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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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Glutamine is both a global activator and repressor of gene expression. Scatter plots show pairwise comparisons of gene expression profiles of S288c cells grown in the presence of glutamine, urea, or glutamine + urea. (A) MD-glutamine versus MD-urea. (B) MD-urea versus MD-glutamine + urea. (C) MD-glutamine versus MD-glutamine + urea. (D) Control experiment comparing MD-glutamine with itself. For each plot, the x axis depicts cDNA samples labeled with Cy5 dye and the y axis depicts samples labeled with Cy3 dye.
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Figure 2: Glutamine is both a global activator and repressor of gene expression. Scatter plots show pairwise comparisons of gene expression profiles of S288c cells grown in the presence of glutamine, urea, or glutamine + urea. (A) MD-glutamine versus MD-urea. (B) MD-urea versus MD-glutamine + urea. (C) MD-glutamine versus MD-glutamine + urea. (D) Control experiment comparing MD-glutamine with itself. For each plot, the x axis depicts cDNA samples labeled with Cy5 dye and the y axis depicts samples labeled with Cy3 dye.

Mentions: Strain S288c was grown with vigorous shaking to 0.5 OD600/ml in 1 liter of MD media containing appropriate nitrogen sources, as indicated in the text. Cells were immediately harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80°C. Relative mRNA levels were determined by hybridizing fluorescently labeled cDNAs to microarrays containing cDNAs representing virtually every yeast open reading frame (DeRisi et al. 1997; Lashkari et al. 1997). Arrays were produced under the auspices of J. Derisi at UCSF by a consortium of laboratories affiliated with the Department of Biochemistry and Biophysics. Primers for amplification of the yeast genome were purchased from Research Genetics and were provided by the laboratory of P. Walter. Arrays were scanned using a GenePix 4000a Microarray Scanner (Axon Instruments, Inc.) and analyzed using software provided by the manufacturer. Data were also analyzed using software available through the web site affiliated with the laboratories of P. Brown and D. Botstein at Stanford University (Stanford, CA; http://rana.Stanford.EDU/software/). Scatterplots shown in Fig. 2 (below) were constructed using Excel software (Microsoft Corp.). The complete data set for the nitrogen source experiments presented here will be made available upon request.


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)

Glutamine is both a global activator and repressor of gene expression. Scatter plots show pairwise comparisons of gene expression profiles of S288c cells grown in the presence of glutamine, urea, or glutamine + urea. (A) MD-glutamine versus MD-urea. (B) MD-urea versus MD-glutamine + urea. (C) MD-glutamine versus MD-glutamine + urea. (D) Control experiment comparing MD-glutamine with itself. For each plot, the x axis depicts cDNA samples labeled with Cy5 dye and the y axis depicts samples labeled with Cy3 dye.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169436&req=5

Figure 2: Glutamine is both a global activator and repressor of gene expression. Scatter plots show pairwise comparisons of gene expression profiles of S288c cells grown in the presence of glutamine, urea, or glutamine + urea. (A) MD-glutamine versus MD-urea. (B) MD-urea versus MD-glutamine + urea. (C) MD-glutamine versus MD-glutamine + urea. (D) Control experiment comparing MD-glutamine with itself. For each plot, the x axis depicts cDNA samples labeled with Cy5 dye and the y axis depicts samples labeled with Cy3 dye.
Mentions: Strain S288c was grown with vigorous shaking to 0.5 OD600/ml in 1 liter of MD media containing appropriate nitrogen sources, as indicated in the text. Cells were immediately harvested by centrifugation, flash frozen in liquid nitrogen, and stored at −80°C. Relative mRNA levels were determined by hybridizing fluorescently labeled cDNAs to microarrays containing cDNAs representing virtually every yeast open reading frame (DeRisi et al. 1997; Lashkari et al. 1997). Arrays were produced under the auspices of J. Derisi at UCSF by a consortium of laboratories affiliated with the Department of Biochemistry and Biophysics. Primers for amplification of the yeast genome were purchased from Research Genetics and were provided by the laboratory of P. Walter. Arrays were scanned using a GenePix 4000a Microarray Scanner (Axon Instruments, Inc.) and analyzed using software provided by the manufacturer. Data were also analyzed using software available through the web site affiliated with the laboratories of P. Brown and D. Botstein at Stanford University (Stanford, CA; http://rana.Stanford.EDU/software/). Scatterplots shown in Fig. 2 (below) were constructed using Excel software (Microsoft Corp.). The complete data set for the nitrogen source experiments presented here will be made available upon request.

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