Heteromeric Amino Acid Transporters Research Articles

Structure and mechanisms of transport of human Asc1/CD98hc amino acid transporter

Recent cryoEM studies elucidated details of the structural basis for the substrate selectivity and translocation of heteromeric amino acid transporters. However, Asc1/CD98hc is the only neutral heteromeric amino acid transporter that can function through facilitated diffusion, and the only one that efficiently transports glycine and D-serine, and thus has a regulatory role in the central nervous system. Here we use cryoEM, ligand-binding simulations, mutagenesis, transport assays, and molecular dynamics to define human Asc1/CD98hc determinants for substrate specificity and gain insights into the mechanisms that govern substrate translocation by exchange and facilitated diffusion. The cryoEM structure of Asc1/CD98hc is determined at 3.4–3.8 Å resolution, revealing an inward-facing semi-occluded conformation. We find that Ser 246 and Tyr 333 are essential for Asc1/CD98hc substrate selectivity and for the exchange and facilitated diffusion modes of transport. Taken together, these results reveal the structural bases for ligand binding and transport features specific to human Asc1.

Structure, Function and Pharmacology of SLC7 Family Members and Homologues

Amino acids are essential components of all living cells serving as building blocks of proteins, as energy source, and as precursors of metabolites and signaling molecules. Amino acid transporters are membrane proteins that mediate the transfer of amino acids across the plasma membrane, and between compartments in cells, different cells and organs. The absence, overexpression or malfunction of specific amino acid transporters have been associated with human disease. One of the projects within the Swiss National Centre of Competence in Research (NCCR) TransCure was directed at SLC7 family amino acid transporters, with a particular focus on the heteromeric amino acid transporters 4F2hc-LAT1 (SLC3A2-SLC7A5) and 4F2hc-LAT2 (SLC3A2-SLC7A8), and the bacterial homologue AdiC. The project addressed questions of basic research (function and structure), pharmacology (identification of potent inhibitors and activators), and pre-clinical medicine (e.g., physiological role in the placenta) and disease models (e.g., tumor progression) of specific SLC7 family amino acid transporters. This review presents, summarizes and discusses selected main results obtained in this NCCR TransCure project.

Amino Acid Solute Carrier Transporters in Inflammation and Autoimmunity.

The past decade exposed the importance of many homeostasis and metabolism related proteins in autoimmunity disease and inflammation. Solute carriers (SLCs) are a group of membrane channels that can transport amino acids, the building blocks of proteins, nutrients, and neurotransmitters. This review summarizes the role of SLCs amino acid transporters in inflammation and autoimmunity disease. In detail, the importance of Glutamate transporters SLC1A1, SLC1A2, and SLC1A3, mainly expressed in the brain where they help prevent glutamate excitotoxicity, is discussed in the context of central nervous system disorders such as multiple sclerosis. Similarly, the cationic amino acid transporter SLC7A1 (CAT1), which is an important arginine transporter for T cells, and SLC7A2 (CAT2), essential for innate immunity. SLC3 family proteins, which bind with light chains from the SLC7 family (SLC7A5, SLC7A7 and SLC7A11) to form heteromeric amino acid transporters, are also explored to describe their roles in T cells, NK cells, macrophages and tumor immunotherapies. Altogether, the link between SLC amino acid transporters with inflammation and autoimmunity may contribute to a better understanding of underlying mechanism of disease and provide novel potential therapeutic avenues. Significance Statement SIGNIFICANCE STATEMENT In this review, we summarize the link between SLC amino acid transporters and inflammation and immune responses, specially SLC1 family members and SLC7 members. Studying the link may contribute to a better understanding of related diseases and provide potential therapeutic targets and useful to the researchers who have interest in the involvement of amino acids in immunity.

Structural basis for substrate specificity of heteromeric transporters of neutral amino acids

Despite having similar structures, each member of the heteromeric amino acid transporter (HAT) family shows exquisite preference for the exchange of certain amino acids. Substrate specificity determines the physiological function of each HAT and their role in human diseases. However, HAT transport preference for some amino acids over others is not yet fully understood. Using cryo-electron microscopy of apo human LAT2/CD98hc and a multidisciplinary approach, we elucidate key molecular determinants governing neutral amino acid specificity in HATs. A few residues in the substrate-binding pocket determine substrate preference. Here, we describe mutations that interconvert the substrate profiles of LAT2/CD98hc, LAT1/CD98hc, and Asc1/CD98hc. In addition, a region far from the substrate-binding pocket critically influences the conformation of the substrate-binding site and substrate preference. This region accumulates mutations that alter substrate specificity and cause hearing loss and cataracts. Here, we uncover molecular mechanisms governing substrate specificity within the HAT family of neutral amino acid transporters and provide the structural bases for mutations in LAT2/CD98hc that alter substrate specificity and that are associated with several pathologies.

The Ectodomains of rBAT and 4F2hc Are Fake or Orphan α-Glucosidases.

It is known that 4F2hc and rBAT are the heavy subunits of the heteromeric amino acid transporters (HATs). These heavy subunits are N-glycosylated proteins, with an N-terminal domain, one transmembrane domain and a bulky extracellular domain (ectodomain) that belongs to the α-amylase family. The heavy subunits are covalently linked to a light subunit from the SLC7 family, which is responsible for the amino acid transport activity, forming a heterodimer. The functions of 4F2hc and rBAT are related mainly to the stability and trafficking of the HATs in the plasma membrane of vertebrates, where they exert the transport activity. Moreover, 4F2hc is a modulator of integrin signaling, has a role in cell fusion and it is overexpressed in some types of cancers. On the other hand, some mutations in rBAT are found to cause the malfunctioning of the b0,+ transport system, leading to cystinuria. The ectodomains of 4F2hc and rBAT share both sequence and structure homology with α-amylase family members. Very recently, cryo-EM has revealed the structure of several HATs, including the ectodomains of rBAT and 4F2hc. Here, we analyze available data on the ectodomains of rBAT and 4Fhc and their relationship with the α-amylase family. The physiological relevance of this relationship remains largely unknown.

SLC3 and SLC7 families of heteromeric amino acid transporters (HATs) in GtoPdb v.2021.3

The SLC3 and SLC7 families combine to generate functional transporters, where the subunit composition is a disulphide-linked combination of a heavy chain (SLC3 family) with a light chain (SLC7 family) [1].

The zebrafish cationic amino acid transporter/glycoprotein-associated family: sequence and spatiotemporal distribution during development of the transport system b0,+ (slc3a1/slc7a9)

System b0,+ absorbs lysine, arginine, ornithine, and cystine, as well as some (large) neutral amino acids in the mammalian kidney and intestine. It is a heteromeric amino acid transporter made of the heavy subunit SLC3A1/rBAT and the light subunit SLC7A9/b0,+AT. Mutations in these two genes can cause cystinuria in mammals. To extend information on this transport system to teleost fish, we focused on the slc3a1 and slc7a9 genes by performing comparative and phylogenetic sequence analysis, investigating gene conservation during evolution (synteny), and defining early expression patterns during zebrafish (Danio rerio) development. Notably, we found that slc3a1 and slc7a9 are non-duplicated in the zebrafish genome. Whole-mount in situ hybridization detected co-localized expression of slc3a1 and slc7a9 in pronephric ducts at 24 h post-fertilization and in the proximal convoluted tubule at 3 days post-fertilization (dpf). Notably, both the genes showed co-localized expression in epithelial cells in the gut primordium at 3 dpf and in the intestine at 5 dpf (onset of exogenous feeding). Taken together, these results highlight the value of slc3a1 and slc7a9 as markers of zebrafish kidney and intestine development and show promise for establishing new zebrafish tools that can aid in the rapid screening(s) of substrates. Importantly, such studies will help clarify the complex interplay between the absorption of dibasic amino acids, cystine, and (large) neutral amino acids and the effect(s) of such nutrients on organismal growth.

Plasmodium vivax binds host CD98hc (SLC3A2) to enter immature red blood cells.

More than one-third of the world's population is exposed to Plasmodium vivax malaria, mainly in Asia1. P. vivax preferentially invades reticulocytes (immature red blood cells)2-4. Previous work has identified 11 parasite proteins involved in reticulocyte invasion, including erythrocyte binding protein 2 (ref. 5) and the reticulocyte-binding proteins (PvRBPs)6-10. PvRBP2b binds to the transferrin receptor CD71 (ref. 11), which is selectively expressed on immature reticulocytes12. Here, we identified CD98 heavy chain (CD98), a heteromeric amino acid transporter from the SLC3 family (also known as SLCA2), as a reticulocyte-specific receptor for the PvRBP2a parasite ligand using mass spectrometry, flow cytometry, biochemical and parasite invasion assays. We characterized the expression level of CD98 at the surface of immature reticulocytes (CD71+) and identified an interaction between CD98 and PvRBP2a expressed at the merozoite surface. Our results identify CD98 as an additional host membrane protein, besides CD71, that is directly associated with P. vivax reticulocyte tropism. These findings highlight the potential of using PvRBP2a as a vaccine target against P. vivax malaria.

Mechanism of substrate transport and inhibition of the human LAT1-4F2hc amino acid transporter

LAT1 (SLC7A5) is one of the representative light chain proteins of heteromeric amino acid transporters, forming a heterodimer with its heavy chain partner 4F2hc (SLC3A2). LAT1 is overexpressed in many types of tumors and mediates the transfer of drugs and hormones across the blood-brain barrier. Thus, LAT1 is considered as a drug target for cancer treatment and may be exploited for drug delivery into the brain. Here, we synthesized three potent inhibitors of human LAT1, which inhibit transport of leucine with IC50 values between 100 and 250 nM, and solved the cryo-EM structures of the corresponding LAT1-4F2hc complexes with these inhibitors bound at resolution of up to 2.7 or 2.8 Å. The protein assumes an outward-facing occluded conformation, with the inhibitors bound in the classical substrate binding pocket, but with their tails wedged between the substrate binding site and TM10 of LAT1. We also solved the complex structure of LAT1-4F2hc with 3,5-diiodo-l-tyrosine (Diiodo-Tyr) at 3.4 Å overall resolution, which revealed a different inhibition mechanism and might represent an intermediate conformation between the outward-facing occluded state mentioned above and the outward-open state. To our knowledge, this is the first time that the outward-facing conformation is revealed for the HAT family. Our results unveil more important insights into the working mechanisms of HATs and provide a structural basis for future drug design.

Heteromeric Amino Acid Transporters in Brain: from Physiology to Pathology.

In humans, more than 50 transporters are responsible for the traffic and balance of amino acids within and between cells and tissues, and half of them have been associated with disease [1]. Covering all common amino acids, Heteromeric Amino acid Transporters (HATs) are one class of such transporters. This review first highlights structural and functional studies that solved the atomic structure of HATs and revealed molecular clues on substrate interaction. Moreover, this review focuses on HATs that have a role in the central nervous system (CNS) and that are related to neurological diseases, including: (i) LAT1/CD98hc and its role in the uptake of branched chain amino acids trough the blood brain barrier and autism. (ii) LAT2/CD98hc and its potential role in the transport of glutamine between plasma and cerebrospinal fluid. (iii) y+LAT2/CD98hc that is emerging as a key player in hepatic encephalopathy. xCT/CD98hc as a potential therapeutic target in glioblastoma, and (iv) Asc-1/CD98hc as a potential therapeutic target in pathologies with alterations in NMDA glutamate receptors.

The Heavy Chain 4F2hc Modulates the Substrate Affinity and Specificity of the Light Chains LAT1 and LAT2

The human L-type amino acid transporters LAT1 and LAT2 mediate the transport of amino acids and amino acid derivatives across plasma membranes in a sodium-independent, obligatory antiport mode. In mammalian cells, LAT1 and LAT2 associate with the type-II membrane N-glycoprotein 4F2hc to form heteromeric amino acid transporters (HATs). The glycosylated ancillary protein 4F2hc is known to be important for successful trafficking of the unglycosylated transporters to the plasma membrane. The heavy (i.e., 4F2hc) and light (i.e., LAT1 and LAT2) chains belong to the solute carrier (SLC) families SLC3 and SLC7, and are covalently linked by a conserved disulfide bridge. Overexpression, absence, or malfunction of certain HATs is associated with human diseases and HATs are therefore considered therapeutic targets. Here, we present a comparative, functional characterization of the HATs 4F2hc-LAT1 and 4F2hc-LAT2, and their light chains LAT1 and LAT2. For this purpose, the HATs and the light chains were expressed in the methylotrophic yeast Pichia pastoris and a radiolabel transport assay was established. Importantly and in contrast to mammalian cells, P. pastoris has proven useful as eukaryotic expression system to successfully express human LAT1 and LAT2 in the plasma membrane without the requirement of co-expressed trafficking chaperone 4F2hc. Our results show a novel function of the heavy chain 4F2hc that impacts transport by modulating the substrate affinity and specificity of corresponding LATs. In addition, the presented data confirm that the light chains LAT1 and LAT2 constitute the substrate-transporting subunits of the HATs, and that light chains are also functional in the absence of the ancillary protein 4F2hc.

Structural basis for amino acid exchange by a human heteromeric amino acid transporter

Heteromeric amino acid transporters (HATs) comprise a group of membrane proteins that belong to the solute carrier (SLC) superfamily. They are formed by two different protein components: a light chain subunit from an SLC7 family member and a heavy chain subunit from the SLC3 family. The light chain constitutes the transport subunit whereas the heavy chain mediates trafficking to the plasma membrane and maturation of the functional complex. Mutation, malfunction, and dysregulation of HATs are associated with a wide range of pathologies or represent the direct cause of inherited and acquired disorders. Here we report the cryogenic electron microscopy structure of the neutral and basic amino acid transport complex (b[0,+]AT1-rBAT) which reveals a heterotetrameric protein assembly composed of two heavy and light chain subunits, respectively. The previously uncharacterized interaction between two HAT units is mediated via dimerization of the heavy chain subunits and does not include participation of the light chain subunits. The b(0,+)AT1 transporter adopts a LeuT fold and is captured in an inward-facing conformation. We identify an amino-acid-binding pocket that is formed by transmembrane helices 1, 6, and 10 and conserved among SLC7 transporters.

Computational analysis identifies druggable mutations in human rBAT mediated Cystinuria

Culex quinquefasciatus Cqm1 protein acts as the receptor for Lysinibacillus sphaericus mosquito-larvicidal binary (BinAB) toxin that is used worldwide for mosquito control. We found amino acid transporter protein, rBAT, as phylogenetically closest Cqm1 homolog in humans. The present study reveals large evolutionary distance between Cqm1 and rBAT, and rBAT ectodomain lacks the sequence motif which serves as binding-site for the BinAB toxin. Thus, BinAB toxin can be expected to remain safe for humans. rBAT (heavy subunit; SLC3A1) and catalytic b0,+AT (light subunit; SLC7A9), linked by single disulfide bond, mediate renal reabsorption of cystine and dibasic amino acids in Na+ independent manner. Mutations in rBAT cause type I Cystinuria disease which shows global prevalence, and rBAT can be thought as an important pharmacological target. However, 3D structures of rBAT and b0,+AT, the two components of b0,+ heteromeric amino acid transporter systems, are not available. We constructed a reliable homology model of rBAT using Cqm1 coordinates and that of transmembrane b0,+AT subunit using LAT1 coordinates. Mapping of pathogenic mutations onto rBAT ectodomain revealed their scattered distribution throughout the rBAT protein. Further, our computational simulations-based scoring of several known deleterious mutations of rBAT revealed that mutations those do not compromise the protein fold and stability, are localized on the same face of the molecule. These residues are expected to interact with the b0,+AT transporter. The present study thus identifies druggable sites on rBAT that could be targeted for the treatment of type I Cystinuria. Communicated by Ramaswamy H. Sarma

SLC3A2 mutation as a genetic biomarker for dMMR in colorectal cancer.

e13545 Background: SLC3A2 (solute carrier family member 2) 3 is a member of the SLC3 family Ⅱ transmembrane glycoprotein, which is a key component of heteromeric amino acid transporters (HATs). The association between SLC3A2 and cancer was established, given that the amino acid supply by SLC3A2 is required for the resistance of oxidative stress and the maintenance of cell proliferation. However, whether SLC3A2 mutation plays a role in carcinogenesis remains unclear. Methods: This study enrolled 1000 colorectal cancer patients. Tumor tissues and paired peripheral blood mononuclear cells (PBMC) were collected for the sequencing of cancer-related genes by target capture and next-generation sequencing. Results: p.Lys301Argfs*31 mutation in SLC3A2 were detected in 40(40/1000, 4.0%) patients. low TMB and MSS were reported in 2 cases while the remaining 38 cases exhibited high TMB and MSI-H. 41(41/1000, 4.1%) DNA mismatch repair (dMMR) were identified in 41(41/1000, 4.1%) tumor samples, 27(27/41, 65.9%) of which had p.Lys301Argfs*31 mutation. Moreover, this frameshift mutation (COSM253097) was observed in 28 cases of large intestine cancer, 1 case of peritoneum cancer, 1 case of Stomach cancer, 1 case of biliary tract cancer and 1 case of ovary cancer in the COSMIC database. Conclusions: p.Lys301Argfs*31 mutation of SLC3A2 was identified in the majority of colorectal cancer cases with dMMR events, suggesting that this mutation may be a biomarker of dMMR.

The Role of Large Neutral Amino Acid Transporter (LAT1) in Cancer.

The solute carrier family 7 (SLC7) can be categorically divided into two subfamilies, the L-type amino acid transporters (LATs) including SLC7A5-13, and SLC7A15, and the cationic amino acid transporters (CATs) including SLC7A1-4 and SLC7A14. Members of the CAT family transport predominantly cationic amino acids by facilitating diffusion with intracellular substrates. LAT1 (also known as SLC7A5), is defined as a heteromeric amino acid transporter (HAT) interacting with the glycoprotein CD98 (SLC3A2) through a conserved disulfide to uptake not only large neutral amino acids, but also several pharmaceutical drugs to cells. In this review, we provide an overview of the interaction of the structure-function of LAT1 and its essential role in cancer, specifically, its role at the blood-brain barrier (BBB) to facilitate the transport of thyroid hormones, pharmaceuticals (e.g., I-DOPA, gabapentin), and metabolites into the brain. LAT1 expression increases as cancers progress, leading to higher expression levels in highgrade tumors and metastases. In addition, LAT1 plays a crucial role in cancer-associated reprogrammed metabolic networks by supplying tumor cells with essential amino acids. The increasing understanding of the role of LAT1 in cancer has led to an increase in interest surrounding its potential as a drug target for cancer treatment.

CD98hc (SLC3A2) sustains amino acid and nucleotide availability for cell cycle progression

CD98 heavy chain (CD98hc) forms heteromeric amino acid (AA) transporters by interacting with different light chains. Cancer cells overexpress CD98hc-transporters in order to meet their increased nutritional and antioxidant demands, since they provide branched-chain AA (BCAA) and aromatic AA (AAA) availability while protecting cells from oxidative stress. Here we show that BCAA and AAA shortage phenocopies the inhibition of mTORC1 signalling, protein synthesis and cell proliferation caused by CD98hc ablation. Furthermore, our data indicate that CD98hc sustains glucose uptake and glycolysis, and, as a consequence, the pentose phosphate pathway (PPP). Thus, loss of CD98hc triggers a dramatic reduction in the nucleotide pool, which leads to replicative stress in these cells, as evidenced by the enhanced DNA Damage Response (DDR), S-phase delay and diminished rate of mitosis, all recovered by nucleoside supplementation. In addition, proper BCAA and AAA availability sustains the expression of the enzyme ribonucleotide reductase. In this regard, BCAA and AAA shortage results in decreased content of deoxynucleotides that triggers replicative stress, also recovered by nucleoside supplementation. On the basis of our findings, we conclude that CD98hc plays a central role in AA and glucose cellular nutrition, redox homeostasis and nucleotide availability, all key for cell proliferation.

LAT1 (SLC7A5) and CD98hc (SLC3A2) complex dynamics revealed by single-particle cryo-EM.

Solute carriers are a large class of transporters that play key roles in normal and disease physiology. Among the solute carriers, heteromeric amino-acid transporters (HATs) are unique in their quaternary structure. LAT1-CD98hc, a HAT, transports essential amino acids and drugs across the blood-brain barrier and into cancer cells. It is therefore an important target both biologically and therapeutically. During the course of this work, cryo-EM structures of LAT1-CD98hc in the inward-facing conformation and in either the substrate-bound or apo states were reported to 3.3-3.5 Å resolution [Yan et al. (2019), Nature (London), 568, 127-130]. Here, these structures are analyzed together with our lower resolution cryo-EM structure, and multibody 3D auto-refinement against single-particle cryo-EM data was used to characterize the dynamics of the interaction of CD98hc and LAT1. It is shown that the CD98hc ectodomain and the LAT1 extracellular surface share no substantial interface. This allows the CD98hc ectodomain to have a high degree of movement within the extracellular space. The functional implications of these aspects are discussed together with the structure determination.

Structure of the human LAT1-4F2hc heteromeric amino acid transporter complex.

The L-type amino acid transporter 1 (LAT1; also known as SLC7A5) catalyses the cross-membrane flux of large neutral amino acids in a sodium- and pH-independent manner1-3. LAT1, an antiporter of the amino acid-polyamine-organocation superfamily, also catalyses the permeation of thyroid hormones, pharmaceutical drugs, and hormone precursors such as L-3,4-dihydroxyphenylalanine across membranes2-6. Overexpression of LAT1 has been observed in a wide range of tumour cells, and it is thus a potential target for anti-cancer drugs7-11. LAT1 forms a heteromeric amino acid transporter complex with 4F2 cell-surface antigen heavy chain (4F2hc; also known as SLC3A2)-a type II membrane glycoprotein that is essential for the stability of LAT1 and for its localization to the plasma membrane8,9. Despite extensive cell-based characterization of the LAT1-4F2hc complex and structural determination of its homologues in bacteria, the interactions between LAT1 and 4F2hc and the working mechanism of the complex remain largely unknown12-19. Here we report the cryo-electron microscopy structures of human LAT1-4F2hc alone and in complex with the inhibitor 2-amino-2-norbornanecarboxylic acid at resolutions of 3.3 Å and 3.5 Å, respectively. LAT1 exhibits an inward open conformation. Besides a disulfide bond association, LAT1 also interacts extensively with 4F2hc on the extracellular side, within the membrane, and on the intracellular side. Biochemical analysis reveals that 4F2hc is essential for the transport activity of the complex. Together, our characterizations shed light on the architecture of the LAT1-4F2hc complex, and provide insights into its function and the mechanisms through which it might be associated with disease.

Volta Phase Plate Cryo-EM Structure of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2.

Heteromeric amino acid transporters (HATs) are protein complexes that catalyze the transport of amino acids across plasma membranes. HATs are composed of two subunits, a heavy and a light subunit, which belong to the solute carrier (SLC) families SLC3 and SLC7. The two subunits are linked by a conserved disulfide bridge. Several human diseases are associated with loss of function or overexpression of specific HATs making them drug targets. The human HAT 4F2hc-LAT2 (SLC3A2-SLC7A8) is specific for the transport of large neutral L-amino acids and specific amino acid-related compounds. Human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris and pure recombinant protein purified. Here we present the first cryo-electron microscopy (cryo-EM) 3D-map of a HAT, i.e., of the human 4F2hc-LAT2 complex. The structure could be determined at ~13 Å resolution using direct electron detector and Volta phase plate technologies. The 3D-map displays two prominent densities of different sizes. The available X-ray structure of the 4F2hc ectodomain fitted nicely into the smaller density revealing the relative position of 4F2hc with respect to LAT2 and the membrane plane.

The Reproduction Rate of Peptide Transporter PEPT-1 Deficient C. elegans Is Dependent on Dietary Glutamate Supply.

Intestinal absorption of dietary amino acids is mediated via two routes. Free amino acids released by hydrolysis of dietary proteins are taken up by a multitude of amino acid transporters while di- and tripeptides released are taken up by the peptide transporter PEPT-1. Loss of PEPT-1 impairs growth, post-embryonic development and reproduction in Caenorhabditis elegans, and supplementation with a mixture of all L-amino acids only partially rescues fertility. In the present study, we demonstrate that dietary L-glutamate is the responsible amino acid that can increase fertility in hermaphrodite pept-1 worms. This effect was associated with a significantly higher uptake of glutamate/aspartate in pept-1 than in wildtype C. elegans. Furthermore, we found that the intestinal transporter proteins SNF-5 of the solute carrier SLC6 family of nutrient amino acid transporters (NAT) and AAT-6 of the SLC7 family as the light subunit of a heteromeric amino acid transporter (HAT) play a key role in glutamate homeostasis in pept-1 C. elegans. Genes encoding these transporters are highly expressed and upon silencing a 95% reduced fertility (snf-5) and sterility (aat-6) was observed. A subsequent L-glutamate supplementation failed to rescue these phenotypes. Dietary glutamate supplementation did neither influence the feeding frequency, nor did it improve mating efficiency of pept-1 males. Most strikingly, pept-1 were more prone to habituation to repeated gentle touch stimuli than wildtype C. elegans, and dietary glutamate supply was sufficient to alter this behavioral output by restoring the mechanosensory response to wildtype levels. Taken together, our data demonstrate a key role of L-glutamate in amino acid homeostasis in C. elegans lacking the peptide transporter in the intestine and demonstrate its distinct role in reproduction and for neural circuits mediating touch sensitivity.