title: Nitrogen starvation and TorC1 inhibition differentially affect nuclear localization of the Gln3 and Gat1 transcription factors through the rare glutamine tRNACUG in Saccharomyces cerevisiae.
abstract: A leucine, leucyl-tRNA synthetase-dependent pathway activates TorC1 kinase and its downstream stimulation of protein synthesis, a major nitrogen consumer. We previously demonstrated, however, that control of Gln3, a transcription activator of catabolic genes whose products generate the nitrogenous precursors for protein synthesis, is not subject to leucine-dependent TorC1 activation. This led us to conclude that excess nitrogen-dependent down-regulation of Gln3 occurs via a second mechanism that is independent of leucine-dependent TorC1 activation. A major site of Gln3 and Gat1 (another GATA-binding transcription activator) control occurs at their access to the nucleus. In excess nitrogen, Gln3 and Gat1 are sequestered in the cytoplasm in a Ure2-dependent manner. They become nuclear and activate transcription when nitrogen becomes limiting. Long-term nitrogen starvation and treatment of cells with the glutamine synthetase inhibitor methionine sulfoximine (Msx) also elicit nuclear Gln3 localization. The sensitivity of Gln3 localization to glutamine and inhibition of glutamine synthesis prompted us to investigate the effects of a glutamine tRNA mutation (sup70-65) on nitrogen-responsive control of Gln3 and Gat1. We found that nuclear Gln3 localization elicited by short- and long-term nitrogen starvation; growth in a poor, derepressive medium; Msx or rapamycin treatment; or ure2Δ mutation is abolished in a sup70-65 mutant. However, nuclear Gat1 localization, which also exhibits a glutamine tRNACUG requirement for its response to short-term nitrogen starvation or growth in proline medium or a ure2Δ mutation, does not require tRNACUG for its response to rapamycin. Also, in contrast with Gln3, Gat1 localization does not respond to long-term nitrogen starvation. These observations demonstrate the existence of a specific nitrogen-responsive component participating in the control of Gln3 and Gat1 localization and their downstream production of nitrogenous precursors. This component is highly sensitive to the function of the rare glutamine tRNACUG, which cannot be replaced by the predominant glutamine tRNACAA. Our observations also demonstrate distinct mechanistic differences between the responses of Gln3 and Gat1 to rapamycin inhibition of TorC1 and nitrogen starvation.
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Tate
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163.
authors.forename: Rajendra
authors.initials: R
authors.lastname: Rai
authors.affiliation: Department of Microbiology, Immunology, and Biochemistry, University of Tennessee Health Science Center, Memphis, Tennessee 38163 tcooper@uthsc.edu.
authors.forename: Terrance G
authors.initials: TG
authors.lastname: Cooper
mesh_terms: D021581:Active Transport, Cell Nucleus; D004843:Epistasis, Genetic; D050980:GATA Transcription Factors / Q000378:metabolism*; D015870:Gene Expression; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D017930:Genes, Reporter; D008717:Methionine Sulfoximine / Q000494:pharmacology; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism*; D010641:Phenotype; D021381:Protein Transport; D012351:RNA, Transfer, Gln / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D020123:Sirolimus / Q000494:pharmacology; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: C096100:GAT1 protein, S cerevisiae; D050980:GATA Transcription Factors; C071664:GLN3 protein, S cerevisiae; D012351:RNA, Transfer, Gln; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C561842:TORC1 protein complex, S cerevisiae; D014157:Transcription Factors; D008717:Methionine Sulfoximine; D009584:Nitrogen; D020123:Sirolimus
keywords: Gat1; Gln3; glutamine tRNA; nitrogen starvation