RESUMO
The GATOR1 (SEACIT) complex consisting of Iml1-Npr2-Npr3 inhibits target of rapamycin complex 1 (TORC1) in response to amino acid insufficiency. In glucose medium, Saccharomyces cerevisiae mutants lacking the function of this complex grow poorly in the absence of amino acid supplementation, despite showing hallmarks of increased TORC1 signaling. Such mutants sense that they are amino acid replete and thus repress metabolic activities that are important for achieving this state. We found that npr2Δ mutants have defective mitochondrial tricarboxylic acid (TCA)-cycle activity and retrograde response. Supplementation with glutamine, and especially aspartate, which are nitrogen-containing forms of TCA-cycle intermediates, rescues growth of npr2Δ mutants. These amino acids are then consumed in biosynthetic pathways that require nitrogen to support proliferative metabolism. Our findings revealed that negative regulators of TORC1, such as GATOR1 (SEACIT), regulate the cataplerotic synthesis of these amino acids from the TCA cycle, in tune with the amino acid and nitrogen status of cells.
Assuntos
Ciclo do Ácido Cítrico , Mitocôndrias/metabolismo , Complexos Multiproteicos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Ácido Aspártico/metabolismo , Glutamina/metabolismo , Complexos Multiproteicos/genética , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Nitrogênio/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Proteínas de Saccharomyces cerevisiae/genética , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Cells must be capable of switching between growth and autophagy in unpredictable nutrient environments. The conserved Npr2 protein complex (comprising Iml1, Npr2, and Npr3; also called SEACIT) inhibits target of rapamycin complex 1 (TORC1) kinase signaling, which inhibits autophagy in nutrient-rich conditions. In yeast cultured in media with nutrient limitations that promote autophagy and inhibit growth, loss of Npr2 enables cells to bypass autophagy and proliferate. We determined that Npr2-deficient yeast had a metabolic state distinct from that of wild-type yeast when grown in minimal media containing ammonium as a nitrogen source and a nonfermentable carbon source (lactate). Unlike wild-type yeast, which accumulated glutamine, Npr2-deficient yeast metabolized glutamine into nitrogen-containing metabolites and maintained a high concentration of S-adenosyl methionine (SAM). Moreover, in wild-type yeast grown in these nutrient-limited conditions, supplementation with methionine stimulated glutamine consumption for synthesis of nitrogenous metabolites, demonstrating integration of a sulfur-containing amino acid cue and nitrogen utilization. These data revealed the metabolic basis by which the Npr2 complex regulates cellular homeostasis and demonstrated a key function for TORC1 in regulating the synthesis and utilization of glutamine as a nitrogen source.