TOR (Target of Rapamycin) is a Ser/Thr kinase that was originally identified by genetic screening using the budding yeast Saccharomyces cerevisiae. The TOR protein forms two structurally and functionally distinct complexes (TOR complex 1, TORC1, and TOR complex 2, TORC2). TORC1 is involved in various cellular activities, such as cell growth, ribosome biogenesis, translation initiation, metabolism, stress response, aging, and autophagy. TORC2 is involved in actin organization, sphingolipid biogenesis, and endocytosis. TORC1 plays a central role in the signaling network in response to stimuli coupled to internal and external nutrient conditions, particularly an amino acid sufficiency. A dimeric complex of Rag GTPases, the activity of which is regulated by the guanine nucleotide-loading status, and some regulator proteins communicating with Rag GTPases are involved in the activation of TORC1 by amino acids. In TORC2 signaling, membrane stress appears to be a cue, in which some proteins associated with respective membrane compartments, such as eisosomes, play a role.
- TOR (Target of Rapamycin)
- small GTPase
- signal transduction
- protein kinase
- Saccharomyces cerevisiae
All heterotrophs must take organic compounds from outside of cells to gain energy for various biological activities. For example, since amino acids are components of proteins, an insufficiency in amino acids has serious effects on cellular functions. The bacterial feedback regulation of amino acid biosynthesis at the enzyme and gene expression levels is a well-known mechanism that controls intracellular amino acid levels. In this feedback-regulatory mechanism, an amino acid functions as a signaling molecule in the closed metabolic loop for the production of respective amino acids.
On the other hand, in higher Eukarya, an insufficiency in amino acids has been linked to various metabolic diseases; therefore, sensing amino acid amounts inside and outside of cells through the transmission of signals needs to be strictly controlled. TOR (
2. TOR: a master regulator for cell growth
2.1. Rapamycin and FKBP12
Rapamycin is a macrolide antifungal chemical that was identified from the bacterium
The first approach to investigating the mode of action of rapamycin was biochemical. Since rapamycin was found to inhibit the mammalian immune system, molecule(s) with the ability to bind to rapamycin may be involved in the action of this drug as an immunosuppressant. Rapamycin was shown to bind to a peptidyl-prolyl
2.2. Discovery of TOR
In order to identify the target of the Fpr1-rapamycin complex, genetic screening using
Rapamycin itself does not directly bind to the TOR protein, whereas the Fpr1-rapamycin complex binds to the Tor1 or Tor2 protein, thereby inhibiting the protein kinase activity of TOR [14, 15, 16, 17].
2.3. Domain structure of TOR
The domain structures and amino acid sequences of all TOR proteins are evolutionarily conserved. Both Yeast Tor1 and Tor2 contain the following domains (in the direction from the N-terminus to the C-terminus): HEAT repeats, FAT, FRB, kinase, FIT, and FATC (Figure 1). These domains are also found in the mTOR protein in the same order. Each HEAT motif (originally identified in
2.4. TOR complexes
Although the primary structures of Tor1 and Tor2 share strong similarities, their cellular functions are distinct [14, 22]. The
3.1. Subunit components
The following components constitute TORC1: Kog1, Tco89, Lst8, and either Tor1 or Tor2 [26, 27, 28] (Figure 1). Mammalian TORC1 (mTORC1) contains counterparts of each subunit of yeast TORC1, except for Tco89, instead mTORC1 contains PRAS40 (
Kog1 and mammalian ortholog Raptor (
3.2. Activation of TORC1 signaling
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
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 . Similarly, Gtr1 and Gtr2 form a heterodimer . Heterodimers with GTP-bound RagA/RagB and GDP-bound RagC/RagD exhibit full activity. This is also the case for
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
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,
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 . Vam6/Vps39 exhibited Gtr1 GEF activity
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 . 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 Gtr2GTP in order to enhance the hydrolytic activity of its GTPase activity, thereby yielding Gtr2GDP 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 . Npr2 and Npr3 form a heterodimer . 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 . A biochemical analysis revealed that Iml1/Sea1 exhibited GAP activity toward Gtr1
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 (
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  and CASTOR as an Arg sensor . 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.  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 . 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 . Both amino acid-uncharged and amino acid-charged (aminoacylated) tRNAs inhibited TORC1 kinase activity in an
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 (
3.3. The TSC1/2-Rheb branch in the activation of mTORC1
In mammalian cells, mTORC1 is activated by another small GTPase Rheb (
4.1. Subunit components
The following components constitute the budding yeast TORC2: Tor2, Avo1, Avo2, Avo3, Bit61, and Lst8 (Figure 1). Avo1 has several conserved domains. Avo1 contains an RBD (a
Avo3 is the largest subunit of TORC2. It functions as a scaffold protein in order to maintain the integrity and function of TORC2 because the loss of Avo3 induced the disassembly of TORC2 . Avo3 contains the ARM (
Bit61 has a paralog Bit2. Although Bit61 binds to TORC2 through Avo1 and Avo3, it is not an essential subunit for the assembly of TORC2 [25, 75]. The specific functions of Bit61 have not yet been elucidated; however, mammalian orthologs of Bit61 and Bit2 exist (PRR5 also known as Protor-1, and PRR5L also known as Protor-2) and possess an HbrB domain that was found in a fungal
Avo2 is a yeast TORC2-specific subunit, but is not essential. Avo2 contains ankyrin repeats. Avo2 and Bit61 have been reported to bind to Slm1 and Slm2 proteins, which are involved in the recruitment of Ypk1/Ypk2 to TORC2, thereby phosphorylating them .
The core subunits of mammalian TORC2 (mTORC2) include mTOR as the TOR protein, mSin1 (
The ARM-like domain is conserved in Rictor and Avo3, while the RasGEFN domain is not conserved in Rictor. mTORC2 is also insensitive to an acute treatment with rapamycin, the mechanism of which is presumably the same as that elucidated in yeast TORC2. However, in some mammalian cell lines, a prolonged treatment with rapamycin was found to inhibit the interaction between newly synthesized mTOR and Rictor, and mTORC2-Akt signaling was subsequently reduced .
4.2. Activation of TORC2
4.2.1. Implication of GTPases
Small GTPase Rag complexes (RagAGTP/RagBGTP-RagCGDP/RagDGDP in metazoans, and Gtr1GTP-Gtr2GDP 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
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 . More than 20 potential phosphorylation sites have been assigned in Rictor . 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 .
4.3. Activation of TORC2 signaling
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 . 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 . The curvature of the membrane in eisosomes is formed by proteins possessing the BAR (
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 .
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 . 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. Rho1GTP 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 Rho1GTP [98, 99]. On the other hand, methylglyoxal, a typical 2-oxoaldehyde derived from glycolysis , 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 . Besides Ypk1 and Ypk2, Pkc1 has also been identified as a direct substrate of TORC2 in
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 . 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 , 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 . Upon the capture of ligands by tyrosine kinase-type receptors, tyrosine-phosphorylated IRS (
5. Concluding remarks
Laboratory conditions for culturing yeast may be adequate for yeast cells to maintain cellular activities because ample amounts of glucose and amino acids are typically supplied in media. By contrast, nutritional conditions surrounding yeast cells that exist in the natural world are harsh and variable. Yeast cells have evolved mechanisms for sensing changes in nutritional conditions and transitioning the metabolic status and gene expression profile to adapt efficiently and survive inhospitable conditions. The TOR signaling system had been acquired as one of these signal network systems and has been evolutionarily conserved among eukaryotes. In higher eukaryotes, such as humans, dysfunctions in the TOR signaling network closely correlate with pathological conditions including diabetes, cancer, obesity, and neurodegeneration ; therefore, TOR is a target from a clinical point of view. Upstream and downstream processes of TORC1 signaling have been extensively investigated because rapamycin, a potent inhibitor for TORC1, was available. By contrast, studies on TORC2 signaling appear to be challenging because of the absence of TORC2-specific inhibitors. However, yeast was always a vanguard from the beginning of TOR studies (TOR was discovered by genetic screening using yeast) and will continue to be so in the future. Many issues remain to be solved in TOR signaling; however, since TOR is a central player in cell growth, studies on TOR will be nothing less than a study of the living system itself. Investigations on TOR will provide many insights for understanding “life.”