The amino acid transporter SLC38A9 regulates MTORC1 and autophagy

The mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a master regulator of macroautophagy (hereafter autophagy) that responds to different environmental nutrients, including amino acids, glucose, and growth factors. The identity of the amino acid-sensing component of the MTORC1 machinery had remained elusive until a lysosomal low-affinity amino acid transporter, SLC38A9 (solute carrier family 38, member 9), was recently characterized as a novel component of the Ragulator-RRAG GTPase complex by 3 independent research groups.     

Amino Acid-Dependent mTORC1 Regulation by the Lysosomal Membrane Protein SLC38A9

The serine/threonine kinase mTORC1 regulates cellular homeostasis in response to many cues, such as nutrient status and energy level. Amino acids induce mTORC1 activation on lysosomes via the small Rag GTPases and the Ragulator complex, thereby controlling protein translation and cell growth. Here, we identify the human 11-pass transmembrane protein SLC38A9 as a novel component of the Rag-Ragulator complex. SLC38A9 localizes with Rag-Ragulator complex components on lysosomes and associates with Rag GTPases in an amino acid-sensitive and nucleotide binding state-dependent manner. Depletion of SLC38A9 inhibits mTORC1 activity in the presence of amino acids and in response to amino acid replenishment following starvation. Conversely, SLC38A9 overexpression causes RHEB (Ras homolog enriched in brain) GTPase-dependent hyperactivation of mTORC1 and partly sustains mTORC1 activity upon amino acid deprivation. Intriguingly, during amino acid starvation mTOR is retained at the lysosome upon SLC38A9 depletion but fails to be activated. Together, the findings of our study reveal SLC38A9 as a Rag-Ragulator complex member transducing amino acid availability to mTORC1 activity.     

Insights into the transport side of the human SLC38A9 transceptor

The lysosomal amino acid transporter SLC38A9 is referred to as transceptor, i.e. a transporter with a receptor function. The protein is responsible for coupling amino acid transport across the lysosomal membrane according to the substrate availability to mTORC1 signal transduction. This process allows cells to sense amino acid level responding to growth stimuli in physiological and pathological conditions triggering mTOR regulation. The main substrates underlying this function are glutamine and arginine. The functional and kinetic characterization of glutamine and arginine transport was performed using human SLC38A9 produced in E. coli, purified by affinity chromatography and reconstituted in liposomes. A cooperative behaviour for the wild type protein was revealed for both the substrates. A novel Na⁺ binding site, namely T453, was described by combined approaches of bioinformatics, site-directed mutagenesis and transport assay. Stimulation by cholesterol of glutamine and arginine transport was observed. The biological function of SLC38A9 relies on the interaction between its N-terminus and components of the mTOR complex; a deletion mutant of the N-terminus tail was produced and transport of glutamine was assayed revealing that this portion does not play any role in the intrinsic transport function of the human SLC38A9. Different features for glutamine and arginine transport were revealed: human SLC38A9 is competent for glutamine efflux, while that of arginine is negligible. In line with these results, imposed ?pH stimulated glutamine, not arginine transport. Arginine plays, on the contrary, a modulatory function and is able to stimulate glutamine efflux. Interestingly, reciprocal inhibition experiments also supported by bioinformatics, suggested that glutamine and arginine may bind to different sites in the human SLC38A9 transporter.     

Deciphering the mechanisms of intestinal imino (and amino) acid transport: the redemption of SLC36A1

The absorption of zwitterionic imino and amino acids, and related drugs, is an essential function of the small intestinal epithelium. This review focuses on the physiological roles of transporters recently identified at the molecular level, in particular SLC36A1, by identifying how they relate to the classical epithelial imino and amino acid transporters characterised in mammalian small intestine in the 1960s-1990s. SLC36A1 transports a number of D- and L-imino and amino acids, beta- and gamma-amino acids and orally-active neuromodulatory and antibacterial agents. SLC36A1 (or PAT1) functions as a proton-coupled imino and amino acid symporter in cooperation with the Na+/H+ exchanger NHE3 (SLC9A3) to produce the imino acid carrier identified in rat small intestine in the 1960s but subsequently ignored because of confusion with the IMINO transporter. However, it is the sodium/imino and amino acid cotransporter SLC6A20 which corresponds to the betaine carrier (identified in hamster, 1960s) and IMINO transporter (identified in rabbit and guinea pig, 1980s). This review summarises evidence for expression of SLC36A1 and SLC6A20 in human small intestine, highlights the differences in functional characteristics of the imino acid carrier and IMINO transporter, and explains the confusion surrounding these two distinct transport systems.     

Genetic and biochemical analysis of the yeast plasma membrane Ssy1p-Ptr3p-Ssy5p sensor of extracellular amino acids

Ssy1p and Ptr3p are known components of a yeast plasma membrane system that functions to sense the presence of amino acids in the extracellular environment. In response to amino acids, this sensing system initiates metabolic signals that ultimately regulate the functional expression of several amino acid-metabolizing enzymes and transport proteins, including multiple, genetically distinct amino acid permeases. We have found that SSY5 encodes a third component of this amino acid sensing system. Mutations in SSY5 manifest phenotypes that are indistinguishable from those resulting from either single ssy1 and ptr3 mutations or ssy5 ssy1 and ssy5 ptr3 double mutations. Although Ssy5p is predicted to be a soluble protein, it exhibits properties indicating that it is a peripherally associated plasma membrane protein. Each of the three sensor components, Ssy1p, Ptr3p, and Ssy5p, adopts conformations and modifications that are dependent upon the availability of amino acids and on the presence of the other two components. These results suggest that these components function as part of a sensor complex localized to the plasma membrane. Consistent with a sensor complex, the overexpression of SSY1 or the unique N-terminal extension of this amino acid permease homologue inactivates the amino acid sensor in a dominant-negative manner. Each of the components of the Ssy1p-Ptr3p-Ssy5p (SPS) signaling system undergoes rapid physical changes, reflected in altered electrophoretic mobility, when leucine is added to cells grown in media lacking amino acids. Furthermore, the levels of each SPS sensor component present in whole-cell extracts diminish upon leucine addition. The rapid physical alterations and reduced levels of sensor components are consistent with their being downregulated in response to amino acid availability. These results reveal the dynamic nature of the amino acid-initiated signals transduced by the SPS sensor.     

LPAR5, GNAT3 and partial amino acid transporters messenger RNA expression patterns in digestive tracts,metabolic organs and muscle tissues of growing goats

Sufficient amino acid (AA) transport is essential to ensure the normal physiological function and growth of growing animals. The processes of AA sensing and transport in humans and murine animals, but rarely in goats, have been arousing great interest recently. This study was conducted to investigate the messenger RNA expression patterns of lysophosphatidic acid receptor 5 (LPAR5), guanine nucleotide-binding protein α-transducing 3 (GNAT3) and important partial AA transporters in digestive tracts, metabolic organs and muscles of growing goats. The results showed that these genes were widely expressed in goats, and had different expression patterns. LPAR5, GNAT3, solute carrier (SLC38A2), SLC7A7, SLC7A1 and SLC3A1 were rarely expressed in the rumen, but were highly expressed in the abomasum and intestine which are the main sites of AA absorption. GNAT3, SLC38A1, SLC38A2, SLC6A19, SLC7A7 and SLC7A1 showed comparatively high expression in the pancreas and the vital digestive glands, and the relatively high expression of these nine genes were noted in the tibialis posterior, the active muscle in energy metabolism. The correlation analysis showed that there were certain positive correlation among most genes. The current results indicate that the AA sensing and transport occur extensively in the abomasum and small intestine, metabolic organs and muscle tissues of ruminants, and that related genes have tissue specificity.     

Deciphering the mechanisms of intestinal imino (and amino) acid transport: The redemption of SLC36A1

The absorption of zwitterionic imino and amino acids, and related drugs, is an essential function of the small intestinal epithelium. This review focuses on the physiological roles of transporters recently identified at the molecular level, in particular SLC36A1, by identifying how they relate to the classical epithelial imino and amino acid transporters characterised in mammalian small intestine in the 1960s-1990s. SLC36A1 transports a number of d- and l-imino and amino acids, β- and γ-amino acids and orally-active neuromodulatory and antibacterial agents. SLC36A1 (or PAT1) functions as a proton-coupled imino and amino acid symporter in cooperation with the Na⁺/H⁺ exchanger NHE3 (SLC9A3) to produce the imino acid carrier identified in rat small intestine in the 1960s but subsequently ignored because of confusion with the IMINO transporter. However, it is the sodium/imino and amino acid cotransporter SLC6A20 which corresponds to the betaine carrier (identified in hamster, 1960s) and IMINO transporter (identified in rabbit and guinea pig, 1980s). This review summarises evidence for expression of SLC36A1 and SLC6A20 in human small intestine, highlights the differences in functional characteristics of the imino acid carrier and IMINO transporter, and explains the confusion surrounding these two distinct transport systems.     

Highly conserved asparagine 82 controls the interaction of Na+ with the sodium-coupled neutral amino acid transporter SNAT2

The neutral amino acid transporter 2 (SNAT2), which belongs to the SLC38 family of solute transporters, couples the transport of amino acid to the cotransport of one Na(+) ion into the cell. Several polar amino acids are highly conserved within the SLC38 family. Here, we mutated three of these conserved amino acids, Asn(82) in the predicted transmembrane domain 1 (TMD1), Tyr(337) in TMD7, and Arg(374) in TMD8; and we studied the functional consequences of these modifications. The mutation of N82A virtually eliminated the alanine-induced transport current, as well as amino acid uptake by SNAT2. In contrast, the mutations Y337A and R374Q did not abolish amino acid transport. The K(m) of SNAT2 for its interaction with Na(+), K(Na(+)), was dramatically reduced by the N82A mutation, whereas the more conservative mutation N82S resulted in a K(Na(+)) that was in between SNAT2(N82A) and SNAT2(WT). These results were interpreted as a reduction of Na(+) affinity caused by the Asn(82) mutations, suggesting that these mutations interfere with the interaction of SNAT2 with the sodium ion. As a consequence of this dramatic reduction in Na(+) affinity, the apparent K(m) of SNAT2(N82A) for alanine was increased 27-fold compared with that of SNAT2(WT). Our results demonstrate a direct or indirect involvement of Asn(82) in Na(+) coordination by SNAT2. Therefore, we predict that TMD1 is crucial for the function of SLC38 transporters and that of related families.     

SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes

The SLC36 family of transporters consists of four genes, two of which, SLC36A1 and SLC36A2, have been demonstrated to code for human proton-coupled amino acid transporters or hPATs. Here we report the characterization of the fourth member of the family, SLC36A4 or hPAT4, which when expressed in Xenopus laevis oocytes also encodes a plasma membrane amino acid transporter, but one that is not proton-coupled and has a very high substrate affinity for the amino acids proline and tryptophan. hPAT4 in Xenopus oocytes mediated sodium-independent, electroneutral uptake of [(3)H]proline, with the highest rate of uptake when the uptake medium pH was 7.4 and an affinity of 3.13 μM. Tryptophan was also an excellently transported substrate with a similarly high affinity (1.72 μM). Other amino acids that inhibited [(3)H]proline were isoleucine (K(i) 0.23 mM), glutamine (0.43 mM), methionine (0.44 mM), and alanine (1.48 mM), and with lower affinity, glycine, threonine, and cysteine (K(i) >5 mM for all). Of the amino acids directly tested for transport, only proline, tryptophan, and alanine showed significant uptake, whereas glycine and cysteine did not. Of the non-proteogenic amino acids and drugs tested, only sarcosine produced inhibition (K(i) 1.09 mM), whereas γ-aminobutyric acid (GABA), β-alanine, L-Dopa, D-serine, and δ-aminolevulinic acid were without effect on [(3)H]proline uptake. This characterization of hPAT4 as a very high affinity/low capacity non-proton-coupled amino acid transporter raises questions about its physiological role, especially as the transport characteristics of hPAT4 are very similar to the Drosophila orthologue PATH, an amino acid "transceptor" that plays a role in nutrient sensing.     

Pharmacological inhibition of lysosomes activates the MTORC1 signaling pathway in chondrocytes in an autophagy-independent manner

Mechanistic target of rapamycin (serine/threonine kinase) complex 1 (MTORC1) is a protein-signaling complex at the fulcrum of anabolic and catabolic processes, which acts depending on wide-ranging environmental cues. It is generally accepted that lysosomes facilitate MTORC1 activation by generating an internal pool of amino acids. Amino acids activate MTORC1 by stimulating its translocation to the lysosomal membrane where it forms a super-complex involving the lysosomal-membrane-bound vacuolar-type H⁺-ATPase (v-ATPase) proton pump. This translocation and MTORC1 activation require functional lysosomes. Here we found that, in contrast to this well-accepted concept, in epiphyseal chondrocytes inhibition of lysosomal activity by v-ATPase inhibitors bafilomycin A₁ or concanamycin A potently activated MTORC1 signaling. The activity of MTORC1 was visualized by phosphorylated forms of RPS6 (ribosomal protein S6) and EIF4EBP1, 2 well-known downstream targets of MTORC1. Maximal RPS6 phosphorylation was observed at 48-h treatment and reached as high as a 12-fold increase (p < 0.018). This activation of MTORC1 was further confirmed in bone organ culture and promoted potent stimulation of longitudinal growth (p < 0.001). Importantly, the same effect was observed in ATG5 (autophagy-related 5)-deficient bones suggesting a macroautophagy-independent mechanism of MTORC1 inhibition by lysosomes. Thus, our data show that in epiphyseal chondrocytes lysosomes inhibit MTORC1 in a macroautophagy-independent manner and this inhibition likely depends on v-ATPase activity.     

Identification of SLC38A7 (SNAT7) protein as a glutamine transporter expressed in neurons

The SLC38 family of transporters has in total 11 members in humans and they encode amino acid transporters called sodium-coupled amino acid transporters (SNAT). To date, five SNATs have been characterized and functionally subdivided into systems A (SLC38A1, SLC38A2, and SLC38A4) and N (SLC38A3 and SLC38A5) showing the highest transport for glutamine and alanine. Here we present identification of a novel glutamine transporter encoded by the Slc38a7 gene, which we propose should be named SNAT7. This transporter has L-glutamine as the preferred substrate but also transports other amino acids with polar side chains, as well as L-histidine and L-alanine. The expression pattern and substrate profile for SLC38A7 shows highest similarity to the known system N transporters. Therefore, we propose that SLC38A7 is a novel member of this system. We used in situ hybridization and immunohistochemistry with a custom-made antibody to show that SLC38A7 is expressed in all neurons, but not in astrocytes, in the mouse brain. SLC38A7 is unique in being the first system N transporter expressed in GABAergic and also other neurons. The preferred substrate and axonal localization of SLC38A7 close to the synaptic cleft indicates that SLC38A7 could have an important function for the reuptake and recycling of glutamate.     

氨基酸转运载体SLC38A1研究进展

氨基酸转运载体是介导氨基酸跨膜转运的膜蛋白,在医学、营养等生命科学领域有重要的研究意义。氨基酸转运载体SLC38A1选择性、生理性表达于人体正常大脑和胎盘组织,研究表明,SLC38A1在恶性肿瘤中呈过表达,可以促进肿瘤细胞的增殖、侵袭和迁移。SLC38A1有望成为新的肿瘤靶点之一,本文就SLC38A1在肿瘤中的研究进展作一综述。     

Stimulation of the amino acid transporter SLC6A19 by JAK2

JAK2 (Janus kinase-2) is expressed in a wide variety of cells including tumor cells and contributes to the proliferation and survival of those cells. The gain of function mutation ^V617FJAK2 mutant is found in the majority of myeloproliferative diseases. Cell proliferation depends on the availability of amino acids. Concentrative cellular amino acid uptake is in part accomplished by Na⁺ coupled amino acid transport through SLC6A19 (B(0)AT). The present study thus explored whether JAK2 activates SLC6A19. To this end, SLC6A19 was expressed in Xenopus oocytes with or without wild type JAK2, ^V617FJAK2 or inactive ^K882EJAK2 and electrogenic amino acid transport determined by dual electrode voltage clamp. In SLC6A19-expressing oocytes but not in oocytes injected with water or JAK2 alone, the addition of leucine (2 mM) to the bath generated a current (I_le), which was significantly increased following coexpression of JAK2 or ^V617FJAK2, but not by coexpression of ^K882EJAK2. Coexpression of JAK2 enhanced the maximal transport rate without significantly modifying the affinity of the carrier. Exposure of the oocytes to the JAK2 inhibitor AG490 (40 μM) resulted in a gradual decline of I_le. According to chemiluminescence JAK2 enhanced the carrier protein abundance in the cell membrane. The decline of I_le following inhibition of carrier insertion by brefeldin A (5 μM) was similar in the absence and presence of JAK2 indicating that JAK2 stimulates carrier insertion into rather than inhibiting carrier retrival from the cell membrane. In conclusion, JAK2 up-regulates SLC6A19 activity which may foster amino acid uptake into JAK2 expressing cells.     

SLC6A14 (ATB0,+) protein, a highly concentrative and broad specific amino acid transporter, is a novel and effective drug target for treatment of estrogen receptor-positive breast cancer

SLC6A14, also known as ATB(0,+), is an amino acid transporter with unique characteristics. It transports 18 of the 20 proteinogenic amino acids. However, this transporter is expressed only at low levels in normal tissues. Here, we show that the transporter is up-regulated specifically in estrogen receptor (ER)-positive breast cancer, demonstrable with primary human breast cancer tissues and human breast cancer cell lines. SLC6A14 is an estrogen/ER target. The transport features of SLC6A14 include concentrative transport of leucine (an activator of mTOR), glutamine (an essential amino acid for nucleotide biosynthesis and substrate for glutaminolysis), and arginine (an essential amino acid for tumor cells), suggesting that ER-positive breast cancer cells up-regulate SLC6A14 to meet their increased demand for these amino acids. Consequently, treatment of ER-positive breast cancer cells in vitro with α-methyl-DL-tryptophan (α-MT), a selective blocker of SLC6A14, induces amino acid deprivation, inhibits mTOR, and activates autophagy. Prolongation of the treatment with α-MT causes apoptosis. Addition of an autophagy inhibitor (3-methyladenine) during α-MT treatment also induces apoptosis. These effects of α-MT are specific to ER-positive breast cancer cells, which express the transporter. The ability of α-MT to cause amino acid deprivation is significantly attenuated in MCF-7 cells, an ER-positive breast cancer cell line, when SLC6A14 is silenced with shRNA. In mouse xenograft studies, α-MT by itself is able to reduce the growth of the ER-positive ZR-75-1 breast cancer cells. These studies identify SLC6A14 as a novel and effective drug target for the treatment of ER-positive breast cancer.     

SLC7A9 cDNA cloning and mutational analysis of SLC3A1 and SLC7A9 in canine cystinuria

Cystinuria is a genetic disorder in the domestic dog that leads to recurrent urolith formation. The genetic basis of the disorder is best characterized in humans and is caused by mutations in one of the amino acid transporter genes SLC3A1 or SLC7A9, which results in hyperexcretion of cystine and the dibasic amino acids in the urine and subsequent precipitation of cystine due to its low solubility in urine. In this study we describe the cloning of the canine SLC7A9 cDNA and present a thorough mutation analysis of the coding SLC3A1 and SLC7A9 regions in cystinuric dogs of different breeds. Mutation analysis of the two cystinuria disease genes revealed one SLC7A9 mutation (A217T) and two SLC3A1 mutations (I192V and S698G) in French and English Bulldogs that affect nonconserved amino acid residues, arguing against functional impact on the proteins. The absence of deleterious mutations linked to cystinuria in the remainder of our panel of cystinuric dogs is surprising because SLC3A1 or SLC7A9 mutations explain approximately 70% of all human cystinuria cases studied. The present study, along with previous investigations of canine and human cystinuria, implies that regulatory parts of the SLC3A1 and SLC7A9 genes as well as other unknown genes may harbor mutations causing cystinuria.