Function and structure of heterodimeric amino acid transporters

2001 ◽  
Vol 281 (4) ◽  
pp. C1077-C1093 ◽  
Author(s):  
Carsten A. Wagner ◽  
Florian Lang ◽  
Stefan Bröer
Keyword(s):  
Amino Acid ◽  
Membrane Protein ◽  
Heavy Chain ◽  
Light Chain ◽  
Disulfide Bridge ◽  
Amino Acid Transporter ◽  
Light Chains ◽  
Amino Acid Transporters ◽  
Amino Acid Transport System ◽  
Acid Transporter

Heterodimeric amino acid transporters are comprised of two subunits, a polytopic membrane protein (light chain) and an associated type II membrane protein (heavy chain). The heavy chain rbAT (related to b0,+ amino acid transporter) associates with the light chain b0,+AT (b0,+ amino acid transporter) to form the amino acid transport system b0,+, whereas the homologous heavy chain 4F2hc interacts with several light chains to form system L (with LAT1 and LAT2), system y+L (with y+LAT1 and y+LAT2), system x[Formula: see text](with xAT), or system asc (with asc1). The association of light chains with the two heavy chains is not unambiguous. rbAT may interact with LAT2 and y+LAT1 and vice versa; 4F2hc may interact with b0,+AT when overexpressed. 4F2hc is necessary for trafficking of the light chain to the plasma membrane, whereas the light chains are thought to determine the transport characteristics of the respective heterodimer. In contrast to 4F2hc, mutations in rbAT suggest that rbAT itself takes part in the transport besides serving for the trafficking of the light chain to the cell surface. Heavy and light subunits are linked together by a disulfide bridge. The disulfide bridge, however, is not necessary for the trafficking of rbAT or 4F2 heterodimers to the membrane or for the functioning of the transporter. However, there is experimental evidence that the disulfide bridge in the 4F2hc/LAT1 heterodimer plays a role in the regulation of a cation channel. These results highlight complex interactions between the different subunits of heterodimeric amino acid transporters and suggest that despite high grades of homology, the interactions between rbAT and 4F2hc and their respective partners may be different.

scholarly journals Downregulation of Placental Amino Acid Transporter Expression and mTORC1 Signaling Activity Contributes to Fetal Growth Retardation in Diabetic Rats

2020 ◽  
Vol 21 (5) ◽  
pp. 1849
Author(s):  
Jie Xu ◽  
Jiao Wang ◽  
Yang Cao ◽  
Xiaotong Jia ◽  
Yujia Huang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Rat Model ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Amino Acid Transport System ◽  
Intrauterine Growth ◽  
System L ◽  
Acid Transporter ◽  
Transporter Expression

Alterations in placental transport may contribute to abnormal fetal intrauterine growth in pregnancies complicated by diabetes, but it is not clear whether the placental amino acid transport system is altered in diabetic pregnancies. We therefore studied the changes in the expressions of placental amino acid transporters in a rat model of diabetes induced by streptozotocin, and tested the effects of hyperglycemia on trophoblast amino acid transporter in vitro. Our results showed that the expressions for key isoforms of system L amino acid transporters were significantly reduced in the placentas of streptozotocin-induced diabetic pregnant rats, which was associated with the decreased birthweight in the rats. A decreased placental efficiency and decreased placental mammalian target of rapamycin (mTOR) complex 1 (mTORC1) activity were also found in the rat model. In addition, hyperglycemia in vitro could inhibit amino acid transporter expression and mTORC1 activity in human trophoblast. Inhibition of mTORC1 activity led to reduced amino acid transporter expression in placental trophoblast. We concluded that reduced placental mTORC1 activity during pregnancy resulted in decreased placental amino acid transporter expression and, subsequently, contributed to fetal intrauterine growth restriction in pregnancies complicated with diabetes.

A traffic signal for heterodimeric amino acid transporters to transfer from the ER to the Golgi

2008 ◽  
Vol 417 (2) ◽  
pp. e9-e11 ◽  
Author(s):  
Vadivel Ganapathy
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Amino Acid Transporter ◽  
Traffic Signal ◽  
Transport Systems ◽  
Amino Acid Transporters ◽  
Sequence Motif ◽  
Prevailing Paradigm ◽  
New Findings ◽  
Acid Transporter

Heterodimeric amino acid transporters represent a unique class of transport systems that consist of a light chain that serves as the ‘transporter proper’ and a heavy chain that is necessary for targeting the complex to the plasma membrane. The currently prevailing paradigm assigns no role for the light chains in the cellular processing of these transporters. In this issue of the Biochemical Journal, Sakamoto et al. provide evidence contrary to this paradigm. Their studies with the rBAT –b0,+AT (related to b0,+ amino acid transporter–b0,+-type amino acid transporter) heterodimeric amino acid transporter show that the C-terminus of the light chain b0,+AT contains a sequence motif that serves as the traffic signal for the transfer of the heterodimeric complex from the endoplasmic reticulum to the Golgi. This is a novel function for the light chain in addition to its already established role as the subunit responsible for the transport activity. These new findings also seem to be applicable to other heterodimeric amino acid transporters as well.

A novel role of the C-terminus of b0,+AT in the ER–Golgi trafficking of the rBAT–b0,+AT heterodimeric amino acid transporter

2008 ◽  
Vol 417 (2) ◽  
pp. 441-448 ◽  
Author(s):  
Shinichi Sakamoto ◽  
Arthit Chairoungdua ◽  
Shushi Nagamori ◽  
Pattama Wiriyasermkul ◽  
Kanyarat Promchan ◽  
...  
Keyword(s):  
Amino Acid ◽  
Light Chain ◽  
Amino Acid Transporter ◽  
Site Directed Mutagenesis ◽  
Amino Acid Transporters ◽  
Hek 293 ◽  
C Terminus ◽  
Heterodimeric Complex ◽  
Acid Transporter ◽  
Membrane Spanning

The heterodimeric complex composed of rBAT (related to b0,+ amino acid transporter), a single-membrane-spanning glycosylated heavy chain, and b0,+AT, a putative 12-membrane-spanning non-glycosylated light chain, is an amino acid transporter that mediates the activity of system b0,+, a major apical transport system for cystine and dibasic amino acids in renal proximal tubule and small intestine. The C-terminus of b0,+AT has been proposed to play an important role in the functional expression of the heterodimeric transporters. In the present study, to reveal the roles of the C-terminus, we analysed b0,+AT mutants whose C-termini were sequentially deleted or replaced by site-directed mutagenesis in polarized MDCKII (Madin–Darby canine kidney II), non-polarized HEK-293 (human embryonic kidney-293) and HeLa cells. Although the deletion of C-terminus of b0,+AT did not affect the formation of a heterodimer with rBAT, it resulted in the loss of apparent transport function, owing to the failure of the plasma-membrane targeting of rBAT–b0,+AT heterodimeric complex associated with incomplete glycosylation of rBAT. A motif-like sequence Val480-Pro481-Pro482 was identified in the C-terminus of b0,+AT to be responsible for the C-terminus action in promoting the trafficking of rBAT–b0,+AT heterodimeric complex from the ER (endoplasmic reticulum) to Golgi apparatus. This is, to our knowledge, the first demonstration of the active contribution of the C-terminus of a light-chain subunit to the intracellular trafficking of heterodimeric transporters. Because the motif-like sequence Val480-Pro481-Pro482 is well conserved among the C-termini of light-chain subunits, common regulatory mechanisms could be proposed among heterodimeric amino acid transporters.

scholarly journals Sub-Nanometer Cryo-EM Density Map of the Human Heterodimeric Amino Acid Transporter 4F2hc-LAT2

2020 ◽  
Vol 21 (19) ◽  
pp. 7094 ◽  
Author(s):  
Jean-Marc Jeckelmann ◽  
Dimitrios Fotiadis
Keyword(s):  
Amino Acid ◽  
Protein Complexes ◽  
Point Mutations ◽  
Methylotrophic Yeast ◽  
Homology Model ◽  
Disulfide Bridge ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Density Map ◽  
Acid Transporter

Heterodimeric amino acid transporters (HATs) are protein complexes mediating the transport of amino acids and derivatives thereof across biological membranes. HATs are composed of two subunits, a heavy and a light chain subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. The human HAT 4F2hc-LAT2 is composed of the type-II membrane N-glycoprotein 4F2hc (SCL3A2) and the L-type amino acid transporter LAT2 (SLC7A8), which are covalently linked to each other by a conserved disulfide bridge. Whereas LAT2 catalyzes substrate transport, 4F2hc is important for the successful trafficking of the transporter to the plasma membrane. The overexpression, malfunction, or absence of 4F2hc-LAT2 is associated with human diseases, and therefore, this heterodimeric complex represents a potential drug target. The recombinant human 4F2hc-LAT2 can be functionally overexpressed in the methylotrophic yeast Pichia pastoris, and the protein can be purified. Here, we present the cryo-EM density map of the human 4F2hc-LAT2 amino acid transporter at sub-nanometer resolution. A homology model of 4F2hc-LAT2 in the inward-open conformation was generated and fitted into the cryo-EM density and analyzed. In addition, disease-causing point mutations in human LAT2 were mapped on the homology model of 4F2hc-LAT2, and the possible functional implications on the molecular level are discussed.

scholarly journals Cryo-EM structure of the human heteromeric amino acid transporter b0,+AT-rBAT

2020 ◽  
Vol 6 (16) ◽  
pp. eaay6379 ◽  
Author(s):  
Renhong Yan ◽  
Yaning Li ◽  
Yi Shi ◽  
Jiayao Zhou ◽  
Jianlin Lei ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Membrane Trafficking ◽  
Binding Pocket ◽  
Disulfide Bridge ◽  
Ligand Molecule ◽  
Amino Acid Transporters ◽  
Cationic Amino Acids

Heteromeric amino acid transporters (HATs) catalyze the transmembrane movement of amino acids, comprising two subunits, a heavy chain and a light chain, linked by a disulfide bridge. The b0,+AT (SLC7A9) is a representative light chain of HATs, forming heterodimer with rBAT, a heavy chain which mediates the membrane trafficking of b0,+AT. The b0,+AT-rBAT complex is an obligatory exchanger, which mediates the influx of cystine and cationic amino acids and the efflux of neutral amino acids in kidney and small intestine. Here, we report the cryo-EM structure of the human b0,+AT-rBAT complex alone and in complex with arginine substrate at resolution of 2.7 and 2.3 Å, respectively. The overall structure of b0,+AT-rBAT exists as a dimer of heterodimer consistent with the previous study. A ligand molecule is bound to the substrate binding pocket, near which an occluded pocket is identified, to which we found that it is important for substrate transport.

scholarly journals Pichia pastoris and the Recombinant Human Heterodimeric Amino Acid Transporter 4F2hc-LAT1: From Clone Selection to Pure Protein

2021 ◽  
Vol 4 (3) ◽  
pp. 51
Author(s):  
Satish Kantipudi ◽  
Daniel Harder ◽  
Sara Bonetti ◽  
Dimitrios Fotiadis ◽  
Jean-Marc Jeckelmann
Keyword(s):  
Amino Acid ◽  
Pichia Pastoris ◽  
Protein Complexes ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Expression Host ◽  
Amino Acids And Derivatives ◽  
Heterodimeric Complex ◽  
Acid Transporter ◽  
And Function

Heterodimeric amino acid transporters (HATs) are protein complexes composed of two subunits, a heavy and a light subunit belonging to the solute carrier (SLC) families SLC3 and SLC7. HATs transport amino acids and derivatives thereof across the plasma membrane. The human HAT 4F2hc-LAT1 is composed of the type-II membrane N-glycoprotein 4F2hc (SLC3A2) and the L-type amino acid transporter LAT1 (SLC7A5). 4F2hc-LAT1 is medically relevant, and its dysfunction and overexpression are associated with autism and tumor progression. Here, we provide a general applicable protocol on how to screen for the best membrane transport protein-expressing clone in terms of protein amount and function using Pichia pastoris as expression host. Furthermore, we describe an overexpression and purification procedure for the production of the HAT 4F2hc-LAT1. The isolated heterodimeric complex is pure, correctly assembled, stable, binds the substrate L-leucine, and is thus properly folded. Therefore, this Pichia pastoris-derived recombinant human 4F2hc-LAT1 sample can be used for downstream biochemical and biophysical characterizations.

scholarly journals Amino Acid Transport Defects in Human Inherited Metabolic Disorders

2019 ◽  
Vol 21 (1) ◽  
pp. 119 ◽  
Author(s):  
Raquel Yahyaoui ◽  
Javier Pérez-Frías
Keyword(s):  
Amino Acid ◽  
Metabolic Disorders ◽  
Strong Association ◽  
Cell Types ◽  
Amino Acid Transporter ◽  
Molecular Defect ◽  
Amino Acid Transporters ◽  
Inherited Disorders ◽  
Laboratory Findings ◽  
Acid Transporter

Amino acid transporters play very important roles in nutrient uptake, neurotransmitter recycling, protein synthesis, gene expression, cell redox balance, cell signaling, and regulation of cell volume. With regard to transporters that are closely connected to metabolism, amino acid transporter-associated diseases are linked to metabolic disorders, particularly when they involve different organs, cell types, or cell compartments. To date, 65 different human solute carrier (SLC) families and more than 400 transporter genes have been identified, including 11 that are known to include amino acid transporters. This review intends to summarize and update all the conditions in which a strong association has been found between an amino acid transporter and an inherited metabolic disorder. Many of these inherited disorders have been identified in recent years. In this work, the physiological functions of amino acid transporters will be described by the inherited diseases that arise from transporter impairment. The pathogenesis, clinical phenotype, laboratory findings, diagnosis, genetics, and treatment of these disorders are also briefly described. Appropriate clinical and diagnostic characterization of the underlying molecular defect may give patients the opportunity to avail themselves of appropriate therapeutic options in the future.

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

2020 ◽  
Vol 117 (35) ◽  
pp. 21281-21287 ◽  
Author(s):  
Di Wu ◽  
Tamara N. Grund ◽  
Sonja Welsch ◽  
Deryck J. Mills ◽  
Max Michel ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heavy Chain ◽  
Light Chain ◽  
Basic Amino Acid ◽  
Binding Pocket ◽  
Structural Basis ◽  
Amino Acid Transporters ◽  
Wide Range ◽  
Functional Complex ◽  
Protein Components

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.

192 AMINO ACID TRANSPORTER EXPRESSION IN BOVINE OVIDUCT EPITHELIAL CELLS

2008 ◽  
Vol 20 (1) ◽  
pp. 175
Author(s):  
S. L. Whitear ◽  
H. J. Leese
Keyword(s):  
Amino Acid ◽  
Epithelial Cells ◽  
Amino Acid Transporter ◽  
Positive Control ◽  
Amino Acid Transporters ◽  
Oviduct Epithelial Cells ◽  
Acid Transporter ◽  
Oviduct Fluid ◽  
Transporter Expression

Oviduct fluid provides the environment for the gametes and early embryo but little is known about the mechanisms underlying its formation. Components of oviduct fluid have been shown to be present at concentrations different from that in blood, indicative of selective transport by the epithelial cells lining the lumen. For example, amino acid concentrations in oviduct fluid differ from those in extracellular fluid and have also been shown to be important to preimplantation embryos in vitro, enhancing development, especially when added at physiological concentrations. However, little is known about amino acid transport systems in the oviduct, and the aim of this work was to search for mRNA transcripts for amino acid transporters in bovine oviduct epithelial cells. Contra- and ipsi-lateral oviducts were removed from abattoir-derived reproductive tracts at specific stages of the reproductive cycle. Oviducts were trimmed of surrounding tissue and fat and slit longitudinally to expose the luminal surface. Bovine oviduct epithelial cells (bOEC) were scraped from the surface using a sterile glass coverslip and washed by centrifugation. mRNA was isolated using Trizol-chloroform extraction and lithium chloride precipitation methods. PCR was used to detect cDNA encoding the amino acid transporters CAT-1, CAT-4, and LAT1. A negative control (water) and a positive control (human placental cDNA) were included in each experiment and β-actin expression was used as a positive control for cDNA library generation. Products were separated by agarose gel electrophoresis. PCR for β-actin resulted in the presence of a positive band in all samples, showing successful extraction of mRNA and generation of cDNA libraries. mRNA for CAT-1 and LAT1 was detected in bOEC from contra- and ipsi-lateral oviducts and from each cycle stage tested. There was, however, no detectable mRNA for CAT-4 in any of the samples. To our knowledge, this is the first report of amino acid transporter expression in the mammalian oviduct. CAT-1 is a ubiquitous sodium-independent uniporter of cationic amino acids that has been localized to the basolateral membrane of epithelial cells. The presence of mRNA for this amino acid transporter in all samples tested is therefore to be expected. LAT1 is a obligatory exchanger which exports glutamine and cystine and imports large uncharged branched-chain amino acids. This transporter may be partly responsible for the high concentration of glutamate in the basal compartment of in vitro cell cultures reported in our previous work (Whitear and Leese 2007 Biennial Meet. Joint Fertil. Soc., York, UK). CAT-4 shares only 40% sequence homology with CAT-1 and its function is unknown. Its expression appears to be restricted to brain, testis, and placenta, and the absence of mRNA for the oviduct was, perhaps, not surprising. Further experiments will investigate expression levels of other amino acid transporters in bOEC and transporter localization using immunohistochemistry. This work was funded by the BBSRC and ANGLE Technology Ltd.

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