Upstream regulation of yeast TOR complexes

Nitrogen is an essential component of living organism. Protein synthesis and biosynthesis of nitrogen containing molecules essential for growth, such as amino acids and NAD, strictly depend on the availability of the nitrogen source. Limiting amount of nitrogen limit cell growth. Exogenous amino acids and other nitrogenous compounds such as ammonium constitute the nitrogen source. The budding yeast S. cerevisiae can sense and utilize a total of 21 different nitrogenous compounds to sustain growth. The uptake and utilization of different nitrogen sources is hierarchical and subject to strict and complex regulation at the transcriptional, translation and post-translational level. In general, preferred nitrogen sources inhibit the uptake and catabolism of non-preferred nitrogen sources.
The target of rapamycin (TOR) is a conserved Ser/Thr protein kinase among eukaryotes controlling growth in response to nutrients and growth factors. TOR is found in two essential conserved multiprotein complexes named TOR complex 1 (TORC1) and TORC2. In yeast, TORC1 signaling is sensitive to nutrients, particularly to availability of the nitrogen source. Thus, TORC1 by promoting anabolic processes, such as protein synthesis and ribosome biogenesis, couples growth to the availability of the nitrogen source.
The sensing mechanism activating TORC1 in response to nitrogen source sufficiency is only poorly understood. Recent studies identified the EGO complex as intermediary component of the amino acid sensing pathway. In the presence of amino acids, particularly leucine, the EGO complex is activated and promotes TORC1 activity. The small GTP binding proteins Gtr1 and Gtr2 are part of the EGO complex. Gtr1 and Gtr2 form a heterodimeric complex. In response to amino acids Gtr1 is loaded with GTP and Gtr2 with GDP, leading to activation of the EGO complex. Several factors determine EGO complex activation by dictating the Gtr1/2 guanosine loading. Vam6 was proposed to act as guanosine exchange factor (GEF) towards Gtr1. The Npr2/Npr3/Iml1 complex was reported to act as GTPase activating protein (GAP) for Gtr1. Therefore, amino acid stimulation of TORC1 is decreased in the absence of Vam6 and increased in the absence of Iml1. Several observations point out that EGO complex signaling, alone, is not sufficient to explain TORC1 activation by the nitrogen source. For instance, 1) components of the EGO complex signaling are not essential; 2) ammonium starvation down-regulates growth even in cells where EGO complex signaling is hyperactivated. Therefore, nitrogen source and amino acids sufficiency might signal to TORC1 via distinct mechanisms.
In this study we analyze the effect of different nitrogen sources on TORC1 activity. We use the phosphorylation state of the direct TORC1 target Sch9 as readout for TORC1 activity. We describe that preferred nitrogen sources activate TORC1 signaling stronger and better than non-preferred nitrogen source. TORC1 activation by preferred nitrogen sources is paralleled by an increase in glutamine synthesis and accumulation. Growth is increased in the presence of preferred nitrogen sources in a glutamine synthesis dependent way. Therefore, glutamine constitutes a metabolic input linking TORC1 activation in response to the quality of the nitrogen source to growth capacity. We find that EGO complex signaling is dispensable when a preferred nitrogen source is provided. TORC1 activation and growth increase are still induced in cells compromised for EGO complex signaling. Taken together, we demonstrate that nitrogen source and amino acid sufficiency act via discrete mechanisms to activate TORC1.