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.

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 Multiple components of arginine and phenylalanine transport induced in neutral and basic amino acid transporter-cRNA-injected Xenopus oocytes

1996 ◽  
Vol 318 (3) ◽  
pp. 915-922 ◽  
Author(s):  
George J PETER ◽  
Iain G. DAVIDSON ◽  
Aamir AHMED ◽  
Lynn McILROY ◽  
Alexander R. FORRESTER ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Competitive Inhibition ◽  
Protein Complexes ◽  
Basic Amino Acid ◽  
Amino Acid Transporter ◽  
Transport Processes ◽  
Arginine Transport ◽  
Acid Transporter ◽  
Phenylalanine Transport

The induced uptakes of l-[3H]phenylalanine and l-[3H]arginine in oocytes injected with clonal NBAT (neutral and basic amino acid transporter) cRNA show differential inactivation by pre-treatment with N-ethylmaleimide (NEM), revealing at least two distinct transport processes. NEM-resistant arginine transport is inhibited by leucine and phenylalanine but not by alanine or valine; mutual competitive inhibition of NEM-resistant uptake of arginine and phenylalanine indicates that the two amino acids share a single transporter. NEM-senstive arginine transport is inhibited by leucine, phenylalanine, alanine and valine. At least two NEM-sensitive transporters may be expressed because we have been unable to confirm mutual competitive inhibition between arginine and phenylalanine transport. The NEM-resistant transport mechanism appears to involve distinct but overlapping binding sites for cationic and zwitterionic substrates. NBAT is known to form oligomeric protein complexes in cell membranes, and its functional roles when expressed in Xenopus oocytes may include interaction with oocyte proteins, leading to increased native amino acid transport activities; these resemble NBAT-expressed activities in terms of NEM-sensitivity and apparent substrate range (including an unusual inhibition by β-phenylalanine).

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.

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.

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.

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.

scholarly journals Skeletal muscle amino acid transporter expression is increased in young and older adults following resistance exercise

2011 ◽  
Vol 111 (1) ◽  
pp. 135-142 ◽  
Author(s):  
Micah J. Drummond ◽  
Christopher S. Fry ◽  
Erin L. Glynn ◽  
Kyle L. Timmerman ◽  
Jared M. Dickinson ◽  
...  
Keyword(s):  
Older Adults ◽  
Amino Acid ◽  
Resistance Exercise ◽  
Amino Acid Transporter ◽  
Initiation Factor ◽  
Amino Acid Transporters ◽  
Α Subunit ◽  
Mtorc1 Signaling ◽  
Acid Transporter ◽  
Transporter Expression

Amino acid transporters and mammalian target of rapamycin complex 1 (mTORC1) signaling are important contributors to muscle protein anabolism. Aging is associated with reduced mTORC1 signaling following resistance exercise, but the role of amino acid transporters is unknown. Young ( n = 13; 28 ± 2 yr) and older ( n = 13; 68 ± 2 yr) subjects performed a bout of resistance exercise. Skeletal muscle biopsies ( vastus lateralis) were obtained at basal and 3, 6, and 24 h postexercise and were analyzed for amino acid transporter mRNA and protein expression and regulators of amino acid transporter transcription utilizing real-time PCR and Western blotting. We found that basal amino acid transporter expression was similar in young and older adults ( P > 0.05). Exercise increased L-type amino acid transporter 1/solute-linked carrier (SLC) 7A5, CD98/SLC3A2, sodium-coupled neutral amino acid transporter 2/SLC38A2, proton-assisted amino acid transporter 1/SLC36A1, and cationic amino acid transporter 1/SLC7A1 mRNA expression in both young and older adults ( P < 0.05). L-type amino acid transporter 1 and CD98 protein increased only in younger adults ( P < 0.05). eukaryotic initiation factor 2 α-subunit (S52) increased similarly in young and older adults postexercise ( P < 0.05). Ribosomal protein S6 (S240/244) and activating transcription factor 4 nuclear protein expression tended to be higher in the young, while nuclear signal transducer and activator of transcription 3 (STAT3) (Y705) was higher in the older subjects postexercise ( P < 0.05). These results suggest that the rapid upregulation of amino acid transporter expression following resistance exercise may be regulated differently between the age groups, but involves a combination of mTORC1, activating transcription factor 4, eukaryotic initiation factor 2 α-subunit, and STAT3. We propose an increase in amino acid transporter expression may contribute to enhanced amino acid sensitivity following exercise in young and older adults. In older adults, the increased nuclear STAT3 phosphorylation may be indicative of an exercise-induced stress response, perhaps to export amino acids from muscle cells.

scholarly journals Cloning, functional expression and dietary regulation of the mouse neutral and basic amino acid transporter (NBAT)

1997 ◽  
Vol 328 (2) ◽  
pp. 657-664 ◽  
Author(s):  
Hiroko SEGAWA ◽  
Ken-ichi MIYAMOTO ◽  
Yoshio OGURA ◽  
Hiromi HAGA ◽  
Kyoko MORITA ◽  
...  
Keyword(s):  
Amino Acid ◽  
Xenopus Oocytes ◽  
Physiological Role ◽  
Basic Amino Acid ◽  
Functional Expression ◽  
Amino Acid Transporter ◽  
Amino Acid Transporters ◽  
Transport Activity ◽  
Acid Transporter ◽  
Mouse Ileum

The Na+-independent dibasic and neutral amino acid transporter NBAT is among the least hydrophobic of mammalian amino acid transporters. The transporter contains one to four transmembrane domains and induces amino acid transport activity via a b0,+-like system when expressed in Xenopus oocytes. However, the physiological role of NBAT remains unclear. Complementary DNA clones encoding mouse NBAT have now been isolated. The expression of mouse NBAT in Xenopus oocytes also induced an obligatory amino acid exchange activity similar to that of the b0,+-like system. The amount of NBAT mRNA in mouse kidney increased during postnatal development, consistent with the increase in renal cystine and dibasic transport activity. Dietary aspartate induced a marked increase in cystine transport via the b0,+ system in mouse ileum. A high-aspartate diet also increased the amount of NBAT mRNA in mouse ileum. In the ileum of mice fed on the aspartate diet, the extent of cystine transport was further increased by preloading brush border membrane vesicles with lysine. Hybrid depletion of NBAT mRNA from ileal polyadenylated RNA revealed that the increase in cystine transport activity induced by the high-aspartate diet, as measured in Xenopus oocytes, was attributable to NBAT. These results demonstrate that mouse NBAT has an important role in cystine transport.

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