The structurally and functionally conserved TOR pathway has for a long time been suggested to play a role in the regulation of cell growth and many related properties by nutrient availability (Fig. 4). However, no clear mechanisms have been identified by which the TOR pathway would detect extracellular nutrients and the more recent work suggests that the TOR proteins rather sense intracellular nitrogen availability, in particular mobilization of nitrogen reserves from the vacuole/lysosome. From yeast to humans, inactivation of TOR affects multiple processes involved in cell metabolism, growth, and longevity (most recently reviewed by Kim & Guan, 2011; Loewith & Hall, 2011). Quantitative phosphoproteomics revealed that mammalian TOR controls the phosphorylation of 335 proteins (Robitaille et al., 2013). Saccharomyces cerevisiae differs from almost all other eukaryotes by possessing two TOR genes instead of one (Helliwell et al., 1994). Tor1 and Tor2 are 282 kDa in size, 67% identical and also highly similar in sequence to the mammalian TOR protein (37%). They are also the founding members of the family of phosphatidylinositol protein kinases (or phosphatidyl inositol 3′ kinase-related kinases, PIKK; Keith & Schreiber, 1995). Although they contain a catalytic domain resembling that of lipid kinases (PI3K and PI4K), no PIKK has shown lipid kinase activity. All Tor proteins have the same essential features: From N- to C-terminus, they contain the HEAT (Huntington, elongation factor 3, regulatory subunit A of PP2A, TOR1) repeats, the FAT (FRAP, ATM, TTRAP) domain, the FRB (FKBP12-rapamycin-binding) domain, the kinase domain, and the FATC (FAT C-terminus) domain (Schmelzle & Hall, 2000). The HEAT repeats are the binding region for subunits of the TOR complexes (Wullschleger et al., 2005). The central and C-terminal FAT domains are conserved in PIKK (Dames et al., 2005). The FRB domain is responsible for binding to FKBP (FK506 binding protein)-rapamycin (Loewith & Hall, 2011). Tor proteins act in complex with different protein subsets, which provides functional versatility (Helliwell et al., 1994). TOR complex 1 (TORC1) consists of either Tor1 or Tor2, associated with Kog1, Lst8, and Tco89 (Loewith et al., 2002; Wedaman et al., 2003; Reinke et al., 2004). TORC1 is rapamycin sensitive, and its inactivation affects protein synthesis, ribosome biogenesis, transcription, cell cycle, meiosis, nutrient uptake, and autophagy. TORC2 complex contains exclusively Tor2, associated with the subunits Avo1-3, Bit61, and Lst8 (Loewith et al., 2002; Wedaman et al., 2003; Reinke et al., 2004). TORC2 is rapamycin insensitive and affects actin cytoskeleton organization, endocytosis, lipid synthesis, and cell survival. The mechanisms by which the immunosuppressant lipid macrolide rapamycin inhibits TORC1, but not TORC2, are now starting to be understood. Rapamycin hijacks the cytosolic peptidyl-prolyl cis-trans isomerase, also known as immunophilin, FKBP12, or its yeast homolog, Fpr1 (FK506-binding protein 12; Schreiber, 1991). This Fpr1 association with rapamycin causes Fpr1 to interact with TOR resulting in its inhibition. But FKBP-rapamycin can only bind TORC1, apparently because in TORC2 the FRB domain, to which it binds, is protected by Avo1 (Loewith et al., 2002; Wullschleger et al., 2005). This lack of interaction between Fpr1-rapamycin and TORC2 accounts for the previously observed insensitivity of TORC2 to rapamycin.
Figure 4. Role of TORC1 in the NCR and RTG pathways. (a) Preferred nitrogen sources for yeast are these that can easily be converted into glutamate (Glu) and glutamine (Gln), major precursors for amino acid biosynthesis. Their presence in the medium results in increased levels of intracellular glutamate and glutamine. This causes repression of genes involved in the metabolism of less preferred nitrogen sources, nitrogen catabolite repression (NCR). This transcriptional repression is achieved mainly by hyperphosphorylation of Ure2 and Gln3, causing their association and preventing nuclear localization of the transcription factor Gln3. The Gat1 transcription factor is regulated in a similar way. High glutamine levels as well as other amino acids stimulate the vacuolar/endosome membrane-located, EGO complex. This complex is composed of the two Ras-like GTPases, Gtr1, Gtr2, and the Ragulator-like, Ego3 and Ego1. Activation of EGO is stimulated by GTP-bound Gtr1 and GDP-bound Gtr2. GTP loading of Gtr1 is stimulated by the guanine nucleotide-exchange factor (GEF) activities of Vam6/Vps39 and the L-Leu-tRNA synthetase. SEACAT prevents GAP activity of SEACIT on Gtr1. Activated EGO stimulates in turn the vacuolar membrane-associated fraction of the TORC1 complex. TORC1 also phosphorylates Sch9 and Tap42, the latter leading to the inhibition of several protein phosphatases (PPA2, Sit4, etc.). As a result, the protein phosphatases can no longer dephosphorylate the Ure2 complexes with Gln3 and Gat1, reinforcing their hyperphosphorylation. Synthesis of glutamine and glutamate occurring via anaplerotic reactions shared with the TCA cycle is also downregulated. In this case, TORC1 phosphorylated Mks1 bound to Bmh1,2 proteins prevents nuclear localization of the RTG transcription factors, Rtg1 and Rtg3. TORC1-dependent phosphorylation of Npr1 causes Npr1 inactivation, which in a yet not completely understood manner increases plasma membrane stabilization of specific AAPs like Tat2, while stimulating endocytosis of the alternative general AAP, Gap1. (b) Under poor nitrogen conditions, intracellular glutamate and glutamine levels drop. GAPs like the SEACIT increase GDP loading of Gtr1, which inactivates the EGO complex. An inactive EGO complex can no longer stimulate TORC1, which leads to release into the cytosol and activation of Tap42–protein phosphatase complexes. They reduce phosphorylation of Ure2, Gln3, and Gat1 causing nuclear localization of the latter two and subsequent stimulation of NCR gene expression. The phosphatases also dephosphorylate Mks1, which then complexes with Rtg2. This allows Rtg1,3 nuclear localization resulting in stimulation of the expression of RTG genes, sustaining amino acid biosynthesis through the synthesis of glutamate and glutamine. The phosphatases also dephosphorylate Npr1, which then phosphorylates the Rsp5-associated arrestins Bul1 and Bul2 provoking their association with Bmh1/2 proteins, which in turn leads to the stabilization of Gap1 at the plasma membrane. Metabolic reactions are depicted by dotted arrows; regulatory and signaling interactions by full arrows.
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