TOR Signaling in Budding Yeast

3.2.1. Rag GTPases (Gtr1 and Gtr2)

When cells are exposed to conditions that are unfavorable for growth, they cease division and remodel cellular metabolism and gene expression profiles to survive under these stressful conditions. The treatment of yeast cells with rapamycin causes multiple phenomena resembling those occurring in cells starved of nutrients, particularly amino acids. Therefore, one of the physiological cues for the activation of TORC1 signaling may be amino acid(s). Upstream module(s) that communicate with TORC1 were revealed by a genetic approach using S. cerevisiae. Mutants that are unable to recover cell growth when transferred from nutrient-depleted conditions to nutrient-rich conditions are expected to be defective in module(s) that communicate with TORC1. Since rapamycin mimics amino acid-starved conditions, mutants with the ability to recover from rapamycin-induced growth arrest were screened. EGO(Exit from rapamycin-induced GrOwth arrest) mutants were identified, in which the Ras-related GTPase (Rag) Gtr2 and the vacuolar membrane-associated proteins Ego1 and Ego3 were included [30]. Another study revealed that modules involved in the trafficking of the general amino acid permease to the cytoplasmic membrane were Gtr1, Gtr2, Ego1, Ego3, and Ltv1 [31].

Gtr1 and Gtr2 belong to the Rag family. Orthologs of Gtr1 and Gtr2 in mammalian cells are RagA/RagB for Gtr1 and RagC/RagD for Gtr2. Amino acid-sequence similarities between RagA and RagB (90% identity) and between RagA/RagB and Gtr1 (48%) are high. This is also the case between RagC and RagD (81%) and between RagC/RagD and Gtr2 (46%). However, amino acid-sequence similarities between RagA/RagB and RagC/RagD and between Gtr1 and Gtr2 are low (approximately <25%) [32, 33, 34]. Rag GTPases function as heterodimers that are formed by a combination of one monomer of either RagA or RagB and one monomer of either RagC or RagD [33]. Similarly, Gtr1 and Gtr2 form a heterodimer [34]. Heterodimers with GTP-bound RagA/RagB and GDP-bound RagC/RagD exhibit full activity. This is also the case for S. cerevisiaeRag GTPase, that is, Gtr1^GTP and Gtr2^GDP are a dynamic combination that activate TORC1 in yeast (Figure 2).

3.2.2. EGO complex (ragulator)

Small GTPases are generally lipid-linked proteins, and lipid modifications enable these proteins to anchor to biological membranes. However, neither Gtr1 nor Gtr2 is modified by lipids. Ego1, Ego3, and Ego2 were recently found to form the EGO complex, which serves as a scaffold for the Gtr1-Gtr2 heterodimer to anchor to the vacuolar membrane in order to activate TORC1 in response to amino acids in S. cerevisiae[35]. Ego1 is a myristoylated and palmitoylated protein that is anchored to the vacuolar membrane through such lipids [36, 37, 38, 39]. Ego2 and Ego3 bind to vacuolar membrane-anchored Ego1 [35].

Mammalian cells also contain a large protein complex that functions together with the heterodimeric Rag GTPase, designated “Ragulator” (Rag regulator). Ragulator consists of five subunits, that is, LAMTOR1-5 (LAMTOR, Lysosomal Adaptor, and Mitogen-activated protein kinase (MAPK), and mTOR). Ragulator is anchored to lysosomal membranes through lipid-modified LAMTOR1 [40]; therefore, LAMTOR1 may be a functional homolog of Ego1. LAMTOR 2 and LAMTOR3 form a heterodimer each with a monomer protein, which are structurally and functionally homologous to Ego3 [41]. LAMTOR4 and LAMTOR5 show high structural similarities with Ego2 and Ego4, a paralog of Ego2 [35, 42]. Ragulator and heterodimeric Rag GTPases, which consist of GTP-bound RagA/RgB and GDP-bound RagC/RagD, communicate the signal of an amino acid sufficiency to mTORC1 on lysosomal membranes in mammalian cells (Figure 2).

3.2.3. GEF and GAP for Rag GTPases

The activities of small GTPases are generally regulated by the status of the guanine nucleotide loaded, which is controlled by the guanine nucleotide exchange factor (GEF) and GTPase-activating protein (GAP). Gtr1-activating factors were screened using a genetic approach, and, as a result, Vam6 (also known as Vps39) was obtained [36]. Vam6/Vps39 exhibited Gtr1 GEF activity in vitro[36].

On the other hand, a breakthrough regarding the regulation of Gtr2 was achieved by the discovery that Folliculin (FLCN) tumor suppressor functioned as a positive regulator of RagC/RagD [43, 44]. FLCN forms a complex with either FNIP1 or FNIP2 and has the ability to recruit mTORC1 to lysosomal membranes in response to an amino acid stimulation, thereby activating mTORC1. FLCN-FNIP1/2 complexes are GAPs toward RagC/RagD. A similar mechanism was conserved in yeast, that is, Lst7 and Lst4 are counterparts of FLCN and FNIP1/2, respectively [45]. Lst4 and Lst7 form a stable complex, both of which are necessary for the activation of TORC1 in the presence of amino acids. The Lst4-Lst7 complex preferentially binds to Gtr2^GTP in order to enhance the hydrolytic activity of its GTPase activity, thereby yielding Gtr2^GDP to activate TORC1 in yeast upon an amino acid stimulation (Figure 2).

3.2.4. SEACIT and SEACAT (GATOR1 and GATOR2)

What is an upstream regulator(s) of Rag GTPases? In order to solve this question, genome-wide screening was conducted in yeast to discover negative effectors of TORC1 activity, and, as a result, Npr2 and Npr3 were identified [46]. Npr2 and Npr3 form a heterodimer [46]. The coatomer-related Seh1-associated complex (SEAC) that associates with vacuolar membranes was implicated in responses to nitrogen starvation [47, 48, 49]. Npr2 and Npr3 together with Iml1/Sea1 form a SEAC subcomplex, which negatively regulates Gtr1 within the EGO complex [50]. A biochemical analysis revealed that Iml1/Sea1 exhibited GAP activity toward Gtr1 in vitro. Npr2 is phosphorylated by an unknown protein kinase and dephosphorylated by PP2A, the phosphorylation status of which correlates with the assembly of this SEAC subcomplex. The Iml1/Sea1-Npr2-Npr3 SAEC subcomplex, which was named SEACIT (for SEACsubcomplex-Inhibiting TORC1 signaling), functions as GAP toward Gtr1, thereby inhibiting TORC1 signaling [50]. The GAP activity of SEACIT is conserved in higher eukaryotes, such as Drosophilaand humans, that is, DEPDC5-NPRL2-NPRL3 corresponds to the yeast Iml1/Sea1-Npr2-Npr3, and DEPDC5 (ortholog of yeast Iml1/Sea1) directly binds to RagA and enhances the hydrolytic activity of GTP-bound RagA; therefore, the DEPDC5-NPRL2-NPRL3 complex was designated GATOR1 (for GAP Activity TOward Rags 1). GATOR1 inactivates mTORC1 in the absence of amino acids (Figure 2).

The SEAC of yeast is an octameric complex, that is, SEAC contains Sea2, Sea3, Sea4, Seh1, and Sec13 besides Iml1/Sea1-Npr2-Npr3, which constitutes SEACIT. These proteins constitute the other SEAC subcomplex, which binds to SEACIT in order to inhibit its Gtr1 GAP activity, and, thus, has been designated SEACAT (SEACsubcomplex-Activating TORC1 signaling) [49]. Orthologs of components in SEACAT also exist in Drosophilaand mammals, that is, WDR24, WDR59, Mios, Seh1L, and Sec13 in flies and humans, respectively, are Sea2, Sea3, Sea4, Seh1, and Sec13 in yeast, and this complex is referred to as GATOR2. All components in SEACAT and GATOR2 contain beta propeller-forming WD40 motifs, which are characteristic in membrane-coating proteins [51]. Sec13 is a component of COPII, which controls vesicle transport. In addition, Seh1/Seh1L and Sec13 are components of the nuclear pore complex [49].

3.2.5. Upstream modules of GATOR2 (Sestrins and CASTOR)

Amino acid sensors that function upstream of GATOR2 were identified in 2016, that is, Sestrin1/2 as a Leu sensor [52] and CASTOR as an Arg sensor [53]. Previous studies reported that Sestrins interacted with GATOR2 in order to inhibit mTORC1 signaling under amino acid-depleted conditions [54, 55, 56]. Wolfson et al. [52] demonstrated that Leu directly bound to Sestrin2 with a dissociation constant of 20 μM, and the binding of Leu to Sestrin2 disrupted the Sestrin2-GATOR2 interaction, thereby enabling GATOR2 to interact with GATOR1. The interaction between GATOR2 and GATOR1 inhibits the GAP activity of GATOR1 toward RagA/RagB, and, consequently, mTORC1 is activated.

The uncharacterized protein CASTOR1 binds to GATOR2, which inhibits GATOR2 binding to GATOR1. CASTOR1 forms a homodimer with CASTOR1 and a heterodimer with CASTOR2, a CASTOR1-related protein. Arginine specifically binds to CASTOR1 with a dissociation constant of ~30 μM, and the binding of Arg to CASTOR1 disrupts the CASTOR1-GATOR2 interaction, which turns CASTOR1 into a homodimer [53]. Liberated GATOR2 interacts with GATOR1 in order to inhibit its GAP activity toward RagA/RagB, which leads to the activation of mTORC1 (Figure 2).

Since no orthologs of Sestrins or CASTOR have been found in yeast, the mechanisms by which yeast senses intracellular amino acid availability currently remain unclear. A model in which tRNA functions as a negative regulator of TORC1 kinase activity in yeast was recently proposed [57]. Both amino acid-uncharged and amino acid-charged (aminoacylated) tRNAs inhibited TORC1 kinase activity in an in vitrokinase assay. Under nutrition-sufficient conditions, aminoacylated tRNAs predominantly bind to ribosomes for protein synthesis; therefore, tRNAs have fewer opportunities to interact with TORC1 (i.e., TORC1 is active). Upon nutrition starvation, uncharged tRNAs are released from ribosomes and interact with TORC1 in order to inhibit its kinase activity.

Human and yeast cells depleted for Rag GTPase/Gtr remained the ability to respond to amino acid, particularly glutamine [58, 59, 60]. It was recently reported that phosphatidylinositol 3-kinase complex Vps34-Vps15, and a vacuolar membrane protein Pib2, which contains a phosphatidylinositol 3-phosphate-binding FYVE (Fab1, YOTB, Vac1, and EEA1) domain, played a role in sensing glutamine in the Gtr-independent activation of TORC1 in S. cerevisiae[61].

4.2.1. Implication of GTPases

Small GTPase Rag complexes (RagA^GTP/RagB^GTP-RagC^GDP/RagD^GDP in metazoans, and Gtr1^GTP-Gtr2^GDP in yeast) play pivotal roles in the amino acid-induced activation of TORC1, as described in the previous sections. The other small GTPase Rheb is also involved in the growth factor-mediated activation of mTORC1. Do any small GTPases play roles in the activation of TORC2? In the fission yeast S. pombe, genetic screening revealed that the human Rab6 GTPase ortholog Ryh1 was involved in TORC2-Gad8 signaling [81]. S. pombeTORC2 phosphorylated Gad8, a member of the AGC kinase family, and a genetic mutation in ryh1⁺ markedly decreased the phosphorylation level of Gad8. sat1⁺ and sat4⁺ genes were predicted to code for GEFs toward Ryh1, and the mutational inactivation of these genes also induced a decrease in the phosphorylation level of Gad8, suggesting that Rhy1^GTP is an active form in terms of the activation of TORC2-Gad8 signaling. GTP-locked Rhy1 facilitated the physical interaction between TORC2 and its substrate Gad8. Furthermore, the expression of human Rab6 functionally compensated for the loss of ryh1⁺ in S. pombein terms of TORC2 signaling, which implied that Rab GTPase is involved in mTORC2-Akt signaling in mammals, similar to fission yeast. However, since S. cerevisiaedoes not possess the Rab6 ortholog, it currently remains unclear whether this regulatory system is generally conserved in eukaryotes. However, Avo1 and Avo3 contain the RBD and RasGEFN domains, respectively, both of which are related to Ras GTPase; therefore, some small GTPases may be involved in TORC2 signaling in S. cerevisiae. Previous studies demonstrated the participation of small GTPases in mTORC2 signaling. Rac1 GTPase was reported to bind directly to mTOR within mTORC1 and mTORC2, which led to the appropriate localization of these TOR complexes to the respective cellular membranes [82]. Rit, a Ras family GTPase, was shown to bind to mTORC2 and subsequently activate it in response to oxidative stress [83]. Since the oxidative stress-responsive activation of TORC2 was also observed in S. cerevisiae[84], a similar mechanism by which Ras family GTPase activates TORC2 may be conserved in budding yeast.

4.2.2. Posttranslational modifications in TORC2 components

mTOR is phosphorylated in the growth factor-mediated activation of mammalian TOR signaling. For example, Thr²¹⁷³ in the kinase domain of the mTOR protein is phosphorylated by Akt, which appears to be the negative feedback regulation of mTORC2 signaling. This feedback regulation is also conserved in fission yeast TORC2-Gad8 signaling, that is, Gad8 phosphorylates Thr¹⁹⁷² in the ATP-binding domain to reduce Tor1 activity within TORC2 [85]. More than 20 potential phosphorylation sites have been assigned in Rictor [86]. Ser²⁶⁰ in the CRIM domain and Thr³⁹⁸ in the PH domain in mSin1 are also phosphorylated [87, 88]. A high-throughput phosphoproteomic analysis predicted numerous potential phosphorylation sites in Avo1-3 and Bit61 [89].

Besides phosphorylation, Rictor is known to be acetylated at Lys¹¹¹⁶, Lys¹¹¹⁹, and Lys¹¹²⁵ [90, 91], modifications to which may activate mTORC2 activity.

4.3.1. Relationship between membrane tension and the activation of TORC2 signaling in yeast

When TORC2 was observed using GFP-tagged Avo1 or Avo3, its localization was visible as many dots just beneath the plasma membrane. The plasma membrane regions at which patchy TORC2 is located are referred to as the MCT [78]. Although Avo1 contains the PH domain, which has the potential to associate with membrane phospholipids, the underlying mechanisms by which TORC2 localizes to the plasma membrane remain unclear. Other regions on the yeast plasma membrane, referred to as eisosomes, are characterized by their distinctive shape, that is, they are furrows approximately 50-nm deep and 300-nm long on the surface of the plasma membrane [92]. The curvature of the membrane in eisosomes is formed by proteins possessing the BAR (Bin/amphiphysin/Rvs) domain, that is, Pil1 and Lsp1. Eisosomes exist in close proximity to the MCT, but never overlap.

Slm1 and its paralog Slm2 are eisosome-residential proteins and are effectors as well as substrates of TORC2. Under normal turgor pressure conditions, Slm1 and Slm2 are predominantly localized in eisosomes; however, following an increase in membrane tension caused by, for example, hypotonic shock or some mechanical stress, Slm1 and Slm2 alter their localization from eisosomes to the MCT and then bind to TORC2 via its components Avo2 and Bit61. Slm1 and Slm2 may recruit Ypk1 to TORC2, and the interaction between TORC2 and its substrate Ypk1 promotes the phosphorylation of Ypk1 (Figure 3).

It has not yet been established whether there exist any natural conditions that change the tension of the plasma membrane in yeast. One of these conditions may induce a decrease in the levels of sphingolipids that constitute the yeast plasma membrane together with glycerophospholipids and ergosterols. The initial step in the biosynthetic pathway of sphingolipids is catalyzed by serine palmitoyltransferase. The activity of this enzyme is negatively regulated by Orm1 and its paralog Orm2, the functions of which are controlled through the phosphorylation by Ypk1, a TORC2 substrate, at Ser⁵¹, Ser⁵², and Ser⁵³ in Orm1, and Ser⁴⁶, Ser⁴⁷, and Ser⁴⁸ in Orm2 [93, 94]. Myriocin is a potent inhibitor of serine palmitoyltransferase; therefore, the treatment of yeast cells with this chemical reduces the production of sphingolipids, which causes feedback regulation to activate sphingolipid biosynthesis through the stimulation of TORC2-Ypk1 signaling. Orm1/2 is subsequently phosphorylated, and its inhibitory effects on serine palmitoyltransferase are then compromised. Aureobasidin A, a cyclic depsipeptide antibiotic drug, exerts similar effects on the yeast plasma membrane in terms of altering membrane tension because this chemical inhibits the synthesis of inositol-phosphoceramide, one of the sphingolipid species in yeast. Aureobasidin A and myriocin consistently induce the phosphorylation of Ypk1 at Thr⁶⁶², a target site of TORC2 [94].

4.3.2. Activation of TORC2 signaling by the metabolic cue methylglyoxal

In contrast to mammals, which possess many isozymes of protein kinase C and its related kinases, Pkc1 is the sole protein kinase C in budding yeast. Pkc1 is involved in numerous pivotal biological functions including the organization of the actin cytoskeleton and the maintenance of cell wall integrity (CWI). The Mpk1 MAPK cascade lies downstream of Pkc1, and the Pkc1-Mpk1 MAPK cascade constitutes the main stream of the CWI pathway [95]. Chemicals that provoke cell wall damage such as Congo red or heat-shock stress activate the CWI pathway. The small GTPase Rho1 plays a crucial role in the heat-shock stress-induced activation of the CWI pathway, that is, the transmembrane proteins Wsc1 and Mid2 on the plasma membrane sense heat shock and interact with Rom2, a GEF toward Rho1, to load GTP to Rho1. Rho1^GTP physically interacts with Pkc1 to communicate the signal to the downstream Mpk1 MAPK cascade [96, 97]. A recent study reported that phosphatidylserine, one of the major glycerophospholipids prevailing in the plasma membrane, mediates the physical interaction between Pkc1 and Rho1^GTP [98, 99]. On the other hand, methylglyoxal, a typical 2-oxoaldehyde derived from glycolysis [100], also activates the Pkc1-Mpk1 MAPK cascade; however, the methylglyoxal-induced activation of this pathway is not dependent on Wsc1/Mid2, whereas Rho1 is indispensable [101]. Besides Ypk1 and Ypk2, Pkc1 has also been identified as a direct substrate of TORC2 in S. cerevisiae, that is, Thr¹¹²⁵ within the turn motif and Ser¹¹⁴³ within the hydrophobic motif in Pkc1 are phosphorylated by TORC2 [101]. Methylglyoxal enhanced the phosphorylation levels of Pkc1 at Ser¹¹⁴³ in a TORC2-dependent manner [101] (Figure 3).

The methylglyoxal-induced activation of TORC2 is conserved in mammalian cells, that is, the phosphorylation levels of Ser⁴⁷³ within the hydrophobic motif in Akt, a substrate of mTORC2, were enhanced following the treatment of mouse 3 T3-L1 cells with methylglyoxal [101]. Collectively, these findings demonstrate that methylglyoxal activates (m)TORC2 signaling in yeast and mammalian cells; however, the underlying mechanisms have not yet been elucidated. Since methylglyoxal is a naturally occurring ubiquitous metabolite and is involved in type 2 diabetes and its complications [100], its involvement in the activation of (m)TORC2 signaling is of considerable interest in order to obtain insights into not only novel activation mechanisms of TORC2 but also the physiological significance of methylglyoxal.

4.3.3. Activation of mTORC2 signaling by growth factor

In mammalian cells, physiological cues for the activation of mTORC2 signaling are insulin and insulin-like growth factors [102]. Upon the capture of ligands by tyrosine kinase-type receptors, tyrosine-phosphorylated IRS (insulin receptor substrate) undergoes the activation of phosphatidylinositol 3-kinase, which enhances the levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P₃). Two events are subsequently induced by this phosphoinositide near the plasma membrane: that is, the activation of PDK (phosphoinositide-dependent kinase) and the recruitment of Akt to the plasma membrane in which the PH domain of Akt binds to PtdIns(3,4,5)P₃. In turn, Akt at Thr³⁰⁸ within the activation loop and Ser⁴⁷³ within the hydrophobic motif are phosphorylated by PDK and mTORC2, respectively; however, the mechanisms by which insulin activates mTORC2 remain obscure.