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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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Related in: MedlinePlus

Examples of differences in gene expression during growth of yeast cells in the presence of different sources of assimilable nitrogen. Strain S288c was grown to mid-log phase in MD media containing the indicated nitrogen sources. Total mRNA was isolated and Northern blot analysis was performed, probing for the specified mRNAs. (A) Control transcripts showing no significant differences under the conditions tested. (B) Transcripts displaying similar levels of expression in MD-glutamine and MD-glutamate. (C) Transcripts displaying more complex patterns of expression.
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Figure 1: Examples of differences in gene expression during growth of yeast cells in the presence of different sources of assimilable nitrogen. Strain S288c was grown to mid-log phase in MD media containing the indicated nitrogen sources. Total mRNA was isolated and Northern blot analysis was performed, probing for the specified mRNAs. (A) Control transcripts showing no significant differences under the conditions tested. (B) Transcripts displaying similar levels of expression in MD-glutamine and MD-glutamate. (C) Transcripts displaying more complex patterns of expression.

Mentions: Of the more than 6,200 genes examined, a surprisingly small number (<40) displayed differences in expression threefold or greater under these two nitrogen conditions; 12 were expressed preferentially in MD-glutamine and 24 were expressed preferentially in MD-urea (Table ). We confirmed these results for a representative number of genes directly by Northern blot analysis (Fig. 1). The majority of genes that were expressed better in MD-glutamine encoded permeases specific for amino acids associated with rich nutrient conditions, including GNP1 and TAT2, which encode high affinity permeases specific for glutamine and tryptophan, respectively (Zhu et al. 1996; Beck et al. 1999). In contrast, genes expressed preferentially in MD-urea could be grouped into one of three general classes: (a) transport permeases specific for poor nitrogen conditions, including DUR3, which encodes urea permease; (b) the majority of the DAL genes, which are involved in the uptake and catabolism of allantoin (Cooper et al. 1979; Cox et al. 1999); and (c) metabolic enzymes, including several associated with the citric acid and glyoxylate cycles (Table ). Given the relatively small number of genes identified in this experiment, these results suggest that a limited number of regulatory pathways are likely to be involved in the differential use of these two nitrogen sources.


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)

Examples of differences in gene expression during growth of yeast cells in the presence of different sources of assimilable nitrogen. Strain S288c was grown to mid-log phase in MD media containing the indicated nitrogen sources. Total mRNA was isolated and Northern blot analysis was performed, probing for the specified mRNAs. (A) Control transcripts showing no significant differences under the conditions tested. (B) Transcripts displaying similar levels of expression in MD-glutamine and MD-glutamate. (C) Transcripts displaying more complex patterns of expression.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 1: Examples of differences in gene expression during growth of yeast cells in the presence of different sources of assimilable nitrogen. Strain S288c was grown to mid-log phase in MD media containing the indicated nitrogen sources. Total mRNA was isolated and Northern blot analysis was performed, probing for the specified mRNAs. (A) Control transcripts showing no significant differences under the conditions tested. (B) Transcripts displaying similar levels of expression in MD-glutamine and MD-glutamate. (C) Transcripts displaying more complex patterns of expression.
Mentions: Of the more than 6,200 genes examined, a surprisingly small number (<40) displayed differences in expression threefold or greater under these two nitrogen conditions; 12 were expressed preferentially in MD-glutamine and 24 were expressed preferentially in MD-urea (Table ). We confirmed these results for a representative number of genes directly by Northern blot analysis (Fig. 1). The majority of genes that were expressed better in MD-glutamine encoded permeases specific for amino acids associated with rich nutrient conditions, including GNP1 and TAT2, which encode high affinity permeases specific for glutamine and tryptophan, respectively (Zhu et al. 1996; Beck et al. 1999). In contrast, genes expressed preferentially in MD-urea could be grouped into one of three general classes: (a) transport permeases specific for poor nitrogen conditions, including DUR3, which encodes urea permease; (b) the majority of the DAL genes, which are involved in the uptake and catabolism of allantoin (Cooper et al. 1979; Cox et al. 1999); and (c) metabolic enzymes, including several associated with the citric acid and glyoxylate cycles (Table ). Given the relatively small number of genes identified in this experiment, these results suggest that a limited number of regulatory pathways are likely to be involved in the differential use of these two nitrogen sources.

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