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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 after rapamycin treatment in a TOR1-dependent manner. (A) rtg1Δ (EY0733) or rtg3Δ (EY0735) cells expressing Rtg1-GFP or Rtg3-GFP, respectively, were treated with drug vehicle alone (left) or with 1 μg/ml of rapamycin (right) for 5 min, followed by examination by fluorescence microscopy. Pronounced nuclear accumulation of both Rtg1-GFP and Rtg3-GFP was observed in cells treated with rapamycin. (B) The experiment in A was repeated using cells that carried the dominant rapamycin resistant TOR1-1 allele.
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Figure 5: Rtg1 and Rtg3 are localized within the nucleus after rapamycin treatment in a TOR1-dependent manner. (A) rtg1Δ (EY0733) or rtg3Δ (EY0735) cells expressing Rtg1-GFP or Rtg3-GFP, respectively, were treated with drug vehicle alone (left) or with 1 μg/ml of rapamycin (right) for 5 min, followed by examination by fluorescence microscopy. Pronounced nuclear accumulation of both Rtg1-GFP and Rtg3-GFP was observed in cells treated with rapamycin. (B) The experiment in A was repeated using cells that carried the dominant rapamycin resistant TOR1-1 allele.

Mentions: Strains derived from K699 that were deleted for RTG1, RTG2, or RTG3 were made using the same PCR-based gene disruption technique described above, using the TRP1 gene of Candida glabrata as a selectable marker (Kitada et al. 1995). Primers used for PCR possessed 40 bases of homology that corresponded to the 5′ and 3′ ends of the open reading frame of each target gene. These strains, EY0733, EY0734, and EY0735, were transformed with an appropriate GFP fusion plasmid, described above, and used for the fluorescence microscopy experiments presented in Fig. 4, Fig. 5 A, and 7 (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 localized within the nucleus after rapamycin treatment in a TOR1-dependent manner. (A) rtg1Δ (EY0733) or rtg3Δ (EY0735) cells expressing Rtg1-GFP or Rtg3-GFP, respectively, were treated with drug vehicle alone (left) or with 1 μg/ml of rapamycin (right) for 5 min, followed by examination by fluorescence microscopy. Pronounced nuclear accumulation of both Rtg1-GFP and Rtg3-GFP was observed in cells treated with rapamycin. (B) The experiment in A was repeated using cells that carried the dominant rapamycin resistant TOR1-1 allele.
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Related In: Results  -  Collection

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Figure 5: Rtg1 and Rtg3 are localized within the nucleus after rapamycin treatment in a TOR1-dependent manner. (A) rtg1Δ (EY0733) or rtg3Δ (EY0735) cells expressing Rtg1-GFP or Rtg3-GFP, respectively, were treated with drug vehicle alone (left) or with 1 μg/ml of rapamycin (right) for 5 min, followed by examination by fluorescence microscopy. Pronounced nuclear accumulation of both Rtg1-GFP and Rtg3-GFP was observed in cells treated with rapamycin. (B) The experiment in A was repeated using cells that carried the dominant rapamycin resistant TOR1-1 allele.
Mentions: Strains derived from K699 that were deleted for RTG1, RTG2, or RTG3 were made using the same PCR-based gene disruption technique described above, using the TRP1 gene of Candida glabrata as a selectable marker (Kitada et al. 1995). Primers used for PCR possessed 40 bases of homology that corresponded to the 5′ and 3′ ends of the open reading frame of each target gene. These strains, EY0733, EY0734, and EY0735, were transformed with an appropriate GFP fusion plasmid, described above, and used for the fluorescence microscopy experiments presented in Fig. 4, Fig. 5 A, and 7 (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