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Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

Komeili A, Wedaman KP, O'Shea EK, Powers T - J. Cell Biol. (2000)

Bottom Line: Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases.We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment.Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of California School of Medicine, San Francisco, California 94143, USA.

ABSTRACT
De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

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Rtg1 and Rtg3 are required for expression of distinct metabolic genes in MD-urea. (A) Summary of metabolic genes (bold) subject to glutamine-mediated transcriptional repression (see Table II; note that CIT1 is not listed in Table  as its MD-glutamine/MD-urea expression ratio of ∼2.0 fell below the cut off value of 3.0 required for listing). Genes depicted were similarly repressed in MD-glutamine and MD-glutamate, except for GLN1 (see Fig. 1). (B) Nitrogen source shift experiment. Wild-type (S288c), rtg1Δ (PLY037), and rtg3Δ (PLY039) cells were grown in MD-glutamine until 0.5 OD600/ml and were either harvested (lanes 1, 4, and 7) or transferred to MD-glutamine (lanes 2, 5, and 8) or MD-urea (lanes 3, 6, and 9) media for 30 min before harvesting. RNA was prepared and analyzed by Northern blotting and probed for the specified mRNAs.
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Figure 3: Rtg1 and Rtg3 are required for expression of distinct metabolic genes in MD-urea. (A) Summary of metabolic genes (bold) subject to glutamine-mediated transcriptional repression (see Table II; note that CIT1 is not listed in Table as its MD-glutamine/MD-urea expression ratio of ∼2.0 fell below the cut off value of 3.0 required for listing). Genes depicted were similarly repressed in MD-glutamine and MD-glutamate, except for GLN1 (see Fig. 1). (B) Nitrogen source shift experiment. Wild-type (S288c), rtg1Δ (PLY037), and rtg3Δ (PLY039) cells were grown in MD-glutamine until 0.5 OD600/ml and were either harvested (lanes 1, 4, and 7) or transferred to MD-glutamine (lanes 2, 5, and 8) or MD-urea (lanes 3, 6, and 9) media for 30 min before harvesting. RNA was prepared and analyzed by Northern blotting and probed for the specified mRNAs.

Mentions: Strains derived from DBY7286 that were deleted for RTG1 or RTG3 were constructed using standard gene replacement techniques (Rothstein 1991). The entire coding regions of both genes were replaced with the URA3 gene from pRS306 (Sikorski and Heiter 1989). These strains were used for the experiments presented in Fig. 3 and Fig. 6 (below).


Mechanism of metabolic control. Target of rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription factors.

Komeili A, Wedaman KP, O'Shea EK, Powers T - J. Cell Biol. (2000)

Rtg1 and Rtg3 are required for expression of distinct metabolic genes in MD-urea. (A) Summary of metabolic genes (bold) subject to glutamine-mediated transcriptional repression (see Table II; note that CIT1 is not listed in Table  as its MD-glutamine/MD-urea expression ratio of ∼2.0 fell below the cut off value of 3.0 required for listing). Genes depicted were similarly repressed in MD-glutamine and MD-glutamate, except for GLN1 (see Fig. 1). (B) Nitrogen source shift experiment. Wild-type (S288c), rtg1Δ (PLY037), and rtg3Δ (PLY039) cells were grown in MD-glutamine until 0.5 OD600/ml and were either harvested (lanes 1, 4, and 7) or transferred to MD-glutamine (lanes 2, 5, and 8) or MD-urea (lanes 3, 6, and 9) media for 30 min before harvesting. RNA was prepared and analyzed by Northern blotting and probed for the specified mRNAs.
© Copyright Policy
Related In: Results  -  Collection

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Figure 3: Rtg1 and Rtg3 are required for expression of distinct metabolic genes in MD-urea. (A) Summary of metabolic genes (bold) subject to glutamine-mediated transcriptional repression (see Table II; note that CIT1 is not listed in Table as its MD-glutamine/MD-urea expression ratio of ∼2.0 fell below the cut off value of 3.0 required for listing). Genes depicted were similarly repressed in MD-glutamine and MD-glutamate, except for GLN1 (see Fig. 1). (B) Nitrogen source shift experiment. Wild-type (S288c), rtg1Δ (PLY037), and rtg3Δ (PLY039) cells were grown in MD-glutamine until 0.5 OD600/ml and were either harvested (lanes 1, 4, and 7) or transferred to MD-glutamine (lanes 2, 5, and 8) or MD-urea (lanes 3, 6, and 9) media for 30 min before harvesting. RNA was prepared and analyzed by Northern blotting and probed for the specified mRNAs.
Mentions: Strains derived from DBY7286 that were deleted for RTG1 or RTG3 were constructed using standard gene replacement techniques (Rothstein 1991). The entire coding regions of both genes were replaced with the URA3 gene from pRS306 (Sikorski and Heiter 1989). These strains were used for the experiments presented in Fig. 3 and Fig. 6 (below).

Bottom Line: Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases.We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment.Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

View Article: PubMed Central - PubMed

Affiliation: Howard Hughes Medical Institute, University of California School of Medicine, San Francisco, California 94143, USA.

ABSTRACT
De novo biosynthesis of amino acids uses intermediates provided by the TCA cycle that must be replenished by anaplerotic reactions to maintain the respiratory competency of the cell. Genome-wide expression analyses in Saccharomyces cerevisiae reveal that many of the genes involved in these reactions are repressed in the presence of the preferred nitrogen sources glutamine or glutamate. Expression of these genes in media containing urea or ammonia as a sole nitrogen source requires the heterodimeric bZip transcription factors Rtg1 and Rtg3 and correlates with a redistribution of the Rtg1p/Rtg3 complex from a predominantly cytoplasmic to a predominantly nuclear location. Nuclear import of the complex requires the cytoplasmic protein Rtg2, a previously identified upstream regulator of Rtg1 and Rtg3, whereas export requires the importin-beta-family member Msn5. Remarkably, nuclear accumulation of Rtg1/Rtg3, as well as expression of their target genes, is induced by addition of rapamycin, a specific inhibitor of the target of rapamycin (TOR) kinases. We demonstrate further that Rtg3 is a phosphoprotein and that its phosphorylation state changes after rapamycin treatment. Taken together, these results demonstrate that target of rapamycin signaling regulates specific anaplerotic reactions by coupling nitrogen quality to the activity and subcellular localization of distinct transcription factors.

Show MeSH