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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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Rtg3 is a phosphoprotein and is differentially phosphorylated after rapamycin treatment. (A) Cells expressing Rtg1-HA3 (PLY047), Rtg2-HA3 (PLY089), and Rtg3-HA3 (PLY050) were grown to 0.5 OD600/ml in YPD and were treated either with drug vehicle alone or with rapamycin for 15 min. Extracts were prepared and Western blot analysis was performed using anti–HA monoclonal antibodies to detect each protein. No change in the abundance or relative mobility of Rtg1 or Rtg2 could be detected after rapamycin treatment. In contrast, a portion of Rtg3 showed an increased mobility (arrowhead) after rapamycin treatment, compared with its mobility in the absence of rapamycin (*). (B) Wild-type (K699) and rtg2Δ (EY0734) cells transformed with pRtg3-zz and were grown to 0.5 OD600/ml in SCD media lacking uracil. Cells were then treated with drug vehicle or with rapamycin for 15 min. Extracts were prepared and Rtg3-zz was immunoprecipitated with IgG-Sepharose and either mock-treated or treated with phosphatase before Western blot analysis, as indicated. Increased mobility of a portion of Rtg3-zz after rapamcyin treatment is indicated (arrowhead). (C) Wild-type (K699) cells carrying pRtg3-zz were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and processed as in B. For each experiment in A–C, all samples were from the same gel. Identical results were obtained in three separate experiments.
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Figure 9: Rtg3 is a phosphoprotein and is differentially phosphorylated after rapamycin treatment. (A) Cells expressing Rtg1-HA3 (PLY047), Rtg2-HA3 (PLY089), and Rtg3-HA3 (PLY050) were grown to 0.5 OD600/ml in YPD and were treated either with drug vehicle alone or with rapamycin for 15 min. Extracts were prepared and Western blot analysis was performed using anti–HA monoclonal antibodies to detect each protein. No change in the abundance or relative mobility of Rtg1 or Rtg2 could be detected after rapamycin treatment. In contrast, a portion of Rtg3 showed an increased mobility (arrowhead) after rapamycin treatment, compared with its mobility in the absence of rapamycin (*). (B) Wild-type (K699) and rtg2Δ (EY0734) cells transformed with pRtg3-zz and were grown to 0.5 OD600/ml in SCD media lacking uracil. Cells were then treated with drug vehicle or with rapamycin for 15 min. Extracts were prepared and Rtg3-zz was immunoprecipitated with IgG-Sepharose and either mock-treated or treated with phosphatase before Western blot analysis, as indicated. Increased mobility of a portion of Rtg3-zz after rapamcyin treatment is indicated (arrowhead). (C) Wild-type (K699) cells carrying pRtg3-zz were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and processed as in B. For each experiment in A–C, all samples were from the same gel. Identical results were obtained in three separate experiments.

Mentions: Strains derived from DBY8943 that produced versions of Rtg1-Rtg3 tagged at their COOH termini with three copies of the hemagglutinin (HA) epitope were constructed using the PCR-based method described by Brachmann et al. 1998 and Longtine et al. 1998. As template for PCR, we used the plasmid pFA6a-3HA-HIS3MX6 that contained the Schizosaccharomyces pombe HIS3 homologue (Longtine et al. 1998). The tagged genes were determined to be functional based on the normal growth of each resulting strain, PLY047, PLY050, and PLY089, on MD–ammonia and –urea agar plates. These strains were used for the experiment presented in Fig. 9 A (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)

Rtg3 is a phosphoprotein and is differentially phosphorylated after rapamycin treatment. (A) Cells expressing Rtg1-HA3 (PLY047), Rtg2-HA3 (PLY089), and Rtg3-HA3 (PLY050) were grown to 0.5 OD600/ml in YPD and were treated either with drug vehicle alone or with rapamycin for 15 min. Extracts were prepared and Western blot analysis was performed using anti–HA monoclonal antibodies to detect each protein. No change in the abundance or relative mobility of Rtg1 or Rtg2 could be detected after rapamycin treatment. In contrast, a portion of Rtg3 showed an increased mobility (arrowhead) after rapamycin treatment, compared with its mobility in the absence of rapamycin (*). (B) Wild-type (K699) and rtg2Δ (EY0734) cells transformed with pRtg3-zz and were grown to 0.5 OD600/ml in SCD media lacking uracil. Cells were then treated with drug vehicle or with rapamycin for 15 min. Extracts were prepared and Rtg3-zz was immunoprecipitated with IgG-Sepharose and either mock-treated or treated with phosphatase before Western blot analysis, as indicated. Increased mobility of a portion of Rtg3-zz after rapamcyin treatment is indicated (arrowhead). (C) Wild-type (K699) cells carrying pRtg3-zz were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and processed as in B. For each experiment in A–C, all samples were from the same gel. Identical results were obtained in three separate experiments.
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Figure 9: Rtg3 is a phosphoprotein and is differentially phosphorylated after rapamycin treatment. (A) Cells expressing Rtg1-HA3 (PLY047), Rtg2-HA3 (PLY089), and Rtg3-HA3 (PLY050) were grown to 0.5 OD600/ml in YPD and were treated either with drug vehicle alone or with rapamycin for 15 min. Extracts were prepared and Western blot analysis was performed using anti–HA monoclonal antibodies to detect each protein. No change in the abundance or relative mobility of Rtg1 or Rtg2 could be detected after rapamycin treatment. In contrast, a portion of Rtg3 showed an increased mobility (arrowhead) after rapamycin treatment, compared with its mobility in the absence of rapamycin (*). (B) Wild-type (K699) and rtg2Δ (EY0734) cells transformed with pRtg3-zz and were grown to 0.5 OD600/ml in SCD media lacking uracil. Cells were then treated with drug vehicle or with rapamycin for 15 min. Extracts were prepared and Rtg3-zz was immunoprecipitated with IgG-Sepharose and either mock-treated or treated with phosphatase before Western blot analysis, as indicated. Increased mobility of a portion of Rtg3-zz after rapamcyin treatment is indicated (arrowhead). (C) Wild-type (K699) cells carrying pRtg3-zz were grown to 0.5 OD600/ml in MD-glutamine or MD-urea and processed as in B. For each experiment in A–C, all samples were from the same gel. Identical results were obtained in three separate experiments.
Mentions: Strains derived from DBY8943 that produced versions of Rtg1-Rtg3 tagged at their COOH termini with three copies of the hemagglutinin (HA) epitope were constructed using the PCR-based method described by Brachmann et al. 1998 and Longtine et al. 1998. As template for PCR, we used the plasmid pFA6a-3HA-HIS3MX6 that contained the Schizosaccharomyces pombe HIS3 homologue (Longtine et al. 1998). The tagged genes were determined to be functional based on the normal growth of each resulting strain, PLY047, PLY050, and PLY089, on MD–ammonia and –urea agar plates. These strains were used for the experiment presented in Fig. 9 A (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