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

2019 ◽  
Vol 20 (4) ◽  
pp. 931 ◽  
Author(s):  
Jean-Marc Jeckelmann ◽  
Dimitrios Fotiadis
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Drug Targets ◽  
Plasma Membranes ◽  
Protein Complexes ◽  
Methylotrophic Yeast ◽  
Disulfide Bridge ◽  
Phase Plate ◽  
Amino Acid Transporters ◽  
Loss Of Function

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.

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.

Differential cystine and dibasic amino acid handling after loss of function of the amino acid transporter b0,+AT (Slc7a9) in mice

2013 ◽  
Vol 305 (12) ◽  
pp. F1645-F1655 ◽  
Author(s):  
Andrea Di Giacopo ◽  
Isabel Rubio-Aliaga ◽  
Alessandra Cantone ◽  
Ferruh Artunc ◽  
Rexhep Rexhepaj ◽  
...  
Keyword(s):  
Gene Expression ◽  
Amino Acids ◽  
Amino Acid ◽  
Membrane Vesicle ◽  
Fractional Excretion ◽  
Amino Acid Transporters ◽  
Type I ◽  
Loss Of Function ◽  
Dibasic Amino Acid ◽  
Knockout Animals

Cystinuria is an autosomal recessive disease caused by mutations in SLC3A1 ( rBAT) and SLC7A9 ( b 0,+ AT). Gene targeting of the catalytic subunit ( Slc7a9) in mice leads to excessive excretion of cystine, lysine, arginine, and ornithine. Here, we studied this non-type I cystinuria mouse model using gene expression analysis, Western blotting, clearance, and brush-border membrane vesicle (BBMV) uptake experiments to further characterize the renal and intestinal consequences of losing Slc7a9 function. The electrogenic and BBMV flux studies in the intestine suggested that arginine and ornithine are transported via other routes apart from system b0,+. No remarkable gene expression changes were observed in other amino acid transporters and the peptide transporters in the intestine and kidney. Furthermore, the glomerular filtration rate (GFR) was reduced by 30% in knockout animals compared with wild-type animals. The fractional excretion of arginine was increased as expected (∼100%), but fractional excretions of lysine (∼35%), ornithine (∼16%), and cystine (∼11%) were less affected. Loss of function of b0,+AT reduced transport of cystine and arginine in renal BBMVs and completely abolished the exchanger activity of dibasic amino acids with neutral amino acids. In conclusion, loss of Slc7a9 function decreases the GFR and increases the excretion of several amino acids to a lesser extent than expected with no clear regulation at the mRNA and protein level of alternative transporters and no increased renal epithelial uptake. These observations indicate that transporters located in distal segments of the kidney and/or metabolic pathways may partially compensate for Slc7a9 loss of function.

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.

The immunosuppressant FK506 inhibits amino acid import in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (8) ◽  
pp. 5010-5019 ◽  
Author(s):  
J Heitman ◽  
A Koller ◽  
J Kunz ◽  
R Henriquez ◽  
A Schmidt ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acids ◽  
Amino Acid ◽  
Cyclosporin A ◽  
Secretory Pathway ◽  
Yeast Cells ◽  
Amino Acid Transporters ◽  
Yeast Saccharomyces Cerevisiae ◽  
Eukaryotic Microorganisms

The immunosuppressants cyclosporin A, FK506, and rapamycin inhibit growth of unicellular eukaryotic microorganisms and also block activation of T lymphocytes from multicellular eukaryotes. In vitro, these compounds bind and inhibit two different types of peptidyl-prolyl cis-trans isomerases. Cyclosporin A binds cyclophilins, whereas FK506 and rapamycin bind FK506-binding proteins (FKBPs). Cyclophilins and FKBPs are ubiquitous, abundant, and targeted to multiple cellular compartments, and they may fold proteins in vivo. Previously, a 12-kDa cytoplasmic FKBP was shown to be only one of at least two FK506-sensitive targets in the yeast Saccharomyces cerevisiae. We find that a second FK506-sensitive target is required for amino acid import. Amino acid-auxotrophic yeast strains (trp1 his4 leu2) are FK506 sensitive, whereas prototrophic strains (TRP1 his4 leu2, trp1 HIS4 leu2, and trp1 his4 LEU2) are FK506 resistant. Amino acids added exogenously to the growth medium mitigate FK506 toxicity. FK506 induces GCN4 expression, which is normally induced by amino acid starvation. FK506 inhibits transport of tryptophan, histidine, and leucine into yeast cells. Lastly, several genes encoding proteins involved in amino acid import or biosynthesis confer FK506 resistance. These findings demonstrate that FK506 inhibits amino acid import in yeast cells, most likely by inhibiting amino acid transporters. Amino acid transporters are integral membrane proteins which import extracellular amino acids and constitute a protein family sharing 30 to 35% identity, including eight invariant prolines. Thus, the second FK506-sensitive target in yeast cells may be a proline isomerase that plays a role in folding amino acid transporters during transit through the secretory pathway.

Placental amino acid transport

1995 ◽  
Vol 268 (6) ◽  
pp. C1321-C1331 ◽  
Author(s):  
A. J. Moe
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Human Placenta ◽  
Amino Acid Transport ◽  
Single Layer ◽  
Maternal Blood ◽  
Transport Systems ◽  
Amino Acid Transporters ◽  
Acid Transport ◽  
Principal Mechanism

Normal fetal growth and development depend on a continuous supply of amino acids from the mother to the fetus. The placenta is responsible for the transfer of amino acids between the two circulations. The human placenta is hemomonochorial, meaning that the maternal and fetal circulations are separated by a single layer of polarized epithelium called the syncytiotrophoblast, which is in direct contact with maternal blood. Transport proteins located in the microvillous and basal membranes of the syncytiotrophoblast are the principal mechanism for transfer from maternal blood to fetal blood. Knowledge of the function and regulation of syncytiotrophoblast amino acid transporters is of great importance in understanding the mechanism of placental transport and potentially improving fetal and newborn outcomes. The development of methods for the isolation of microvillous and basal membrane vesicles from human placenta over the past two decades has contributed greatly to this understanding. Now a primary cultured trophoblast model is available to study amino acid transport and regulation as the cells differentiate. The types of amino acid transporters and their distribution between the syncytiotrophoblast microvillous and basal membranes are somewhat unique compared with other polarized epithelia. These differences may reflect the unusual circumstance of this epithelium that is exposed to blood on both sides. The current state of knowledge as to the types of transport systems present in syncytiotrophoblast, their regulation, and the effects of maternal consumption of drugs on transport are discussed.

scholarly journals Rhizobium leguminosarum Has a Second General Amino Acid Permease with Unusually Broad Substrate Specificity and High Similarity to Branched-Chain Amino Acid Transporters (Bra/LIV) of the ABC Family

2002 ◽  
Vol 184 (15) ◽  
pp. 4071-4080 ◽  
Author(s):  
A. H. F. Hosie ◽  
D. Allaway ◽  
C. S. Galloway ◽  
H. A. Dunsby ◽  
P. S. Poole
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Rhizobium Leguminosarum ◽  
Binding Protein ◽  
Amino Acid Transporters ◽  
Acid Uptake ◽  
Amino Acid Permease ◽  
Branched Chain Amino Acid ◽  
General Amino Acid Permease ◽  
Branched Chain

ABSTRACT Amino acid uptake by Rhizobium leguminosarum is dominated by two ABC transporters, the general amino acid permease (Aap) and the branched-chain amino acid permease (BraRl). Characterization of the solute specificity of BraRl shows it to be the second general amino acid permease of R. leguminosarum. Although BraRl has high sequence identity to members of the family of hydrophobic amino acid transporters (HAAT), it transports a broad range of solutes, including acidic and basic polar amino acids (l-glutamate, l-arginine, and l-histidine), in addition to neutral amino acids (l-alanine and l-leucine). While amino and carboxyl groups are required for transport, solutes do not have to be α-amino acids. Consistent with this, BraRl is the first ABC transporter to be shown to transport γ-aminobutyric acid (GABA). All previously identified bacterial GABA transporters are secondary carriers of the amino acid-polyamine-organocation (APC) superfamily. Also, transport by BraRl does not appear to be stereospecific as d amino acids cause significant inhibition of uptake of l-glutamate and l-leucine. Unlike all other solutes tested, l-alanine uptake is not dependent on solute binding protein BraCRl. Therefore, a second, unidentified solute binding protein may interact with the BraDEFGRl membrane complex during l-alanine uptake. Overall, the data indicate that BraRl is a general amino acid permease of the HAAT family. Furthermore, BraRl has the broadest solute specificity of any characterized bacterial amino acid transporter.

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