retrograde Response to Mitochondrial Dysfunction Is Separable from TOR1/2 Regulation of Retrograde Gene Expression* , 2005, Journal of Biological Chemistry.

[15]  T. Cooper.  9 Integrated regulation of the nitrogen-carbon interface , 2004 .

[16]  A. Gronenborn,et al.  The solution structure of a fungal AREA protein-DNA complex: an alternative binding mode for the basic carboxyl tail of GATA factors. , 1998, Journal of molecular biology.

[17]  R. C. Dickson,et al.  Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance , 1987, Journal of bacteriology.

[18]  C. Kaiser,et al.  Nitrogen regulation in Saccharomyces cerevisiae. , 2002, Gene.

[19]  E. Dubois,et al.  Rapamycin-induced Gln3 Dephosphorylation Is Insufficient for Nuclear Localization , 2009, Journal of Biological Chemistry.

[20]  T. Cooper,et al.  Methionine Sulfoximine Treatment and Carbon Starvation Elicit Snf1-independent Phosphorylation of the Transcription Activator Gln3 in Saccharomyces cerevisiae* , 2005, Journal of Biological Chemistry.

[21]  Yu Jiang,et al.  Regulation of the Cell Cycle by Protein Phosphatase 2A in Saccharomyces cerevisiae , 2006, Microbiology and Molecular Biology Reviews.

[22]  T. Cooper,et al.  Stress-responsive Gln3 Localization in Saccharomyces cerevisiae Is Separable from and Can Overwhelm Nitrogen Source Regulation* , 2007, Journal of Biological Chemistry.

[23]  E. Dubois,et al.  Nitrogen Catabolite Repression-Sensitive Transcription as a Readout of Tor Pathway Regulation: The Genetic Background, Reporter Gene and GATA Factor Assayed Determine the Outcomes , 2009, Genetics.

[24]  X. Zheng,et al.  Tripartite Regulation of Gln3p by TOR, Ure2p, and Phosphatases* , 2000, The Journal of Biological Chemistry.

[25]  T. Cooper,et al.  Mks1p Is Required for Negative Regulation of Retrograde Gene Expression in Saccharomyces cerevisiae but Does Not Affect Nitrogen Catabolite Repression-sensitive Gene Expression* , 2002, The Journal of Biological Chemistry.

[26]  J. Crespo,et al.  The TOR-controlled transcription activators GLN3, RTG1, and RTG3 are regulated in response to intracellular levels of glutamine , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[27]  T. Cooper,et al.  Saccharomyces cerevisiae GATA Sequences Function as TATA Elements during Nitrogen Catabolite Repression and When Gln3p Is Excluded from the Nucleus by Overproduction of Ure2p* , 2000, The Journal of Biological Chemistry.

[28]  R Andhare,et al.  Nitrogen Catabolite Repression of DAL80Expression Depends on the Relative Levels of Gat1p and Ure2p Production in Saccharomyces cerevisiae * , 2000, The Journal of Biological Chemistry.

[29]  T. Cooper,et al.  Actin Cytoskeleton Is Required For Nuclear Accumulation of Gln3 in Response to Nitrogen Limitation but Not Rapamycin Treatment in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[30]  A M Gronenborn,et al.  NMR structure of a specific DNA complex of Zn-containing DNA binding domain of GATA-1. , 1993, Science.

[31]  T. Powers,et al.  Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae , 2006, Current Genetics.

[32]  Michael N. Hall,et al.  The TOR signalling pathway controls nuclear localization of nutrient-regulated transcription factors , 1999, Nature.

[33]  T. Cooper,et al.  Gln3p Nuclear Localization and Interaction with Ure2p inSaccharomyces cerevisiae * , 2001, The Journal of Biological Chemistry.

[34]  Serguei Sokol,et al.  Investigating the caffeine effects in the yeast Saccharomyces cerevisiae brings new insights into the connection between TOR, PKC and Ras/cAMP signalling pathways , 2006, Molecular microbiology.

[35]  J. Broach,et al.  Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast , 1999, The EMBO journal.

[36]  B. Magasanik,et al.  Sequence and expression of GLN3, a positive nitrogen regulatory gene of Saccharomyces cerevisiae encoding a protein with a putative zinc finger DNA-binding domain , 1991, Molecular and cellular biology.

[37]  J. Heitman,et al.  The TOR signaling cascade regulates gene expression in response to nutrients. , 1999, Genes & development.

[38]  T. Cooper,et al.  Cytoplasmic Compartmentation of Gln3 during Nitrogen Catabolite Repression and the Mechanism of Its Nuclear Localization during Carbon Starvation in Saccharomyces cerevisiae * , 2002, The Journal of Biological Chemistry.

[39]  M. Cyert,et al.  Yeast calcineurin regulates nuclear localization of the Crz1p transcription factor through dephosphorylation. , 1999, Genes & development.

[40]  Jacob Hofman-Bang,et al.  Nitrogen catabolite repression in Saccharomyces cerevisiae , 1999, Molecular biotechnology.

[41]  Stuart L. Schreiber,et al.  Partitioning the transcriptional program induced by rapamycin among the effectors of the Tor proteins , 2000, Current Biology.

[42]  J. Winderickx,et al.  Nutrient-Induced Responses in Eukaryotic Cells , 2004, Topics in Current Genetics.

[43]  Sean B. Carroll,et al.  Gene duplication and the adaptive evolution of a classic genetic switch , 2007, Nature.

[44]  T. Cooper,et al.  Gln3 Phosphorylation and Intracellular Localization in Nutrient Limitation and Starvation Differ from Those Generated by Rapamycin Inhibition of Tor1/2 in Saccharomyces cerevisiae* , 2004, Journal of Biological Chemistry.

[45]  T. Cooper,et al.  Formalin can alter the intracellular localization of some transcription factors in Saccharomyces cerevisiae. , 2008, FEMS yeast research.

[46]  X. Zheng,et al.  Phosphorylation Regulates the Interaction between Gln3p and the Nuclear Import Factor Srp1p* , 2001, The Journal of Biological Chemistry.

[47]  R. Drillien,et al.  Ureidosuccinic Acid Uptake in Yeast and Some Aspects of Its Regulation , 1972, Journal of bacteriology.

[48]  J. Heitman,et al.  Rapamycin Induces the G0 Program of Transcriptional Repression in Yeast by Interfering with the TOR Signaling Pathway , 1998, Molecular and Cellular Biology.

[49]  Gonghong Yan,et al.  Rapamycin activates Tap42‐associated phosphatases by abrogating their association with Tor complex 1 , 2006, The EMBO journal.

[50]  E. Dubois,et al.  The Yeast GATA Factor Gat1 Occupies a Central Position in Nitrogen Catabolite Repression-Sensitive Gene Activation , 2009, Molecular and Cellular Biology.

[51]  M. Aigle,et al.  Yeast mutants pleiotropically impaired in the regulation of the two glutamate dehydrogenases. , 1973, Biochemical and biophysical research communications.

[52]  M. Cárdenas,et al.  Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae , 2008, Proceedings of the National Academy of Sciences.

[53]  K. Arndt,et al.  TIP41 interacts with TAP42 and negatively regulates the TOR signaling pathway. , 2001, Molecular cell.

[54]  Manolis Kellis,et al.  The evolutionary dynamics of the Saccharomyces cerevisiae protein interaction network after duplication , 2008, Proceedings of the National Academy of Sciences.

[55]  E. Dubois,et al.  Saccharomyces cerevisiae Sit4 Phosphatase Is Active Irrespective of the Nitrogen Source Provided, and Gln3 Phosphorylation Levels Become Nitrogen Source-responsive in a sit4-deleted Strain* , 2006, Journal of Biological Chemistry.

[56]  K. Arndt,et al.  Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. , 1996, Genes & development.