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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 localized within the nucleus under glutamine-limiting conditions. rtg1Δ (EY0733) or rtg3Δ (EY0735) cells were transformed with pRtg1-GFP or pRtg3-GFP, respectively, and were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and examined by fluorescence microscopy. Punctate nuclear fluorescence was observed for both Rtg1-GFP and Rtg3-GFP in MD-urea. The nuclear disposition of this GFP-based fluorescence was confirmed by its colocalization with DAPI-stained nuclear DNA (data not shown).
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Figure 4: Rtg1 and Rtg3 are localized within the nucleus under glutamine-limiting conditions. rtg1Δ (EY0733) or rtg3Δ (EY0735) cells were transformed with pRtg1-GFP or pRtg3-GFP, respectively, and were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and examined by fluorescence microscopy. Punctate nuclear fluorescence was observed for both Rtg1-GFP and Rtg3-GFP in MD-urea. The nuclear disposition of this GFP-based fluorescence was confirmed by its colocalization with DAPI-stained nuclear DNA (data not shown).

Mentions: All strains of S. cerevisiae used in this study are listed in Table . The following culture media was used: YPD (1% yeast extract, 2% peptone, 2% dextrose); minimal dextrose (MD) (0.8% yeast nitrogen base without amino acids and ammonium sulfate, pH 5.5, 2% dextrose); synthetic complete dextrose (SCD) (0.7% yeast nitrogen base without amino acids, pH 5.5, 2% dextrose). MD media contained in addition one or more of the following nitrogen sources: glutamine, glutamate, ammonia, or urea, as indicated in the text, each at 0.2% final concentration. To supplement the auxotrophic requirements of strains used for the fluorescence microscopy experiment presented in Fig. 4 (below), required amino acids, adenine, and uracil were added to MD media at concentrations described by Sherman 1991. SCD media was also supplemented with appropriate amino acids, adenine and uracil as described by Sherman 1991. Yeast cultures were grown at 30°C for all experiments. Yeast transformations were performed using a DMSO-enhanced lithium acetate procedure (Hill et al. 1991). Rapamycin (Sigma-Aldrich) was dissolved in DMSO and added to a final concentration of 0.2 μg/ml unless stated otherwise.


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 localized within the nucleus under glutamine-limiting conditions. rtg1Δ (EY0733) or rtg3Δ (EY0735) cells were transformed with pRtg1-GFP or pRtg3-GFP, respectively, and were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and examined by fluorescence microscopy. Punctate nuclear fluorescence was observed for both Rtg1-GFP and Rtg3-GFP in MD-urea. The nuclear disposition of this GFP-based fluorescence was confirmed by its colocalization with DAPI-stained nuclear DNA (data not shown).
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Rtg1 and Rtg3 are localized within the nucleus under glutamine-limiting conditions. rtg1Δ (EY0733) or rtg3Δ (EY0735) cells were transformed with pRtg1-GFP or pRtg3-GFP, respectively, and were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and examined by fluorescence microscopy. Punctate nuclear fluorescence was observed for both Rtg1-GFP and Rtg3-GFP in MD-urea. The nuclear disposition of this GFP-based fluorescence was confirmed by its colocalization with DAPI-stained nuclear DNA (data not shown).
Mentions: All strains of S. cerevisiae used in this study are listed in Table . The following culture media was used: YPD (1% yeast extract, 2% peptone, 2% dextrose); minimal dextrose (MD) (0.8% yeast nitrogen base without amino acids and ammonium sulfate, pH 5.5, 2% dextrose); synthetic complete dextrose (SCD) (0.7% yeast nitrogen base without amino acids, pH 5.5, 2% dextrose). MD media contained in addition one or more of the following nitrogen sources: glutamine, glutamate, ammonia, or urea, as indicated in the text, each at 0.2% final concentration. To supplement the auxotrophic requirements of strains used for the fluorescence microscopy experiment presented in Fig. 4 (below), required amino acids, adenine, and uracil were added to MD media at concentrations described by Sherman 1991. SCD media was also supplemented with appropriate amino acids, adenine and uracil as described by Sherman 1991. Yeast cultures were grown at 30°C for all experiments. Yeast transformations were performed using a DMSO-enhanced lithium acetate procedure (Hill et al. 1991). Rapamycin (Sigma-Aldrich) was dissolved in DMSO and added to a final concentration of 0.2 μg/ml unless stated otherwise.

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
Related in: MedlinePlus