InteractiveFly: GeneBrief

pathetic: Biological Overview | References

Drosophila neuroblasts (NBs) are neural stem cells whose maintenance relies on Notch activity. NBs proliferate throughout larval stages to generate a large number of adult neurons. Their proliferation is protected under conditions of nutrition restriction. As amino acid transporters (Solute Carrier transporters, SLCs), such as SLC36, have important roles in coupling nutrition inputs to growth pathways, they may have a role in this process. For example, an SLC36 family transporter Pathetic (Path) that supports body size and neural dendrite growth in Drosophila, was identified as a putative Notch target in genome-wide studies. This study examined expression and regulation of Path in the Drosophila larval brain. Path function in NB proliferation and overall brain growth was investigated under different nutrition conditions by depleting it from specific cell types in the CNS, using mitotic recombination to generate mutant clones or by directed RNA-interference. Path is expressed in both NBs and glial cells in the Drosophila CNS. In NBs, path is directly targeted by Notch signalling via Su(H) binding at an intronic enhancer, PathNRE. This enhancer is responsive to Notch regulation both in cell lines and in vivo. Loss of path in neural stem cells delayed proliferation, consistent with it having a role in NB maintenance. Expression from pathNRE (Notch responsive element) was compromised in conditions of amino acid deprivation although other Notch regulated enhancers are unaffected. However, NB-expressed Path was not required for brain sparing under amino acid deprivation. Instead, it appears that Path is important in glial cells to help protect brain growth under conditions of nutrient restriction. This study has identified a novel Notch target gene path that is required in NBs for neural stem cell proliferation, while in glia it protects brain growth under nutrition restriction (Feng, 2020).

Drosophila Neuroblasts (NBs) are neural stem cells that divide to give progeny, which differentiate into neurons and glia that later constitute the adult brain. NBs arise from neuroectoderm during embryonic development and enter quiescence at the end of the embryonic stage, until they are reactivated upon feeding during larval stages. After reactivation, around 350 NBs reside on the surface of the brain and constitute the stem cell pool, undergoing multiple rounds of asymmetric cell divisions. During division, each NB generates one larger daughter cell that retains stem cell identity and one smaller daughter cell that divides further to generate progeny that differentiate into certain types of neurons and glia. At the time of metamorphosis, the central nervous system (CNS) contains about 30,000 neurons and 10,000 glial cells. The glial cells fulfil supporting and nurturing function to neurons. Importantly they also ensure NBs receive the correct growth signal at the correct times. For example, signals from glia are necessary for NBs to exit quiescence upon feeding, and to remain proliferative during nutrition deprivation once the larva passes the critical weight time-point (Feng, 2020).

Notch signalling is one of the key regulators in maintaining NSCs and performs a similar function in both Drosophila and vertebrate NSCs. Notch depletion causes loss of NB lineages while Notch over-activation inhibits NBs from differentiating and induces brain tumours. In the canonical Notch signalling model, upon Notch ligand binding to the receptor, the Notch intracellular domain (NICD) is cleaved and released into the nucleus. The nuclear NICD interacts with the DNA-binding protein known as Suppressor of Hairless (Su(H)) in flies, to activate the expression of target genes. The functions of Notch are very context-dependent, making it important to identify the Notch regulated genes in different processes including stem cell maintenance (Feng, 2020).

The brain, like other organs, needs to translate changing nutrition inputs into cell growth decisions. An emerging role of amino acid transporters, especially the SLC family, in coupling the nutrition signalling and growth pathways has been revealed in recent years. SLC38A9 acts as an amino acid sensor in the process of mTORC-activation in mammalian cell lines [Wang, 2015; Rebsamen, 2015]. Similarly, SLC36A4 helps to promote proliferation in colorectal cancer cells through its interaction with mTORC1 (Fan, 2016). A requirement for SLC36A4 in mice retinal pigmented epithelial cells also involved mTORC-activation (Shang, 2017). However, where and how might these transporters function in other cases of nutrient sensing, such as the Drosophila NBs, remains unknown. Also, it is unclear whether the growth-promoting role of these amino acid transporters would be adaptive to starvation. For example, the sparing mechanism in nutrient deprived NBs somehow bypasses the Tor pathway (Feng, 2020).

Pathetic (Path) is the Drosophila orthologue of SLC36A4, having the characteristics of a broad specificity transporter with multiple transmembrane domains. It interacts with Tor (Target of apamycin) pathway components in regulating eye growth and body growth of Drosophila and promotes dendritic growth in C4da neurons (Lin, 2015). path also exhibited the hall marks of a Notch regulated gene in a genome-wide study of genes upregulated during Notch-induced NB hyperplasia (Zacharioudaki, 2016). This study has followed up on this observation by analysing the role and regulation of Path in NBs under normal and abnormal nutrition conditions. path was shown to be a novel direct Notch target in NBs, and it is required for NB proliferation. Further its role in brain sparing was characterised, and it was found to be required to fully protect the CNS from nutrient restriction. However, the evidence indicates that glial-expressed Path is important for protecting brain growth under nutrient restriction, rather than its activity in the NBs themselves (Feng, 2020).

Previously, Path was found to be required for overall body growth and extreme dendrite growth, potentially through interacting with the PI3K/Tor pathway and protein synthesis pathways (Lin, 2015). This study identified an autonomous role of path in maintaining NB proliferation, which is in line with previous findings that path promotes growth. Path expression in NBs is partially dependant on Notch, as it contains an intronic enhancer, which is directly regulated by the pathway. Thus its regulation and involvement in NB proliferation argues that Path contributes to the normal function of Notch in NBs, although it remains to be established whether it has a similar essential role in Notch induced tumours. Furthermore, the striking reduction in pathNRE driven expression under nutrient restriction (NR) suggests that the NBs are sensitive to changes in the internal milieu of the animal. This argues that, while glial cells may shield NBs from many environmental effects, the NBs are nevertheless able to detect altered nutrient levels and may harbour addition pathways that contribute to brain-sparing (Feng, 2020).

Although this study found that Path is required for brain sparing (the maintenance of brain size) during NR, this appears to rely on its expression in Glial cells rather than NBs, despite the fact that the intronic pathNRE enhancer is sensitive to nutrient deprivation. Alk/Jeb is the major pathway that has been linked to brain growth under NR conditions, and glial knockdown of Jeb (repo-gal4 > jebRNAi) resulted in smaller NB-clone size as well as lineage number. It is possible that Path at the membrane of surface glial cells could detect the environmental amino acid levels and in turn regulate Alk/Jeb expression. Path is a potential amino acid transporter with multiple transmembrane domains, which exhibited high affinity for alanine and glycine with low transporting capacity when expressed in Xenopus oocytes. The closest mammalian relatives, proton-assisted transporter 4 (PAT4 or hPAT4), have a high affinity for proline and tryptophan. Although it remains to be fully determined which are the main substrates for Path in vivo, a recent study suggests that Path is required for scavenging proline to promote tumour growth in conditions of obesity enhanced tumorigenesis in Drosophila (Newton, 2020). In these conditions, Path regulation was also involved with nutrient sensing mechanisms albeit the levels of sugars were elevated rather than restricted. Further studies will be needed to determine whether proline is the primary substrate for Path in all conditions and the extent that path expression is differentially regulated by changes in nutrient levels according to the tissue and its proliferative state (Feng, 2020).

The Tor pathway is involved in NB reactivation, growth and proliferation. However, in L3, NB growth is regulated by the Alk/Jeb pathway which activates downstream PI3K/Akt signalling, but appears independent of Tor. One model to explain the current results is that Path functions as a mediator between Alk and the Tor pathway. Alk is required for and acts through the PI3K/Akt pathway. In L3 larvae brains, most Tor pathway components are dispensable for brain growth and brain size is not changed significantly when levels of Insulin-like peptides (Ilp) are manipulated. In contrast, the Tor pathway is activated in younger brains suggesting there may be a mechanism to bypass or switch-off the Tor pathway at later stages. One hypothesis, considering pathNRE is upregulated after NB reactivation, is that path helps to keep activity of the Tor pathway at restricted levels at later stages. In this case, the effects of path in NBs would be through nutrient sensing and protein synthesis, similar to its function in C4da neurons (Feng, 2020).

The regulation and expression of Path in the NBs suggests that, in this context, Path promotes cell growth and lineage size. Further studies will be needed to characterise what the consequences from Path up-regulation are in different circumstances. For example, whether Path has the same role in Notch induced NB tumours, and whether this would differ depending on nutrient status, remains to be established. Likewise, this study has investigated its role in so-called Type I NBs. Not mutant clones were obtained to distinguish whether it makes the same contribution in Type II NBs, which produce a highly proliferative transit amplifying intermediary, or whether its activity there differs. Finally, it is striking that pathNRE-GFP expression is decreased in neuroblasts under nutrient restriction, when neuroblast proliferation becomes spared. Whether this reflects a physiological response, whereby NB Path levels are reduced while glial cell Path persists under NR, or whether it reflects a regulatory feature, whereby a different enhancer becomes activated in these conditions, remains to be established. Nevertheless, these data highlight the importance of distinguishing the different regulatory inputs to Path, and the extent that they are dependent on different nutrient and/or amino acid (Feng, 2020).

Brain expression of Path, which encodes a broad specificity transporter, occurs in both NBs and glial cells where its functions appear to differ. NB expression of path, is partially dependent on Notch activity and is required for NB lineage proliferation but not for brain sparing under nutrient restrictions. In contrast Glial expression of Path appears to be independent of Notch and is essential for protecting the brain growth under nutrition restriction. In conclusion, this study has demonstrate different roles of Path in distinct parts of the brain that would together enable the larval brain to proliferate and grow in both normal and NR conditions (Feng, 2020).

Systemic muscle wasting and coordinated tumour response drive tumourigenesis

Cancer cells demand excess nutrients to support their proliferation, but how tumours exploit extracellular amino acids during systemic metabolic perturbations remain incompletely understood. This study used a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis to uncover a systemic host-tumour metabolic circuit that supports tumour growth. Coordinate induction is demonstrated of systemic muscle wasting with tumour-autonomous Yorkie-mediated SLC36-family amino acid transporter expression as a proline-scavenging programme to drive tumourigenesis. Indole-3-propionic acid was identified as an optimal amino acid derivative to rationally target the proline-dependency of tumour growth. Insights from this whole-animal Drosophila model provide a powerful approach towards the identification and therapeutic exploitation of the amino acid vulnerabilities of tumourigenesis in the context of a perturbed systemic metabolic network (Newton, 2020).

Cancer cells require a constant supply of metabolic intermediates to support their proliferation. To meet the biosynthetic demands associated with tumourigenesis, cancer cells actively acquire nutrients from the extracellular space. Cancer is a systemic disease that associates with a range of host metabolic abnormalities such as obesity, insulin resistance and cancer-associated cachexia; each of which alters the host systemic nutritional environment. These changes in both nutrient composition and availability may have profound effects on cancer development and progression. However, how cancer cells sense and respond to nutritional changes in the context of organismal metabolic alterations remains an underexplored area in cancer biology (Newton, 2020).

Cancer-associated cachexia is a systemic metabolic syndrome of weight loss associated with progressive skeletal muscle wasting. The multifactorial and heterogeneous condition of cachexia involves a complex multi-organ interplay, which has impeded its comprehensive understanding at the molecular level. A series of recent studies using Drosophila melanogaster have shown that tumour-derived factors modulate host metabolism. In addition, tumour-derived factors promote the release of nutrients from the tumour microenvironment to promote tumour growth. This study has leveraged a Drosophila model of high-sugar diet (HSD)-enhanced tumourigenesis and demonstrates that HSD-enhanced tumours induce SLC36-family transporter expression as a coordinated mechanism to exploit exogenous proline for tumourigenesis during systemic muscle wasting. Furthermore, these mechanistic insights were used to rationally target the proline dependency of tumours as an approach to inhibit tumour growth (Newton, 2020).

Muscle wasting is observed in chronic muscle wasting diseases due to cancer (cachexia), ageing/senescence (sarcopenia), myopathies and other metabolic diseases, as well as in acute conditions due to burns and sepsis. This study demonstrates that the combination of diet-induced obesity and tumour growth induces systemic muscle wasting, associated with functional locomotion defect. This muscle phenotype was not due to the inability to properly form muscle during development; the muscles are wholly formed at the early stage of larval development, and waste progressively at the later larval stage, as the tumours develop. In addition, it was demonstrated that the muscle phenotype observed in this model is unrelated to a developmentally related degeneration process (Newton, 2020).

This study reveals a systemic amino acid-utilising circuit whereby HSD-enhanced tumours induce muscle wasting as a systemic metabolic network to drive tumourigenesis. A consequence of muscle wasting in ras1^G12V;csk^-/- animals raised on HSD was increased release of proline into the circulation. Plasma amino acid profiling of patients with sarcopenia - a condition of muscle wasting associated with aging—showed elevated plasma proline levels, indicating that elevated free circulating proline is a common feature of muscle wasting. This study identified a proline vulnerability of HSD-enhanced tumours; SLC36-family amino acid transporter Path is required for tumour growth and exogenous proline promotes tumourigenesis through Path. This study highlights two layers of coordination in tumour metabolic response: (1) at the whole organism level, by promoting muscle wasting and systemic amino acid availability, and (2) at the tumour-autonomous level, by altering amino acid transporter repertoire (Newton, 2020).

Path and CG1139 have different transport characteristics for proline, with Path having a lower transport capacity compared to CG1139. Despite this, labelled proline uptake experiments indicated uptake of extracellular proline in Ras/Src/Path-tumours but not in Ras/Src/CG1139-tumours, suggesting that CG1139 and Path may exhibit different activities in the context of an oncogenic background. Consistent with a previous report (Goberdhan, 2005), the current data support the signalling role of Path through activation of the Tor-S6K pathway. Proline metabolism has been demonstrated to support cancer clonogenicity and metastasis. Furthermore, proline promotes cancer cell survival under nutrient-limited or hypoxic microenvironments. The current data extend these studies to reveal a tumour-promoting role of proline in response to systemic host metabolic changes (Newton, 2020).

Indole-3-propionic acid (IPA) specifically targets the proline dependency of tumour growth in Drosophila. Furthermore, this study demonstrates a functional effect of IPA on human cells. A recent study demonstrated that proline uptake in Ras-driven tumour cells is much higher in spheroids in three-dimensional cultures—which better mimic conditions in vivo—compared to monolayers in two-dimensional cultures, suggestive of a potentially stronger effect in tumours that are dependent on proline for growth in vivo. Proline is a limiting amino acid for protein synthesis in kidney cancers. Therefore, reducing proline uptake with SLC36-inhibitors -- as exemplified here by use of IPA -- may warrant consideration as a therapeutic strategy to break the nutritional circuit between systemic muscle wasting and tumour growth in proline vulnerable cancers (Newton, 2020).

Functions of the SLC36 transporter Pathetic in growth control

Neurons exhibit extreme diversity in size, but whether large neurons have specialized mechanisms to support their growth is largely unknown. The SLC36 amino acid transporter Pathetic (Path) has been identified as a factor required for extreme dendrite growth in neurons. Path is broadly expressed, but only neurons with large dendrite arbors or small neurons that are forced to grow large require path for their growth. To gain insight into the basis of growth control by path, this study generated additional alleles of path and further examined the apparent specificity of growth defects in path mutants. The prior finding that loss of path function imposes an upper limit on neuron growth was conformed, and additionally it was found that path likely limits overall neurite length rather than dendrite length alone. Using a GFP knock-in allele of path, additional tissues were identified where path likely functions in nutrient sensing and possibly growth control. Finally, it was demonstrated that path regulates translational capacity in a cell type that does not normally require path for growth, suggesting that path may confer robustness on growth programs by buffering translational output. Altogether, these studies suggest that Path is a nutrient sensor with widespread function in Drosophila (Lin, 2016).

How growth programs are tailored to meet the demands of large neurons is largely unknown. This study demonstrates that the SLC36 transporter Path is required for growth of axons and dendrites in large neurons. Although the precise mechanism of action remains to be determined, the amino-terminal intracellular domain is essential for Path function in neuron growth control (Lin, 2015) and it is hypothesized that Path transduces nutrient signals via protein-protein interactions between its N-terminal intracellular domain and unidentified signaling proteins to modulate translational output . In light of prior studies suggesting a link between SLC36 transporters and TORC1 signaling and the observation that co-overexpression of Path and Rheb potentiates dendrite growth (Lin, 2015) it is proposed that Path may promote neuron growth, in part, through regulation of TORC1 activity. However, given the observations that Path primarily localizes to the plasma membrane and that mutations in TORC1 components cause more modest neuron growth deficits than mutation of path, it seems likely that Path engages downstream pathways other than TORC1 to promote neuron growth. Although the identity of these pathways is currently unknown, eukaryotic initiation factor 2 (eIF2) activity is regulated by a suite of kinases (eIF2K) that tune translation levels in response to environmental stresses, including amino acid deficiency (Lin, 2016).

Although Path is broadly expressed, path is dispensable for growth in most cell types under well-fed laboratory conditions. One prediction of this observation is that Path selectively affects translation in large neurons. The results from this study refute this notion; cells that do not require path for growth still require path for maximum translational output. How can the disparity in growth phenotypes between neurons and other cells explaned? First, Path may play a more significant role in translational control of neurons than other cell types. Drosophila has several uncharacterized genes that encode SLC36 transporters, and these gene products may function redundantly to control growth together with Path in some cell types. Consistent with this notion, overexpression of CG1139, which encodes another SLC36 transporter, drives ommatidial overgrowth in the eye (Goberdhan, 2005) Second, the residual translational capacity in path mutants is likely sufficient to support normal growth in many cell types, at least under certain conditions. Similarly, loss of Drosophila 4E-BP (Thor) function reduces overall translation without any obvious effect on animal growth in well-fed conditions. Intriguingly, environmental stresses such as hypoxia or starvation uncover a requirement for Thor in growth control. Similarly, it is predicted that stresses that compromise translational efficiency should sensitize cells to a requirement for path function, therefore an exciting future direction will be to determine whether path exerts state-dependent functions on growth control (Lin, 2016).

The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons

Dendrites exhibit enormous diversity in form and can differ in size by several orders of magnitude even in a single animal. However, whether neurons with large dendrite arbors have specialized mechanisms to support their growth demands is unknown. To address this question, a genetic screen was conducted for mutations that differentially affected growth in neurons with different-sized dendrite arbors. From this screen, a mutant was identified that selectively affects dendrite growth in neurons with large dendrite arbors without affecting dendrite growth in neurons with small dendrite arbors or the animal overall. This mutant disrupts a putative amino acid transporter, Pathetic (Path), that localizes to the cell surface and endolysosomal compartments in neurons. Although Path is broadly expressed in neurons and nonneuronal cells, mutation of path impinges on nutrient responses and protein homeostasis specifically in neurons with large dendrite arbors but not in other cells. Altogether, these results demonstrate that specialized molecular mechanisms exist to support growth demands in neurons with large dendrite arbors and define Path as a founding member of this growth program (Lin, 2015).

Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes

Mammalian Target of Rapamycin Complex 1 (mTORC1) is activated by growth factor-regulated phosphoinositide 3-kinase (PI3K)/Akt/Rheb signalling and extracellular amino acids (AAs) to promote growth and proliferation. These AAs induce translocation of mTOR to late endosomes and lysosomes (LELs), subsequent activation via mechanisms involving the presence of intralumenal AAs, and interaction between mTORC1 and a multiprotein assembly containing Rag GTPases and the heterotrimeric Ragulator complex. However, the mechanisms by which AAs control these different aspects of mTORC1 activation are not well understood. Recent work has shown that intracellular Proton-assisted Amino acid Transporter 1 (PAT1)/SLC36A1 is an essential mediator of AA-dependent mTORC1 activation. This study demonstrates in Human Embryonic Kidney (HEK-293) cells that PAT1 is primarily located on LELs, physically interacts with the Rag GTPases and is required for normal AA-dependent mTOR relocalisation. The powerful in vivo genetic methodologies available in Drosophila were used to investigate the regulation of the PAT1/Rag/Ragulator complex. GFP-tagged PATs reside at both the cell surface and LELs in vivo, mirroring PAT1 distribution in several normal mammalian cell types. Elevated PI3K/Akt/Rheb signalling increases intracellular levels of PATs and synergistically enhances PAT-induced growth via a mechanism requiring endocytosis. In light of the recent identification of the vacuolar H(+)-ATPase as another Rag-interacting component, a model is proposed in which PATs function as part of an AA-sensing engine that drives mTORC1 activation from LEL compartments (Ogmundsdottir, 2012).

PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids

Growth in normal and tumour cells is regulated by evolutionarily conserved extracellular inputs from the endocrine insulin receptor (InR) signalling pathway and by local nutrients. Both signals modulate activity of the intracellular TOR kinase, with nutrients at least partly acting through changes in intracellular amino acid levels mediated by amino acid transporters. This study shows that in Drosophila, two molecules related to mammalian proton-assisted SLC36 amino acid transporters (PATs), CG3424 and CG1139, are potent mediators of growth. These transporters genetically interact with TOR and other InR signalling components, indicating that they control growth by directly or indirectly modulating the effects of TOR signalling. A mutation in the CG3424 gene, which was named pathetic (path), reduces growth in the fly. In a heterologous Xenopus oocyte system, PATH also activates the TOR target S6 kinase in an amino acid-dependent way. However, functional analysis reveals that PATH has an extremely low capacity and an exceptionally high affinity compared with characterised human PATs and the CG1139 transporter. PATH and potentially other PAT-related transporters must therefore control growth via a mechanism that does not require bulk transport of amino acids into the cell. As PATH is likely to be saturated in vivo, it is proposed that one specialised function of high-affinity PAT-related molecules is to maintain growth as local nutrient levels fluctuate during development (Goberdhan, 2005).

The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H(+) -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. This study provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding betaA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression (Shang, 2017).

PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer

Tumour cells can use strategies that make them resistant to nutrient deprivation to outcompete their neighbours. A key integrator of the cell's responses to starvation and other stresses is amino-acid-dependent mechanistic target of rapamycin complex 1 (mTORC1). Activation of mTORC1 on late endosomes and lysosomes is facilitated by amino-acid transporters within the solute-linked carrier 36 (SLC36) and SLC38 families. This study analysed the functions of SLC36 family member, SLC36A4, otherwise known as proton-assisted amino-acid transporter 4 (PAT4), in colorectal cancer. Independent of other major pathological factors, high PAT4 expression is associated with reduced relapse-free survival after colorectal cancer surgery. Consistent with this, PAT4 promotes HCT116 human colorectal cancer cell proliferation in culture and tumour growth in xenograft models. Inducible knockdown in HCT116 cells reveals that PAT4 regulates a form of mTORC1 with two distinct properties: first, it preferentially targets eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1), and second, it is resistant to rapamycin treatment. Furthermore, in HCT116 cells two non-essential amino acids, glutamine and serine, which are often rapidly metabolised by tumour cells, regulate rapamycin-resistant mTORC1 in a PAT4-dependent manner. Overexpressed PAT4 is also able to promote rapamycin resistance in human embryonic kidney-293 cells. PAT4 is predominantly associated with the Golgi apparatus in a range of cell types, and in situ proximity ligation analysis shows that PAT4 interacts with both mTORC1 and its regulator Rab1A on the Golgi. These findings, together with other studies, suggest that differentially localised intracellular amino-acid transporters contribute to the activation of alternate forms of mTORC1. Furthermore, the data predict that colorectal cancer cells with high PAT4 expression will be more resistant to depletion of serine and glutamine, allowing them to survive and outgrow neighbouring normal and tumorigenic cells, and potentially providing a new route for pharmacological intervention (Fan, 2016).

Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1

The mechanistic target of rapamycin complex 1 (mTORC1) protein kinase is a master growth regulator that responds to multiple environmental cues. Amino acids stimulate, in a Rag-, Ragulator-, and vacuolar adenosine triphosphatase-dependent fashion, the translocation of mTORC1 to the lysosomal surface, where it interacts with its activator Rheb. This study identified SLC38A9, an uncharacterized protein with sequence similarity to amino acid transporters, as a lysosomal transmembrane protein that interacts with the Rag guanosine triphosphatases (GTPases) and Ragulator in an amino acid-sensitive fashion. SLC38A9 transports arginine with a high Michaelis constant, and loss of SLC38A9 represses mTORC1 activation by amino acids, particularly arginine. Overexpression of SLC38A9 or just its Ragulator-binding domain makes mTORC1 signaling insensitive to amino acid starvation but not to Rag activity. Thus, SLC38A9 functions upstream of the Rag GTPases and is an excellent candidate for being an arginine sensor for the mTORC1 pathway (Wang, 2015).

SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. This study describes the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR (Rebsamer, 2015).

SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1

Cell growth and proliferation are tightly linked to nutrient availability. The mechanistic target of rapamycin complex 1 (mTORC1) integrates the presence of growth factors, energy levels, glucose and amino acids to modulate metabolic status and cellular responses. mTORC1 is activated at the surface of lysosomes by the RAG GTPases and the Ragulator complex through a not fully understood mechanism monitoring amino acid availability in the lysosomal lumen and involving the vacuolar H(+)-ATPase. This study describes the uncharacterized human member 9 of the solute carrier family 38 (SLC38A9) as a lysosomal membrane-resident protein competent in amino acid transport. Extensive functional proteomic analysis established SLC38A9 as an integral part of the Ragulator-RAG GTPases machinery. Gain of SLC38A9 function rendered cells resistant to amino acid withdrawal, whereas loss of SLC38A9 expression impaired amino-acid-induced mTORC1 activation. Thus SLC38A9 is a physical and functional component of the amino acid sensing machinery that controls the activation of mTOR (Rebsamen, 2015).

Search PubMed for articles about Drosophila Pathetic

Fan, S. J., Snell, C., Turley, H., Li, J. L., McCormick, R., Perera, S. M., Heublein, S., Kazi, S., Azad, A., Wilson, C., Harris, A. L. and Goberdhan, D. C. (2016). PAT4 levels control amino-acid sensitivity of rapamycin-resistant mTORC1 from the Golgi and affect clinical outcome in colorectal cancer. Oncogene 35(23): 3004-3015. PubMed ID: 26434594

Feng, S., Zacharioudaki, E., Millen, K. and Bray, S. J. (2020). The SLC36 transporter Pathetic is required for neural stem cell proliferation and for brain growth under nutrition restriction. Neural Dev 15(1): 10. PubMed ID: 32741363

Goberdhan, D. C., Meredith, D., Boyd, C. A. and Wilson, C. (2005). PAT-related amino acid transporters regulate growth via a novel mechanism that does not require bulk transport of amino acids. Development 132(10): 2365-2375. PubMed ID: 15843412 velopment 132, 2365-2375 (2005).

Lin, W. Y., Williams, C., Yan, C., Koledachkina, T., Luedke, K., Dalton, J., Bloomsburg, S., Morrison, N., Duncan, K. E., Kim, C. C. and Parrish, J. Z. (2015). The SLC36 transporter Pathetic is required for extreme dendrite growth in Drosophila sensory neurons. Genes Dev 29(11): 1120-1135. PubMed ID: 26063572

Lin, W.Y., Williams, C.R., Yan, C. and Parrish, J.Z. (2016). Functions of the SLC36 transporter Pathetic in growth control. Fly (Austin) 9(3):99-106. PubMed ID: 26735916

Newton, H., Wang, Y. F., Camplese, L., Mokochinski, J. B., Kramer, H. B., Brown, A. E. X., Fets, L. and Hirabayashi, S. (2020). Systemic muscle wasting and coordinated tumour response drive tumourigenesis. Nat Commun 11(1): 4653. PubMed ID: 32938923

Ogmundsdottir, M. H., Heublein, S., Kazi, S., Reynolds, B., Visvalingam, S. M., Shaw, M. K. and Goberdhan, D. C. (2012). Proton-assisted amino acid transporter PAT1 complexes with Rag GTPases and activates TORC1 on late endosomal and lysosomal membranes. PLoS One 7(5): e36616. PubMed ID: 22574197

Rebsamen, M., Pochini, L., Stasyk, T., de Araujo, M. E., Galluccio, M., Kandasamy, R. K., Snijder, B., Fauster, A., Rudashevskaya, E. L., Bruckner, M., Scorzoni, S., Filipek, P. A., Huber, K. V., Bigenzahn, J. W., Heinz, L. X., Kraft, C., Bennett, K. L., Indiveri, C., Huber, L. A. and Superti-Furga, G. (2015). SLC38A9 is a component of the lysosomal amino acid sensing machinery that controls mTORC1. Nature 519(7544): 477-481. PubMed ID: 25561175

Shang, P., Valapala, M., Grebe, R., Hose, S., Ghosh, S., Bhutto, I. A., Handa, J. T., Lutty, G. A., Lu, L., Wan, J., Qian, J., Sergeev, Y., Puertollano, R., Zigler, J. S., Jr., Xu, G. T. and Sinha, D. (2017). The amino acid transporter SLC36A4 regulates the amino acid pool in retinal pigmented epithelial cells and mediates the mechanistic target of rapamycin, complex 1 signaling. Aging Cell 16(2): 349-359. PubMed ID: 28083894

Wang, S., Tsun, Z. Y., Wolfson, R. L., Shen, K., Wyant, G. A., Plovanich, M. E., Yuan, E. D., Jones, T. D., Chantranupong, L., Comb, W., Wang, T., Bar-Peled, L., Zoncu, R., Straub, C., Kim, C., Park, J., Sabatini, B. L. and Sabatini, D. M. (2015). Metabolism. Lysosomal amino acid transporter SLC38A9 signals arginine sufficiency to mTORC1. Science 347(6218): 188-194. PubMed ID: 25567906

date revised: 8 November 2020

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