title: Gln3 phosphorylation and intracellular localization in nutrient limitation and starvation differ from those generated by rapamycin inhibition of Tor1/2 in Saccharomyces cerevisiae.
abstract: The ability of the cell to sense environmental conditions and alter gene expression in response to them is critical to its survival. In Saccharomyces cerevisiae, the Tor1/2 serine/threonine kinases are global regulators situated at the top of a signal cascade reported to receive and transmit nutritional signals associated with the nitrogen supply of the cell. At the other end of that cascade is Gln3, one of two transcriptional activators responsible for most nitrogen catabolic gene expression. When nitrogen is in excess, Tor1/2 are active, and Gln3 is phosphorylated and localizes to the cytoplasm. If Tor1/2 are inhibited by rapamycin or mutation, Gln3 becomes dephosphorylated, accumulates in the nucleus, and mediates nitrogen catabolite repression (NCR)-sensitive transcription. The observations that Gln3 also accumulates in the nuclei of cells provided with poor nitrogen sources or during nitrogen starvation has led to the conclusion that Tor1/2 control intracellular Gln3 localization and NCR-sensitive transcription by regulating Gln3 phosphorylation/dephosphorylation. To test this model, we compared Gln3 phosphorylation states and intracellular localizations under a variety of physiological conditions known to elicit different levels of NCR-sensitive transcription. Our data indicate that: (i) observable Gln3 phosphorylation levels do not correlate in a consistent way with the quality or quantity of the nitrogen source provided, the intracellular localization of Gln3, or the capacity to support NCR-sensitive transcription. (ii) Gln3-Myc(13) is hyperphosphorylated during nitrogen and carbon starvation, but this uniform response does not correlate with Gln3 intracellular localization. (iii) Gln3-Myc(13) dephosphorylation and nuclear localization correlate with one another at early but not late times after rapamycin treatment. These data suggest that rapamycin treatment and growth with poor nitrogen sources bring about nuclear accumulation of Gln3 but likely do so by different mechanisms or by a common mechanism involving molecules other than Gln3 and/or other than the levels of Gln3-Myc(13) phosphorylation thus far detected by others and ourselves.
authors.affiliation: Department of Molecular Sciences, University of Tennessee, Memphis, Tennessee 38163, USA.
authors.forename: Kathleen H
authors.initials: KH
authors.lastname: Cox
authors.forename: Ajit
authors.initials: A
authors.lastname: Kulkarni
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Tate
authors.forename: Terrance G
authors.initials: TG
authors.lastname: Cooper
mesh_terms: D000469:Alkaline Phosphatase / Q000378:metabolism; D000645:Ammonium Sulfate / Q000494:pharmacology; D000935:Antifungal Agents / Q000494:pharmacology; D015152:Blotting, Northern; D015153:Blotting, Western; D018797:Cell Cycle Proteins; D003593:Cytoplasm / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D017353:Gene Deletion; D009584:Nitrogen / Q000378:metabolism*; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D012097:Repressor Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology*; D013997:Time Factors; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation
chemical_list: D000469:Alkaline Phosphatase; D000645:Ammonium Sulfate; D000935:Antifungal Agents; D018797:Cell Cycle Proteins; D009584:Nitrogen; D019869:Phosphatidylinositol 3-Kinases; D010744:Phosphoric Monoester Hydrolases; D017853:Phosphotransferases (Alcohol Group Acceptor); D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D020123:Sirolimus; D014157:Transcription Factors; C071664:GLN3 protein, S cerevisiae; C083324:TOR1 protein, S cerevisiae; C081135:TOR2 protein, S cerevisiae