mTOR (mammalian target of rapamycin) plays a key role in determining how growth factor, nutrient and oxygen levels modulate intracellular events critical for the viability and growth of the cell. This is reflected in the impact of aberrant mTOR signalling on a number of major human diseases and has helped to drive research to understand how TOR (target of rapamycin) is itself regulated. While it is clear that amino acids can affect TOR signalling, how these molecules are sensed by TOR remains controversial, perhaps because cells use different mechanisms as environmental conditions change. Even the question of whether they have an effect inside the cell or at its surface remains unresolved. The present review summarizes current ideas and suggests ways in which some of the models proposed might be unified to produce an amino acid detection system that can adapt to environmental change.
- amino acid transporter
- mammalian target of rapamycin (mTOR)
- proton-assisted amino acid transporter (PAT)
- Ras-related GTPase (Rag)
- solute carrier 36A1 (SLC36A1)
mTOR (mammalian target of rapamycin): a hub for nutrient and growth factor regulation of cellular processes
mTOR initially came to light as the cellular target for the immunosuppressant drug rapamycin. Since then, interest in rapamycin has extended to a widespread array of human diseases, including cancer, cardiovascular disease, autoimmunity, neurodegenerative diseases and metabolic disorders such as diabetes (see, e.g., [1,2]). This reflects the emerging roles for mTOR as a key regulator of energy homoeostasis, cell physiology and growth within all higher eukaryotes (reviewed in ).
The initial characterization of TOR relied on studies in yeast and mammals. However, subsequent work in Drosophila has played a key role in establishing that the growth-factor-regulated signalling cascade involving the lipid kinase PI3K (phosphoinositide 3-kinase) and Akt/PKB (protein kinase B) controls mTOR through the G-protein Rheb and the TSC (tuberous sclerosis complex) components TSC1 and TSC2 . The demonstration that this regulatory mechanism is conserved in mammalian systems has led to rapamycin being taken into clinical trials to treat patients with specific forms of cancer . Indeed, therapies designed to block the activity of mTOR have already proved to have an effect in restricting tumour growth, whereas treatments directed at PI3K/Akt have been hampered by toxicity issues . This has stimulated interest from pharmaceutical companies in developing more mTOR inhibitors .
TOR (target of rapamycin) exists in two distinct complexes , TORC1 and TORC2, that appear to act downstream and upstream of Akt respectively. TORC1, which is controlled by Rheb, mediates anabolic processes that promote growth by increasing protein synthesis. TORC1 stimulates translation by directly phosphorylating p70S6K (p70 S6 kinase) and 4E-BP1 (eukaryotic initiation factor 4E-binding protein 1) to promote ribosome assembly and translation. TORC1 is inhibited by rapamycin, which was thought not to inhibit TORC2 [9,10]. However, there is now evidence that rapamycin can inhibit TORC2 in some cell types . Although it is currently unclear how TORC2 is regulated, growth-factor-regulated TORC1 activity has also been shown to respond to oxygen levels and cellular energy status, which partially reflects extracellular nutrient levels (Figure 1). Understanding the molecular mechanisms by which these controls are achieved has the potential to provide significant therapeutic benefits.
A variety of mechanisms exist to provide the building blocks for cellular growth. Nutrients can be brought into cells through classical transporters, as is the case for amino acids and sugars . Endocytosis results in membrane and extracellular proteins being brought into the cell, some of which are broken down and recycled. Cytoplasmic material can also be reused to provide the supplies needed for growth. This is particularly important when extracellular nutrients are low and TORC1 signalling is reduced, which leads to enhanced autophagy [13,14], where organelles are destroyed and their component macromolecules recycled through autophagolysosomes.
AMPK (AMP-activated protein kinase) has emerged as a key sensor of energy depletion. It acts not only on TSC2 to suppress TORC1 signalling , but also on a component of TORC1, raptor (regulatory associated protein of mTOR) . AMPK is activated by cellular stresses that reduce ATP levels, such as exercise or reduction in nutrients such as fatty acids and glucose (reviewed in ). In an exciting recent development, activation of AMPK has been shown to delay the progression of tumours in mice .
Importantly, work in cell culture has suggested that intracellular amino acids, particularly leucine, may be involved in regulating TOR signalling (reviewed in ). However, in vivo analysis has also highlighted the role of other amino acids outside the cell [20,21]. Perhaps the most likely scenario is that multiple amino-acid-dependent mechanisms that respond to different environmental and physiological conditions regulate TOR signalling. Recent studies have highlighted a number of candidate regulators, and the challenge is now to assemble these into a coherent amino-acid-sensing model.
Which intracellular players have been linked to amino acid sensing?
A number of different molecules have been implicated in amino acid sensing. The Ste20 kinase family member MAP4K3 (mitogen-activated protein kinase kinase kinase kinase 3) has been highlighted as a regulator of p70S6K and 4E-BP1 activity that modulates cell growth in response to amino acids, but not to growth factors or insulin . However, the functional significance of MAP4K3 has yet to be tested in vivo. In addition, the class III PI3K, Vps34 (vacuolar protein sorting 34), has been suggested to regulate mTOR in mammalian cell culture [23,24], but in vivo analysis in Drosophila with Vps34 mutants has not confirmed this role . This suggests either that Vps34 operates in a mammalian-specific or cell-type-specific fashion or that in vitro experiments in the altered environment of the culture dish do not fully reflect the amino-acid-sensing mechanisms taking place in living animals.
Several studies have suggested that mTOR is associated with intracellular membranes within the cytoplasm, including the endoplasmic reticulum, Golgi, endosome and mitochondrial membranes . Localization of Rheb to endomembranes has been shown to be critical for its functions . However, until recently, it was unclear whether any of the membrane-bound pools of TOR were essential for its activities. It has now been shown in HEK (human embryonic kidney)-293T cells that, in response to increased amino acid levels, mTOR shifts to Rab7-positive compartments where its activator Rheb is located. Furthermore, this relocalization of mTOR is dependent on the Rags (Ras-related GTPases), which have therefore been proposed to act as amino acid sensors transmitting the signal from extracellular amino acids to TOR [28,29]. In Drosophila, the Rags are potent growth regulators, suggesting that they play an evolutionarily conserved role in TOR regulation .
Regulation of growth and TOR signalling by amino acid transporters
In parallel with the identification of intracellular mediators of amino acid sensing discussed above, other groups have focused their energies on finding the cell-surface molecules that might ‘detect’ extracellular amino acids. Since one of the most popular models for amino-acid-dependent TOR activation involves sensing intracellular amino acids, amino acid transporters have been postulated to play an important part in this process. In the 1990s, multiple cellular ‘systems’ responsible for transporting amino acids were characterized [30,31]. With the advent of molecular cloning, families of genes were assigned to these different transport systems. These families have been genetically screened for growth regulatory functions in Drosophila. Although transporters in several classes have been linked to amino acid sensing involved in endocrine signalling (Minidiscs , Slimfast  and some tumour cell growth in culture ), surprisingly, a different class, the PAT (proton-assisted amino acid transporter)/SLC36 (solute carrier 36) transporters (reviewed in [35,36]) were highlighted as particularly potent growth activators in growing tissues in vivo and shown to interact with components of the PI3K/Akt and TOR signalling cascades [37,38]
The PATs were initially identified on the surface of mammalian lysosomes, which led to them being named as LYAATs (lysosomal amino acid transporters)  and also as proton-dependent amino acid transporters . PATs are related to AVTs (amino acid vacuolar transporters) in yeast , which can transport amino acids out of the vacuole, a lysosomal structure. In keeping with a role in promoting transport from acidic lysosomal compartments, PAT-mediated amino acid transport is normally, but not always, enhanced by extracellular acidification [37,39,40]. It is now known that PATs are also found at the plasma membrane and in endosomal compartments [40,42]. However, although the presence of different subcellular pools of PATs is intriguing, the roles of these pools are currently unclear. In mammalian cells, the PATs have been suggested to transport amino acids from the apical surface of the gut . However, some PATs are expressed throughout the body , suggesting that they are likely to have much more widespread functions. The regulation of TOR by PATs represents such a role and opens up the important question of which of the various subcellular pools of this molecule is involved in this process.
PATs and intracellular amino acid sensing: towards a more unified model
The recent discovery that amino acids promote the shuttling of mTOR into Rheb-containing late endosomes via a Rag-dependent mechanism  has led to the proposal that Rags are involved in transmitting an amino-acid-dependent signal from a sensor at the cell surface to these intracellular compartments. An alternative interpretation is that the Rags are involved in regulating a membrane-associated amino acid sensor that must shuttle from the cell surface to the Rheb-containing endosomes. Interestingly, the yeast orthologues of Rags, Gtr1p and Gtr2p , are found in the late endosome and are required for proper shuttling of the amino acid permease Gap1p , suggesting that membrane protein trafficking is one of their key functions. This alternative model might also explain why Vps34, a class III PI3K involved in membrane trafficking, has also been implicated in amino acid sensing . Furthermore, since Rheb and the TSC can influence protein trafficking [45–47], this might explain the synergistic interactions that take place between growth factor signalling and amino acid sensing.
The PATs are excellent candidate amino acid transporters to fulfil this shuttling-dependent amino-acid-sensing role, since they are found at the cell surface and in the endosomes and lysosomes, and they can function in acidic compartments such as the late endosome (Figure 1). Furthermore, some findings in Drosophila suggest that one of the growth regulatory PATs, PATH (pathetic), is a low-capacity transporter and therefore may either function in a transport-independent fashion, acting as a so-called ‘transceptor’ (a receptor that is structurally related to a transporter ), or activates TORC1 by transporting low levels of amino acids to a complex at a specific subcellular site . Either model would fit nicely with a PAT–Rheb–TORC1 complex being specifically located at late endosomal membranes.
How could a model involving PATs and Rags fit with data suggesting cytosolic levels of leucine are important in controlling TORC1? One possibility is that amino-acid-dependent regulation of TORC1 is multifaceted, involving TOR activation in subcellular compartments other than the late endosome, so that, under some conditions, in cell culture for example, the import of amino acids such as leucine into the cytosol can be a critical determining factor. Under other conditions, signalling from the endosomes may be more important, and molecules such as the PATs and Rags will play an increasingly central role. A more unifying alternative is that intracellular amino acids such as leucine can modulate the activity of molecules such as the PATs when they act via a transporter or transceptor mechanism. Fascinatingly, the activity of one of the transceptors characterized in yeast, the amino acid sensor Ssy1, is modulated in precisely this way by specific intracellular amino acids .
The presence of an endosomal amino-acid-sensing mechanism in higher eukaryotic cells might be particularly significant when extracellular nutrients or growth factors are sparse and/or when processes such as autophagy are activated. There is evidence that autophagy has a positive effect on growth under some starvation conditions , and low-level intracellular TOR signalling from late endosomal or lysosomal compartments might be essential for this. Equally significantly, subcellular localization of TOR signalling in endosomes may become important in diseases such as cancer, where cells need to survive and grow in low oxygen, nutrient and growth factor environments.
Thus the finding that multiple mechanisms might be involved in linking amino acid sensing to TOR may really be a reflection of the cell's versatility to adapt to different environments to which it is exposed. What is required now is a careful combination of in vivo and in vitro studies to define the different mechanisms that exist and the conditions under which they are employed. We are still some distance from understanding how amino acids are sensed by TOR. Recent work has, however, been critical in highlighting the importance of dissecting the protein-shuttling mechanisms within specific parts of the cell, in addition to identifying the relevant signalling cascades.
Work on nutrient sensing from the D.C.I.G, C.W. and C.A.R.B. groups is supported currently by grants from Cancer Research Technology [grant number C19591/A9093], Cancer Research UK [grant numbers C7713/A6174 and C19591/A6181] and previously by the Biotechnology and Biological Sciences Research Council.
We apologize to those authors whose work we have been unable to cite owing to limitations on the number of references.
mTOR Signalling, Nutrients and Disease: Biochemical Society Focused Meeting held at Medical Sciences Teaching Centre, University of Oxford, U.K., 15–16 September 2008. Organized and Edited by Richard Boyd (Oxford, U.K.), Deborah Goberdhan (Oxford, U.K.) and Richard Lamb (Cancer Research UK, London, U.K.).
Abbreviations: AMPK, AMP-activated protein kinase; 4E-BP1, eukaryotic initiation factor 4E-binding protein 1; MAP4K3, mitogen-activated protein kinase kinase kinase kinase 3; mTOR, mammalian target of rapamycin; p70S6K, p70 S6 kinase; PAT, proton-assisted amino acid transporter; PI3K, phosphoinositide 3-kinase; Rag, Ras-related GTPase; TOR, target of rapamycin; TORC, TOR complex; TSC, tuberous sclerosis complex; Vps34, vacuolar protein sorting 34
- © The Authors Journal compilation © 2009 Biochemical Society