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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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(A) Msn5 is required for export of Rtg1 and Rtg3 from the nucleus. (A) msn5Δ (EY0736) and msn5Δ rtg2Δ (EY0744) cells were transformed with pRtg1-GFP (top) or pRtg3-GFP (bottom) and grown to 0.5 OD600/ml in SCD media lacking uracil and were examined by fluorescence microscopy. Rtg1-GFP and Rtg3-GFP were localized constitutively within the nucleus in msn5Δ cells but not in msn5Δ rtg2Δ cells. (B) RTG-dependent target genes remain responsive to TOR signaling in msn5Δ cells. Wild-type (K699) and msn5Δ (EY0736) cells were grown in YPD until 0.5 OD600/ml and were treated either with drug vehicle (lanes 1 and 3) or with rapamycin (lanes 2 and 4) for 30 min (this time of incubation corresponded to the peak induction of RTG-dependent gene expression observed in the rapamycin time course in Fig. 6). Cells were then harvested and RNA was prepared and analyzed by Northern blotting, probing for the specified mRNAs.
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Figure 8: (A) Msn5 is required for export of Rtg1 and Rtg3 from the nucleus. (A) msn5Δ (EY0736) and msn5Δ rtg2Δ (EY0744) cells were transformed with pRtg1-GFP (top) or pRtg3-GFP (bottom) and grown to 0.5 OD600/ml in SCD media lacking uracil and were examined by fluorescence microscopy. Rtg1-GFP and Rtg3-GFP were localized constitutively within the nucleus in msn5Δ cells but not in msn5Δ rtg2Δ cells. (B) RTG-dependent target genes remain responsive to TOR signaling in msn5Δ cells. Wild-type (K699) and msn5Δ (EY0736) cells were grown in YPD until 0.5 OD600/ml and were treated either with drug vehicle (lanes 1 and 3) or with rapamycin (lanes 2 and 4) for 30 min (this time of incubation corresponded to the peak induction of RTG-dependent gene expression observed in the rapamycin time course in Fig. 6). Cells were then harvested and RNA was prepared and analyzed by Northern blotting, probing for the specified mRNAs.

Mentions: MSN5 was deleted from K699 using a HIS3-marked disruption vector, as described previously (Kaffman et al. 1998a). The rtg2Δ msn5Δ strain was made by mating the rtg2Δ and msn5Δ strains described above and selecting TRP+ HIS+ segregants after sporulation and tetrad dissection. The resulting msn5Δ and rtg2Δ msn5Δ strains, EY0736 and EY0744, respectively, were used for experiments presented in Fig. 8 (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)

(A) Msn5 is required for export of Rtg1 and Rtg3 from the nucleus. (A) msn5Δ (EY0736) and msn5Δ rtg2Δ (EY0744) cells were transformed with pRtg1-GFP (top) or pRtg3-GFP (bottom) and grown to 0.5 OD600/ml in SCD media lacking uracil and were examined by fluorescence microscopy. Rtg1-GFP and Rtg3-GFP were localized constitutively within the nucleus in msn5Δ cells but not in msn5Δ rtg2Δ cells. (B) RTG-dependent target genes remain responsive to TOR signaling in msn5Δ cells. Wild-type (K699) and msn5Δ (EY0736) cells were grown in YPD until 0.5 OD600/ml and were treated either with drug vehicle (lanes 1 and 3) or with rapamycin (lanes 2 and 4) for 30 min (this time of incubation corresponded to the peak induction of RTG-dependent gene expression observed in the rapamycin time course in Fig. 6). Cells were then harvested and RNA was prepared and analyzed by Northern blotting, probing for the specified mRNAs.
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Related In: Results  -  Collection

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

Figure 8: (A) Msn5 is required for export of Rtg1 and Rtg3 from the nucleus. (A) msn5Δ (EY0736) and msn5Δ rtg2Δ (EY0744) cells were transformed with pRtg1-GFP (top) or pRtg3-GFP (bottom) and grown to 0.5 OD600/ml in SCD media lacking uracil and were examined by fluorescence microscopy. Rtg1-GFP and Rtg3-GFP were localized constitutively within the nucleus in msn5Δ cells but not in msn5Δ rtg2Δ cells. (B) RTG-dependent target genes remain responsive to TOR signaling in msn5Δ cells. Wild-type (K699) and msn5Δ (EY0736) cells were grown in YPD until 0.5 OD600/ml and were treated either with drug vehicle (lanes 1 and 3) or with rapamycin (lanes 2 and 4) for 30 min (this time of incubation corresponded to the peak induction of RTG-dependent gene expression observed in the rapamycin time course in Fig. 6). Cells were then harvested and RNA was prepared and analyzed by Northern blotting, probing for the specified mRNAs.
Mentions: MSN5 was deleted from K699 using a HIS3-marked disruption vector, as described previously (Kaffman et al. 1998a). The rtg2Δ msn5Δ strain was made by mating the rtg2Δ and msn5Δ strains described above and selecting TRP+ HIS+ segregants after sporulation and tetrad dissection. The resulting msn5Δ and rtg2Δ msn5Δ strains, EY0736 and EY0744, respectively, were used for experiments presented in Fig. 8 (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
Related in: MedlinePlus