The Target of Rapamycin (TOR) was first identified in Saccharomyces cerevisiae cells treated with rapamycin, where it results in irreversible cell cycle arrest (Heitman et al., 1991). Several years later, a mammalian TOR (mTOR) homolog (Brown et al., 1994; Sabatini et al., 1994) was identified. mTOR forms the core of two separate multiprotein complexes, mTORC1 and mTORC2, which are differentiated by their complements of accessory proteins (Kim et al., 2002; Loewith et al., 2002; Sarbassov et al., 2004; Laplante and Sabatini, 2012; Shimobayashi and Hall, 2014). Since the discovery of the mTOR complexes, extensive efforts have been made to characterize them and to distinguish their functions. Whereas mTORC2 regulates cell survival, metabolism and cytoskeletal structure (Oh and Jacinto, 2011), mTORC1 functions as a central regulator of metabolism, ensuring that the cell grows only under favorable conditions (Rabanal-Ruiz and Korolchuk, 2018). The dysregulation of the mTORC1 signaling pathway is thereby associated with many forms of cancer and metabolic disorders (Guertin and Sabatini, 2007; Saxton and Sabatini, 2017; Mossmann et al., 2018). The central components of mTORC1 are mTOR, which provides the catalytic core, regulatory-associated protein of mTOR (RAPTOR) (Hara et al., 2002; Kim et al., 2002; Loewith et al., 2002), and mammalian lethal with Sec13 protein 8 (mLST8) (Chen and Kaiser, 2003; Kim et al., 2003), while proline-rich Akt substrate of 40 KDa (PRAS40) and DEP domain containing mTOR interacting protein (DEPTOR) are minor regulatory components. The central components form a dimeric complex interacting through the N-terminal HEAT repeats of the catalytic core, mTOR (Aylett et al., 2016; Anandapadamanaban et al., 2019). mTOR signaling is dependent on its serine/threonine kinase activity toward target substrates. The mTORC1 complex triggers cell proliferation and cell growth by promoting anabolic processes and suppressing catabolic metabolism through the phosphorylation of key effector proteins such as ribosomal protein S6 kinase (S6K) and the translation repressor eukaryotic translation initiation factor 4E-binding protein (4E-BP) (Rabanal-Ruiz and Korolchuk, 2018; Kim and Guan, 2019).

The level of activation of mTORC1 is dependent on the nucleotide state of three small GTPases: mTORC1 is recruited to the lysosome by heterodimeric Ras-related GTP-binding protein (Rag GTPases) (Sancak et al., 2008) where it is catalytically activated by GTP-bound Ras homolog enriched in brain (Rheb) GTPase (Long et al., 2005; Sato et al., 2009). Both pathways are necessary, with neither being sufficient to activate mTORC1 independently. It is widely accepted that multiple intracellular and extracellular inputs are integrated to finely regulate the activation of mTORC1 (Laplante and Sabatini, 2012). While external and survival stimuli, such as growth factors or cellular stress, regulate mTORC1 activity through the Rheb axis, signals corresponding to the intracellular nutrient and energy state, such as amino acid availability, are communicated to the Rag GTPases to control the lysosomal localization of mTORC1. However, whether or not some stimuli override others, and whether or not there are diverse activation mechanisms depending on the cell type and context, remains unclear. The main regulator of Rheb is the TSC complex (TSCC), consisting of the two large tuberous sclerosis complex proteins 1 (TSC1) and TSC2, also known as hamartin and tuberin, respectively (Tomasoni and Mondino, 2011), and a smaller protein called TBC1D7 (Dibble et al., 2012). The largest member of the complex, TSC2, acts as a GTPase activating protein (GAP) for the small GTPase Rheb, promoting its inactive GDP-bound conformation, and therefore as a negative regulator of mTORC1 (Tee et al., 2002; Zhang et al., 2003). Nutrients, in particular amino acids, activate lysosome-associated signaling machinery to recruit mTORC1 through the Rag GTPases, which directly interact with mTORC1 in an amino acid dependent manner (Kim et al., 2008; Sancak et al., 2008). The regulation of the Rag GTPases is highly sophisticated, involving several multiprotein complexes acting as GAPs and guanine nucleotide exchange factors (GEFs): GAP Activity Toward Rags (GATOR) 1, a GAP for RagA/B, FLCN-FNIP, a GAP for RagC/D, GATOR2, an inhibitor of GATOR1, KPTN, IKTFG2, C12orf66 and SZT2 (KICSTOR), which is thought to regulate the GATOR1 complex, and the pentameric Rag and mTORC1 regulator (Ragulator) complex, which acts as a GEF for RagA/B (Ramlaul and Aylett, 2018). Overall, mTORC1 signaling represents a sophisticated pathway through which several signals converge to regulate most major cellular functions involved in cell growth (Figure 1). Therefore, is not unexpected that many years after its discovery, our understanding of the mechanism of action and regulation of mTOR is constantly evolving.

While it is widely accepted that mTORC1 activation depends on nutrient availability (Blommaart et al., 1995; Hara et al., 1998), how these stimuli trigger mTORC1 signaling is less clear. The latest evidence suggests that amino acids are sensed through different mechanisms from inside and outside the lysosome via certain human proteins known to act as metabolic sensors. These include cytoplasmic nutrient sensors (Sestrins, CASTORs and SAMTOR), amino acid transporters (SLC38A9) and the v-ATPase within the lysosomal membrane (Rabanal-Ruiz and Korolchuk, 2018; Ramlaul and Aylett, 2018). All these signals converge upon the Rag GTPases, which are anchored to the lysosome via the Ragulator complex (Sancak et al., 2010; Bar-Peled et al., 2012), and which together make up the essential platform for the recruitment of mTORC1 to the lysosome.

In mammals, there are four Rag GTPases that form heterodimers between two sub-types (Sekiguchi et al., 2001). RagA and RagB were firstly discovered as members of the Ras superfamily of small GTPases and homologs of yeast Gtr1 (Schürmann et al., 1995). RagC and RagD were identified as Gtr2 orthologs, completing the Rag family (Sekiguchi et al., 2001). Human RagA is paralogous to RagB, with 90% sequence identity, while human RagC is paralogous to RagD, with 81% sequence identity (Nicastro et al., 2017). Rag family members possess sequence elements that are common to all GTPases of the Ras family, including the P-loop and switch regions I (SWI) and II (SWII), and guanine binding motifs with some important differences (Supplementary Figure 3A). The switch regions adopt open or closed conformations upon GTP or GDP binding (Wittinghofer and Vetter, 2011; Jeong et al., 2012) in order to transmit to mTORC1 the nutrient status of the cell. Their guanine binding motifs, G2 and G3, are also divergent, with an asparagine substituted for a histidine in G2, and an isoleucine instead of an alanine in G3 (Schürmann et al., 1995; Sekiguchi et al., 2001; Sekiguchi et al., 2014). Another remarkable feature of the Rag GTPases is their large C-terminal domains (CTDs), which are responsible for the assembly of functional heterodimers containing RagA or B combined with RagC or D (Sekiguchi et al., 2001; Gong et al., 2011). The Rag heterodimer is pseudo-two-fold symmetric, with an overall U-shape (Supplementary Figure 3B). Heterodimerization is achieved by direct contacts between the RagA/B and RagC/D CTDs, whereas no direct interaction is established between their nucleotide binding domains (NBDs), a novel architecture previously unreported for any GTPase (Gong et al., 2011; Jeong et al., 2012). The NBDs are located on the same side of the complex but face in opposite directions.

Amino acid availability promotes the active Rag GTPase nucleotide configuration, RagA/B-GTP and RagC/D-GDP, which is capable of recruiting mTORC1 through a direct interaction with Raptor (Sancak et al., 2008). Conversely, when amino acid levels are low, the opposite Rag GTPase nucleotide binding state, in which RagA/B is GDP-bound and RagC/D is GTP-bound, is favored, which results in the release of mTORC1 from the lysosome. The recent determination of the structures of two mTORC1-Rag complexes has shed light on the molecular basis of Rag GTPase signal transduction. The first study determined the Raptor-Rag-Ragulator architecture, revealing that Raptor first binds to RagA-GTP in a transient interaction, and can only attach fully to the Rag GTPase heterodimer when RagC is GDP-bound (Rogala et al., 2019). Concurrently, the structure of the mTORC1-RagA-RagC complex demonstrated that the α-solenoid domain of Raptor contacts the Rag GTPase NBDs, leaving the CTDs with a limited interaction surface with Raptor (Anandapadamanaban et al., 2019). The RagA-GTP switch and inter-switch regions are more ordered than RagC-GDP because they form more extensive contacts with mTORC1 (Supplementary Figure 3C). In the reverse situation (GDP-RagA-RagC-GTP), the Rag GTPase complex would not bind to Raptor, because the switch and inter-switch regions of RagA-GDP would be disordered (Supplementary Figure 3D) and RagC-GTP residues analogous to RagA Raptor-binding regions are not conserved (Anandapadamanaban et al., 2019). Structural analysis of the mTORC1-RagA-RagC complex also revealed that, while the Rag GTPases undergo multiple conformational changes upon Raptor binding, the binding of the Rag heterodimer does not cause any conformational changes within mTORC1. This supports the current hypothesis that mTORC1 is recruited by the Rag-Ragulator platform to lysosomes, where it is allosterically activated by Rheb-GTP. Combination of the new mTORC1-Rag GTPase structure with previously published structures of mTORC1-Rheb (Yang et al., 2017) and Ragulator bound to RagA-RagC (de Araujo et al., 2017; Yonehara et al., 2017) provides sufficient information to build a structural model of the active mTORC1 super-complex at the lysosome. This model would allow the simultaneous interaction of mTORC1 with both the Rag GTPases and Rheb, while leaving the catalytic site facing the cytosol to allow mTORC1 to fulfill its function as a kinase and phosphorylate downstream targets (Anandapadamanaban et al., 2019).

The double nucleotide-bound activating conformation, with both GTP and GDP bound on the Rag heterodimer, especially the requirement for GDP state in one active site and the linked architecture, are features unique to the Rags among GTPases (Mishra and Lambright, 2016). Kinetic assays with radiolabeled nucleotides have implied that this unique architecture allows crosstalk between the two subunits: when one GTPase domain is bound to GTP, the heterodimer is in a locked conformation that suppresses the association of a second GTP in the opposing domain or induces fast hydrolysis if this exchange occurs (Shen et al., 2017). This inter-subunit communication provides a singular mode of regulation of GTPases which provides more intermediate states that participate in the precise regulation of mTORC1 signaling pathway. Cryo-EM structural data combined with hydrogen/deuterium exchange mass spectrometry (HDX-MS) of mTORC1 in complex with the Rag GTPase heterodimer revealed the means by which the nucleotide state of the NBD is sensed by the corresponding CTD (Anandapadamanaban et al., 2019), which undergoes conformational changes depending on the Rag GTPase nucleotide state, providing the structural basis for the Rag inter-subunit crosstalk. Moreover, the heterodimeric setting furnishes the Rag GTPases with additional conformational possibilities in comparison to the situation for monomeric small GTPases, to satisfy their requirements for interactions with multiple regulators. The basal GTPase activity of the Rag GTPase heterodimer is lower than that of other Ras-family members (Frech et al., 1994; Shen et al., 2017), therefore they are more dependent on their GAPs in order to switch between their active and inactive settings in response to nutrient availability. There are two protein complexes with GAP activity toward the Rag GTPases: GATOR1 for RagA/B (Bar-Peled et al., 2013; Panchaud et al., 2013) and FLCN-FNIP for RagC/D (Petit et al., 2013; Tsun et al., 2013).

To exert their amino-acid dependent mTORC1-recruiting function, the Rag GTPases are localized to the lysosome, interacting with the lysosomal membrane protein Late Endosomal/Lysosomal Adaptor, MAPK and mTOR activator/regulator (LAMTOR) 1 (Sancak et al., 2010), a part of the “Ragulator” complex (Bar-Peled et al., 2012). The Ragulator complex is formed by five subunits; LAMTOR1 (p18), LAMTOR2 (p14), LAMTOR3 (MP1), LAMTOR4 (HBXIP) and LAMTOR5 (C7orf59). This complex acts as a scaffold for the Rag GTPases, with LAMTOR1 being the key component required to attach them to the lysosome via myristoylation and palmitoylation sites within its N-terminus (Nada et al., 2009; Sancak et al., 2010; Bar-Peled et al., 2012). LAMTOR 2, 3, 4, and 5 contain roadblock domains that form heterodimers, LAMTOR2 with LAMTOR3 and LAMTOR4 with LAMTOR5. The assembled Ragulator complex has an elongated shape with LAMTOR1 α-helices encircling LAMTOR 2-3 and LAMTOR 4-5 heterodimers (Mu et al., 2017). Ragulator is not merely a platform for the proper localization of the Rag GTPases to the lysosome, but also possesses GEF activity to promote their exchange to the active nucleotide state. Ragulator was firstly identified as a GEF for Rag A/B as it accelerates the release of both GDP and GTP and favors the active Rag GTPase nucleotide state (Bar-Peled et al., 2012). Additionally, recent kinetic studies have found that SLC38A9, a lysosomal amino acid sensor, cooperates with Ragulator through an atypical GEF mechanism (Shen and Sabatini, 2018). The structure of the full complex with the Rag GTPases supports the function of Ragulator; the C-terminal Roadblock domain of RagC binds to the LAMTOR2-3 heterodimer (de Araujo et al., 2017; Su et al., 2017; Yonehara et al., 2017), thereby exposing the NBDs as required for their interaction with Raptor. Interestingly, the Roadblock domains found in LAMTOR subunits 2–5 are very common in GTPase interacting partners (Levine et al., 2013). Ragulator thereby functions both as a platform for, and a component of, the regulatory elements of the nutrient signaling pathway, which includes the FLCN-FNIP complex.

Together with the GATOR complexes, the FLCN complex acts as a GAP for the Rag GTPases (Figure 1). FLCN-FNIP controls the nucleotide state of RagC/D, promoting the RagC/D-GDP state, and therefore acting as a positive modulator of mTORC1. Under starvation conditions, FLCN-FNIP is recruited to lysosomes where it interacts with RagA-GDP, whereas when nutrient levels are recovered, the complex dissociates and returns to the cytosol (Petit et al., 2013; Tsun et al., 2013), allowing GTP-bound RagA/B to recruit mTORC1 to the lysosome where it can be fully activated by GTP-Rheb (Figure 1). RagA and RagC coimmunoprecipitated with FLCN that had been co-expressed with FNIP2, suggesting that a FLCN-FNIP complex is required for either of the two proteins to interact with the Rag GTPases (Petit et al., 2013; Tsun et al., 2013). This behavior is recapitulated in yeast, where the respective orthologs, Lst7 and Lst4, localize to the vacuolar membranes when cells are starved, but rapidly dissociate from this compartment when amino acid levels are restored (Pacitto et al., 2015; Péli-Gulli et al., 2015). Loss of either Lst4 or Lst7 similarly decreases TORC1 activity in yeast cells, suggesting that they share a common biological activity toward TORC1 (Péli-Gulli et al., 2015).

The identification of FLCN-FNIP as a critical component of the lysosome-localized machinery for the proper transduction of signals of amino acid availability to mTORC1 through the Rag GTPases, spurred efforts to understand the functional relevance of the FLCN-FNIP recruitment to this compartment. FLCN-FNIP complex possesses GAP activity toward RagC/D, a fact established through GTP hydrolysis assays using different combination of Rag mutants in HEK293T cells, and hence promotes RagC/D-GDP occupancy, activating mTORC1 in an amino acid dependent manner (Tsun et al., 2013). A FLCN-FNIP2 complex is required to exert GAP activity toward RagC/D, as the individual proteins are insufficient to catalyze nucleotide exchange. This is borne out by the presence of longin domains on both FLCN and FNIP proteins, similar to those found in GATOR1 (Shen et al., 2018) which acts as a GAP for RagA/B. Indeed, a recent study proposed that FLCN-FNIP GAP activity occurs downstream of GATOR1 (Meng and Ferguson, 2018); when amino acid levels are low, the GAP activity of GATOR1 promotes the GDP-RagA/B state, and FLCN/FNIP is then recruited to the lysosome to act as a GAP toward RagC/D. Although the FLCN-FNIP GAP activity toward RagC/D identifies this complex as a positive regulator of mTORC1, renal tumors from BHD patients containing FLCN germline mutations showed increased mTORC1 activity (Baba et al., 2008; Hasumi et al., 2009), as well as higher levels of phosphorylation of mTORC1 substrates (Khabibullin et al., 2014). In light of these counterexamples, it has been suggested that the role of FLCN loss of function in renal tumorigenesis may be more sophisticated, not only involving mTORC1 activation, but also other signaling pathways that in turn can affect mTORC1 signaling.

While we have discussed mTORC1 activation with respect to nutrient availability extensively, this kinase also responds to other stimuli, including energy availability. Low energy conditions are normally characterized by high AMP:ATP ratios, which allosterically activates another central metabolic regulator, the AMP-activated protein kinase (AMPK) (Carling, 2004; Shackelford and Shaw, 2009). AMPK is a heterotrimeric kinase formed by a catalytic core, the α subunit, and two regulatory subunits; β and γ (Figures 2A,B). There are multiple gene products for each subunit (α1, α2; β1, β2; γ1, γ2, γ3) which can combine to form twelve different heterotrimers (Stapleton et al., 1994; Thornton et al., 1998; Cheung et al., 2000). The α subunit contains a typical serine/threonine protein kinase catalytic domain (Hanks et al., 1988) as well as several phosphorylation sites, with α1/α2-Thr172/174 being essential for AMPK activity (Stein et al., 2000) (Figure 2A). Multiple crystal structures of the complete kinase complex support a functional role for Thr172/174: binding of AMP or other synthetic activators protects this threonine residue from dephosphorylation and, therefore, AMPK inactivation through phosphatase activity (Figure 2B, inset) (Xiao et al., 2011, 2013; Calabrese et al., 2014; Yan et al., 2019). Two upstream kinases, Liver kinase B1 (LKB1) and calmodulin kinase kinase (CAMKK), are responsible for the activation of AMPK by phosphorylation of Thr172/174, depending on the cellular context (Woods et al., 2003; Shaw et al., 2004). Activated AMPK increases ATP production through promotion of catabolic pathways and the inhibition of synthetic pathways that consume ATP, in a manner antagonistic to mTORC1 activity. Additionally, AMPK inhibits mTORC1 both directly, through the phosphorylation of its component Raptor (Gwinn et al., 2008), and indirectly, by phosphorylation and activation of TSC2 (Inoki et al., 2002). Crosstalk between mTORC1 and AMPK provides a more specific mechanism by which cell growth may be coordinated according to environmental conditions.

However, inhibition of mTORC1 by AMPK is not the only relationship between these two signaling pathways. Coimmunoprecipitation experiments have demonstrated that FNIP1 can interact with all subunits of AMPK, in vitro, and that FLCN is not essential for FNIP-AMPK binding (Baba et al., 2006). FNIP2 was also identified as a FLCN interacting partner that binds AMPK (Hasumi et al., 2008; Takagi et al., 2008). Although it is widely accepted that FLCN binds to AMPK through the FNIPs, the functional relevance of this interaction remains unexplained. It is notable that FNIPs can be phosphorylated by AMPK and both FLCN and FNIP phosphorylation levels are affected by both AMPK signaling and amino acid starvation, which in turn inhibit mTORC1 (Baba et al., 2006; Takagi et al., 2008). Interestingly, FLCN phosphorylation appears to affect complex formation; FNIP1 and FNIP2 preferentially binding phosphorylated FLCN (Baba et al., 2006; Takagi et al., 2008). Additionally, the interaction between the FLCN complex and AMPK has been linked to the induction of apoptosis (Lim et al., 2012) and increased FLCN expression is associated with AMPK-dependent dephosphorylation of Transcription Factor EB (TFEB), which causes its translocation to the nucleus independently of mTORC1 (Collodet et al., 2019), both examples connecting FLCN-FNIP-AMPK with the coordination of metabolism. Finally, both FLCN and FNIP1 have been suggested as negative regulators of AMPK as depletion of FLCN has been found to constitutively activate AMPK (Possik et al., 2014, 2015; Yan et al., 2014; El-Houjeiri et al., 2019) and mutations affecting FNIP1 are associated with higher AMPK activity (Siggs et al., 2016), suggesting that FLCN and FNIP may cooperate to modulate AMPK. In the same way, phosphorylated FNIP1 can bind to chaperone Hsp90, which indeed, regulates proper folding of AMPK subunits and some mTORC1 pathway components, such as Raptor or mTOR itself, suggesting another function for FNIP1 in the regulation of AMPK and mTORC1 pathways (Woodford et al., 2016; Sager et al., 2018, 2019). All this evidence strongly suggests a role for FLCN-FNIP in coordinating cellular metabolism through its effects on both the mTORC1 and AMPK signaling pathways, however, further studies are eagerly awaited to clarify the functional relevance of the FLCN-FNIP-AMPK interaction.

The consensus view of mTORC1 signaling is that it is only active when both the Rag GTPase and the Rheb GTPase axes are fully activated, meaning that mTORC1 needs to be recruited to lysosomes to be fully operational. However, within the signaling pathway this organelle is not solely a platform for the proper assembly of mTORC1 regulatory elements. It is much more deeply involved in the shift between anabolism and catabolism, a function that has presumably evolved as a result of its pivotal role in autophagy. The relationship between mTORC1 and the lysosome is crucial for the control of lysosomal function and provides this organelle with the capacity to sense nutrient availability and generate a cellular response (Settembre and Ballabio, 2014; Rabanal-Ruiz and Korolchuk, 2018). Lysosomes comprise a single-lipid bilayer membrane containing a set of luminal hydrolases responsible for the degradation of a wide range of substrates, including sugars, lipids proteins and nucleic acids (de Duve et al., 1955; de Duve, 2005; Sleat et al., 2013). Lysosomal hydrolases have an acidic optimum pH, therefore the lysosomal lumen needs to be acidic for the proper function of this organelle, promoting protein unfolding (Xu and Ren, 2015). The acidic pH of the lysosomal lumen is maintained by the v-ATPase, introduced earlier as a putative amino acid sensor, a large channel that pumps protons across the lysosomal membrane toward the lumen (Mindell, 2012). Intracellular and extracellular substrates can both be degraded by lysosomes. While extracellular macromolecules reach the lysosome through endocytosis (Conner and Schmid, 2003), cytoplasmic macromolecules or damaged proteins are processed through the autophagic pathway (He and Klionsky, 2009; Kaur and Debnath, 2015).

The function of lysosomes is dependent on their ability to move bidirectionally between the centrosomal and peripheral areas of the cell along linear tracks formed by microtubules (Matteoni and Kreis, 1987). The balance between centrosomal and peripheral lysosomal transport defines the dynamic cytoplasmic distribution of lysosomes, which are normally well-distributed over the cytoplasm with a small degree of enrichment around the nucleus, close to the microtubule organizing center (MTOC) and the Golgi apparatus (Korolchuk et al., 2011). There are two different well-established pathways that control lysosome transport toward the centrosomal and the peripheral region of the cell respectively. Transport toward the plus end of microtubules, usually located at the cell periphery, is regulated by kinesins. The BLOC-one-related complex (BORC) mediates this process by recruiting the small GTPase Arf-like8b (Arl8b), which in turn binds to the adaptor protein SiFA and kinesin interacting protein (SKIP) to finally recruit kinesin-1, the motor responsible for transport (Rosa-Ferreira and Munro, 2011; Pernigo et al., 2013). In parallel, Rab7 and FYVE and coiled-coil domain containing 1 (FYCO1) can also recruit kinesin-1 to promote plus end movement of lysosomes (Pankiv et al., 2010; Raiborg et al., 2015). Conversely, transport toward the minus end of microtubules, which are located within the perinuclear or centrosomal region of the cell, is regulated by dynein-mediated pathways. Rab7 initiates this process by binding to Rab interacting lysosomal protein (RILP), which ultimately recruits cytoplasmic dynein (Cantalupo et al., 2001; Jordens et al., 2001). Alternatively, other studies have shown that the Golgi-located Rab34 and Rab36 GTPases can also recruit RILP, hence affecting lysosome distribution by promoting perinuclear clustering (Wang and Hong, 2002; Goldenberg et al., 2007; Chen et al., 2010).

It is noticeable that the intracellular distribution of lysosomes correlates with nutrient availability: in HeLa cells, starvation promotes not only the inhibition of mTORC1 activity but also results in a lysosomal clustering on the perinuclear region (Korolchuk et al., 2011). Additionally, withdrawal of nutrients promotes the translocation of the MiT family transcription factors TFEB and Transcription Factor Binding To IGHM Enhancer 3 (TFE3) to the nucleus to induce lysosomal biogenesis and autophagosome formation (Sardiello et al., 2009; Settembre et al., 2011). During autophagy, vesicles containing damaged protein and cell debris are transported to the minus end of microtubules, and therefore the perinuclear region of the cell, where they are finally fused to the lysosomes responsible for the degradation of their contents. The promotion of autophagy links the inhibition of mTORC1 activity during starvation to activation of catabolic processes through lysosomal activity (Rabanal-Ruiz and Korolchuk, 2018; Kim and Guan, 2019). Given the availability of amino acids and nutrients the situation is reversed, the minus end transport of lysosomes being inhibited with the result that instead lysosomes are located close to the plasma membrane (Korolchuk et al., 2011). Amino acid availability activates mTORC1, which in turn phosphorylates TFEB and TFE3, preventing their translocation to the nucleus, and hence inhibiting lysosomal biogenesis (Martina et al., 2012; Roczniak-ferguson et al., 2012; Settembre et al., 2012). Additionally, amino acids promote the recruitment of kinesin-1 adaptor FYVE And Coiled-Coil Domain Containing 1 (FYCO1) to lysosomes through interaction with PtdIns3 (Nobukuni et al., 2005; Hong et al., 2017), and the formation of contact sites between lysosomes and the endoplasmic reticulum (ER) through Rab7-Protrudin-PtdIns3 binding. Within these lysosomes-ER contact sites, the transfer from Protrudin to FYCO1 allows lysosomes to be loaded onto kinesin-1, therefore promoting transport toward to cell periphery (Raiborg et al., 2015). During starvation, it is clear that lysosomes are clustered toward the centrosomal region of the cell to favor catabolic processes such as autophagy, however, the evolutionary reason for their peripheral location upon nutrient addition remains ambiguous. It has been suggested that lysosomes cluster on the plasma membrane to be closer to the site of growth factor signaling for the full activation of mTORC1 (Korolchuk et al., 2011) and that growth factors are insufficient to activate mTORC1 unless lysosomes are close to the plasma membrane (Hong et al., 2017). Supporting such a relationship between lysosome positioning and mTORC1 activation, immunoblotting assays in HEK293 cells demonstrated that depletion of microtubule motors appeared to disrupt Rag GTPase-dependent mTORC1 activation (Li and Guan, 2013). Overall, there is clear evidence that the lysosomal positioning and mTORC1 signaling pathways are intimately linked, however further studies are needed to shed light on the specific mechanisms by which this is achieved.

We have covered the means by which the FLCN-FNIP complex is recruited to the lysosome upon starvation, where it interacts with the Rag GTPases and has GAP activity toward RagC (Petit et al., 2013; Tsun et al., 2013), thus playing a crucial role in the proper recruitment of mTORC1 to the lysosome. However, the interaction with the Rag GTPases to recruit mTORC1 is not the only lysosome-related function of FLCN. The loss of FLCN inhibits the phosphorylation of transcription factors TFEB and TFE3, promoting their nuclear translocation and activating lysosome biogenesis (Hong et al., 2010; Martina et al., 2014). Supporting this notion, structure-function analysis have suggested that the role of FLCN in regulating TFEB and TFE3 localization is dependent on the GAP activity of FLCN toward RagC (Lawrence et al., 2019), whereas immunoblotting assays in mammalian and nematode cells suggested that the loss of FLCN drives TFEB and TFE3 nuclear localization independently from the canonical mTORC1 pathway (El-Houjeiri et al., 2019). On the other hand, FLCN-deficient human cells appear to have impaired autophagosome maturation (Dunlop et al., 2014), suggesting a role for FLCN in autophagy regulation, which has also been reported during studies of the AMPK-FLCN functional relationship (see Section “A Potential Interaction of FLCN-FNIP With AMPK”).

Additionally, immunofluorescence analyses of HeLa cells demonstrated that upon starvation, FLCN is not only recruited to the lysosomes but also that perinuclear clustering of this organelle occurs. Pull-down assays have shown that FLCN can interact with both Rab7 and Rab34, which share RILP as an interaction partner (Starling et al., 2016), suggesting that the formation of a FLCN-RILP-Rab34 complex could be the reason for the perinuclear clustering of lysosomes and identifying a functional connection between lysosomal positioning and the mTORC1 pathway. The formation of a FLCN-Rab34/7-RILP complex is supported by the fact that both FLCN and FNIP have longin and DENN domains, known to be commonly found in GEFs for the Rab GTPases (Levivier et al., 2001). FLCN seems to directly interact with RILP through its C-terminal DENN domain (Starling et al., 2016), a different region from that responsible for its interaction with the Rag GTPases, suggesting a potential role for FLCN-DENN domain in the regulation of lysosomal distribution. The interaction between Rab7 and RILP has previously been studied and a crystal structure is available (Wu et al., 2005), showing that GTP-bound Rab7 binds to RILP through its effector-interacting switch regions, usually recognized by most Rab effectors (Wu et al., 2005) (Figures 2C,D). No GEF or GAP activity has been reported for FLCN-FNIP toward Rab34, although it has been reported that FLCN regulates Rab7 GTPase, acting as a GAP to mediate lysosome-mediated degradation of EGFR (Laviolette et al., 2017). Currently, the functional relevance of the FLCN-RILP-Rab interaction appears to be the promotion of Rab-RILP assembly to promote clustering of lysosomes around the nucleus during nutrient insufficiency, however, structure-function studies are awaited to reveal further details about this significant interaction. Interestingly, there are also reported interactions between FLCN and other Rab GTPases, a family of proteins usually involved in intracellular membrane trafficking (Hutagalung and Novick, 2011). In vitro guanine nucleotide exchange assays have suggested that FLCN possesses GEF activity toward Rab35 (Nookala et al., 2012) and that it can bind to this small GTPase through its C-terminus to Rab35, to regulate EGFR intracellular trafficking (Zheng et al., 2017). Although cell-based assays have been unable to validate FLCN GEF activity toward Rab35, its interaction with Rab35 in EGFR regulation provides another example of FLCN linking membrane trafficking to cell growth. Additionally, FLCN was reported to bind to Rab11 via its C-terminal DENN domain and can also promote the Rab11-PAT1 interaction (Zhao et al., 2018). Although no GEF activity of FLCN toward Rab11 has been identified, the FLCN-Rab11 interaction suggests that, apart from tethering the Rags for a proper recruitment of mTORC1 in the lysosome, FLCN may have other roles in the maintenance of the amino acid signal level within the lysosome through amino acid transporters, such as PAT1.

Interestingly, FLCN is not the only mTORC1 component related to the lysosomal machinery. The Ragulator complex, an essential multiprotein complex for mTORC1 recruitment to the lysosome, has been reported to interact with BORC to inhibit lysosomal transport toward the cell periphery upon starvation (Filipek et al., 2017; Pu et al., 2017). Furthermore, RILP was shown to directly interact with the v-ATPase and regulate its activity (De Luca et al., 2014), and mTORC1, itself, has been suggested to regulate v-ATPase, both examples showing potential mechanisms coupling lysosomal positioning and nutrient signaling (Peña-Llopis et al., 2011). Overall, lysosomal positioning and mTORC1 signaling are clearly interconnected with the nutrient signaling response. Moreover, several studies have reported that they share regulatory components (Figure 3). Accordingly, multiple FLCN-Rab relationships have been reported that directly link the mTORC1 pathway to the lysosomal positioning components, providing a mechanistic explanation for the intimate relationship between this organelle and nutrient signaling. Therefore, structure-function studies of the full FLCN-RILP-Rab complex are urgently needed to fully understand the role of FLCN in the intersection of mTORC1 pathway and lysosome positioning.