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.

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Association of 4F2hc with light chains LAT1, LAT2 or y+LAT2 requires different domains

Biochemical Journal ◽

10.1042/bj3550725 ◽

2001 ◽

Vol 355 (3) ◽

pp. 725-731 ◽

Cited By ~ 30

Author(s):

Angelika BRÖER ◽

Björn FRIEDRICH ◽

Carsten A. WAGNER ◽

Sophie FILLON ◽

Vadivel GANAPATHY ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Heavy Chain ◽

Membrane Surface ◽

Transmembrane Helix ◽

Surface Expression ◽

Light Chains ◽

Amino Acid Transporters ◽

Slight Reduction ◽

Heavy Chains

Heterodimeric amino acid transporters are comprised of a type-II membrane protein named the heavy chain (4F2hc or rBAT) that may associate with a number of different polytopic membrane proteins, called light chains. It is thought that the heavy chain is mainly involved in the trafficking of the complex to the plasma membrane, whereas the transport process itself is catalysed by the light chain. The 4F2heavy chain (4F2hc) associates with at least six different light chains to induce distinct amino acid-transport activites. To test if the light chains are specifically recognized and to identify domains involved in the recognition of light chains, C-terminally truncated mutants of 4F2hc were constructed and co-expressed with the light chains LAT1, LAT2 and y+LAT2. The truncated isoform T1, comprised of only 133 amino acids that form the cytosolic N-terminus and the transmembrane helix, displayed only a slight reduction in its ability to promote LAT1 expression at the membrane surface compared with the 529 amino acid wild-type 4F2hc protein. Co-expression of increasingly larger 4F2hc mutants caused a delayed translocation of LAT1. In contrast to the weak effects of 4F2hc truncations on LAT1 expression, surface expression of LAT2 and y+LAT2 was almost completely lost with all truncated heavy chains. Co-expression of LAT1 together with the other light chains did not result in displacement of LAT2 and y+LAT2. The results suggest that extracellular domains of the heavy chain are responsible mainly for recognition of light chains other than LAT1 and that the extracellular domain ensures proper translocation to the plasma membrane.

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Function and structure of heterodimeric amino acid transporters

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.4.c1077 ◽

2001 ◽

Vol 281 (4) ◽

pp. C1077-C1093 ◽

Cited By ~ 240

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.

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Yeast Cell-Based Transport Assay for the Functional Characterization of Human 4F2hc-LAT1 and ‐LAT2, and LAT1 and LAT2 Substrates and Inhibitors

Frontiers in Molecular Biosciences ◽

10.3389/fmolb.2021.676854 ◽

2021 ◽

Vol 8 ◽

Author(s):

Satish Kantipudi ◽

Dimitrios Fotiadis

Keyword(s):

Amino Acid ◽

Mammalian Cells ◽

Functional Characterization ◽

Methylotrophic Yeast ◽

Cell Types ◽

Amino Acid Transporters ◽

Cell Assays ◽

Transport Assay ◽

Compound Screening

In mammalian cells, the L-type amino acid transporters (LATs) LAT1 (SLC7A5) and LAT2 (SLC7A8) form heterodimeric amino acid transporters (HATs) with the ancillary protein 4F2hc and are involved in the cellular uptake of specific amino acids. The HAT 4F2hc-LAT1 is found upregulated in various cancer cell types, while 4F2hc-LAT2 is a transporter for non-cancer cells. Preclinical studies have highlighted that 4F2hc-LAT1 plays an important role in tumor progression representing a valid anticancer target. Consequently, current research is focusing on the development of potent and specific human 4F2hc-LAT1 inhibitors. On the other hand, 4F2hc-LAT2 is emerging as target of other diseases, thus also gaining clinical interest. To determine affinity and specificity of substrates and inhibitors for 4F2hc-LAT1 or 4F2hc-LAT2, robust transport cell assays are indispensable. We have optimized and validated a transport assay using cells of the methylotrophic yeast Pichia pastoris stably overexpressing the human HATs 4F2hc-LAT1 or -LAT2, and the LATs LAT1 or LAT2 alone. The radioligand [3H]L-leucine was used as reporter and the substrates L-leucine, triiodothyronine (T3) and thyroxine (T4) as well as the inhibitors BCH and JPH203 (KYT-0353) for assay validation. Obtained half-maximal inhibitory concentrations also provided new insights, e.g., into the LAT specificity of the potent inhibitor JPH203 and on the potency of the thyroid hormones T3 and T4 to inhibit transport through human 4F2hc-LAT2. The LAT1 and LAT2 assays are of particular interest to determine possible implications and influences of 4F2hc in ligand binding and transport. In summary, the presented assays are valuable for characterization of ligands, e.g., towards 4F2hc-LAT1 specificity, and can also be applied for compound screening. Finally, our established approach and assay would also be applicable to other HATs and LATs of interest.

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Incorporation of proteins into (Xenopus) oocytes by proteoliposome microinjection: functional characterization of a novel aquaporin

Journal of Cell Science ◽

10.1242/jcs.109.6.1285 ◽

1996 ◽

Vol 109 (6) ◽

pp. 1285-1295

Author(s):

F. Le Caherec ◽

P. Bron ◽

J.M. Verbavatz ◽

A. Garret ◽

G. Morel ◽

...

Keyword(s):

Plasma Membrane ◽

Membrane Proteins ◽

Digestive Tract ◽

Xenopus Oocytes ◽

Plasma Membranes ◽

Expression System ◽

Functional Characterization ◽

Xenopus Laevis Oocytes ◽

Plasma Membrane Proteins ◽

Cicadella Viridis

Xenopus laevis oocytes are widely used as an expression system for plasma membrane proteins, achieved by cytoplasmic microinjection of messenger RNA. In the present study, we propose an alternative system allowing functional insertion of exogenous proteins into the plasma membrane of Xenopus oocytes. We microinjected proteoliposome suspensions into the cytoplasm and then analyzed membrane protein function. The proteins used in this work were members of the MIP family: the human erythrocyte water channel aquaporin 1 (AQP1), the major intrinsic protein (MIP26) from bovine eye lens and a 25 kDa polypeptide (P25) from a water shunting complex found in the digestive tract of an homopteran sap-sucking insect (Cicadella viridis). Proteoliposomes containing either AQP1, MIP26, or P25 were injected into Xenopus oocytes. The subsequent insertion of these proteins into the plasma membrane of oocytes was demonstrated by immunocytochemistry. Oocytes microinjected with either AQP1 or P25-proteoliposomes exhibited significantly increased osmotic membrane water permeabilities (Pf = 3.16 +/- 026 and 4.03 +/- 0.26 × 10(−3) cm/second, respectively) compared to those measured for oocytes injected with liposomes alone or with MIP26-proteoliposomes (Pf = 1.39 +/- 0.07 and 1.44 +/- 0.10 × 10(−3) cm/second, respectively). These effects were inhibited by HgCl2 in a reversible manner. Arrhenius activation energies of water transfer were low when AQP1 or P25 were present in oocyte plasma membranes (Ea = 2.29 and 3.01 kcal/mol, respectively, versus Ea = 11.75 kcal/mol for liposome injected oocytes). The properties observed here for AQP1 are identical to those widely reported following AQP1 cRNA expression in oocytes. From the present study, we conclude that: (1) exogenous plasma membrane proteins incorporated into liposomes and microinjected into the cytoplasm of Xenopus oocytes are subsequently found in the plasma membrane of the oocytes in a functional state; and (2) in this system, the P25 polypeptide from the MIP family found in the digestive tract of Cicadella viridis exhibits properties similar to those described for the archetype of water channels AQP1, and thus is a new member of the aquaporin family.

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Expression of membrane transporters in cane toad Bufo marinus oocytes

Journal of Experimental Biology ◽

10.1242/jeb.202.16.2217 ◽

1999 ◽

Vol 202 (16) ◽

pp. 2217-2223

Author(s):

D. Markovich ◽

R.R. Regeer

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Expression System ◽

Bufo Marinus ◽

Membrane Transporters ◽

Xenopus Laevis Oocytes ◽

Amino Acid Transporters ◽

Acid Uptake ◽

Cane Toad ◽

Toad Bufo

Membrane transport proteins (transporters and ion channels) have been extensively expressed in amphibian oocytes. The aims of this study were to determine whether oocytes from the cane toad Bufo marinus could be used as an alternative expression system to the broadly used Xenopus laevis oocytes. mRNAs encoding plasma membrane transporters NaSi-1 and sat-1 (sulphate transporters), NaDC-1 (dicarboxylate transporter), SGLT-1 (Na(+)/glucose cotransporter) and rBAT and 4F2 hc (amino acid transporters) were injected into B. marinus oocytes. All led to significant induction of their respective transport activities. Uptake rates were comparable with those in X. laevis oocytes, with the exception of rBAT, which was able to induce amino acid uptake only in X. laevis oocytes, suggesting that rBAT may require an endogenous X. laevis oocyte protein that is absent from B. marinus oocytes. Transport kinetics were determined for the NaSi-1 cotransporter in B. marinus oocytes, with identical results to those obtained in X. laevis oocytes. NaSi-1 specificity for the Na(+) cation was determined, and the anions selenate, molybdate, tungstate, oxalate and thiosulphate could all inhibit NaSi-1-induced sulphate transport. This study demonstrates that cane toad oocytes can be used successfully to express plasma membrane proteins, making this a viable heterologous system for the expression of proteins.

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Expression of solute carrier 7A4 (SLC7A4) in the plasma membrane is not sufficient to mediate amino acid transport activity

Biochemical Journal ◽

10.1042/bj20020084 ◽

2002 ◽

Vol 364 (3) ◽

pp. 767-775 ◽

Cited By ~ 40

Author(s):

Sabine WOLF ◽

Annette JANZEN ◽

Nicole VÉKONY ◽

Ursula MARTINÉ ◽

Dennis STRAND ◽

...

Keyword(s):

Plasma Membrane ◽

Amino Acid ◽

Protein Expression ◽

Amino Acid Transport ◽

Fluorescent Protein ◽

Xenopus Laevis Oocytes ◽

Amino Acid Transporters ◽

Acid Transport ◽

Transport Activity ◽

Solute Carrier

Member 4 of human solute carrier family 7 (SLC7A4) exhibits significant sequence homology with the SLC7 subfamily of human cationic amino acid transporters (hCATs) [Sperandeo, Borsani, Incerti, Zollo, Rossi, Zuffardi, Castaldo, Taglialatela, Andria and Sebastio (1998) Genomics 49, 230–236]. It is therefore often referred to as hCAT-4 even though no convincing transport activity has been shown for this protein. We expressed SLC7A4 in Xenopus laevis oocytes, but could not detect any transport activity for cationic, neutral or anionic amino acids or for the polyamine putrescine. In addition, human glioblastoma cells stably overexpressing a fusion protein between SLC7A4 and the enhanced green fluorescent protein (EGFP) did not exhibit an increased transport activity for l-arginine. The lack of transport activity was not due to a lack of SLC7A4 protein expression in the plasma membrane, as in both cell types SLC7A4-EGFP exhibited a similar subcellular localization and level of protein expression as functional hCAT-EGFP proteins. The expression of SLC7A4 can be induced in NT2 teratocarcinoma cells by treatment with retinoic acid. However, also for this endogenously expressed SLC7A4, we could not detect any transport activity for l-arginine. Our data demonstrate that the expression of SLC7A4 in the plasma membrane is not sufficient to induce an amino acid transport activity in X. laevis oocytes or human cells. Therefore, SLC7A4 is either not an amino acid transporter or it needs additional (protein) factor(s) to be functional.

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Na(+)-independent transport (uniport) of amino acids and glucose in mammalian cells.

Journal of Experimental Biology ◽

10.1242/jeb.196.1.93 ◽

1994 ◽

Vol 196 (1) ◽

pp. 93-108

Author(s):

D K Kakuda ◽

C L MacLeod

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Mammalian Cells ◽

Glucose Transporter ◽

Sequence Similarity ◽

Amino Acid Sequence Similarity ◽

Amino Acid Transporters ◽

Cationic Amino Acid ◽

Transporter Family ◽

Share Amino Acid Sequence

Recent advances have made possible the isolation of the genes and their cDNAs encoding Na(+)-independent amino acid transporters. Two classes of amino acid 'uniporters' have been isolated. One class contains the mCAT (murine cationic amino acid transporter) gene family that encodes proteins predicted to span the membrane 12-14 times and exhibits structural properties similar to the GLUT (glucose transporter) family and to other well-known transporters. The other class consists of two known genes, rBAT (related to B system amino acid transporters) and 4F2hc, that share amino acid sequence similarity with alpha-amylases and alpha-glucosidases. They are type II glycoproteins predicted to span the membrane only once, yet they mediate the Na(+)-independent transport of cationic and zwitterionic amino acids in Xenopus oocytes. Mutations in the human rBAT gene have been identified by Palacín and his co-workers in several families suffering from a heritable form of cystinuria. This important finding clearly establishes a key role for rBAT in cystine transport. The two classes of amino acid transporters are compared with the well-studied GLUT family of Na(+)-independent glucose transporters.

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Cloning and functional characterization of a new subtype of the amino acid transport system N

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1757 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1757-C1768 ◽

Cited By ~ 79

Author(s):

Takeo Nakanishi ◽

Ramesh Kekuda ◽

You-Jun Fei ◽

Takahiro Hatanaka ◽

Mitsuru Sugawara ◽

...

Keyword(s):

Amino Acids ◽

Amino Acid ◽

Transport System ◽

Amino Acid Transport ◽

Mammalian Cells ◽

Functional Characterization ◽

Isobutyric Acid ◽

Xenopus Laevis Oocytes ◽

Acid Transport ◽

Amino Acid Transport System

We have cloned a new subtype of the amino acid transport system N2 (SN2 or second subtype of system N) from rat brain. Rat SN2 consists of 471 amino acids and belongs to the recently identified glutamine transporter gene family that consists of system N and system A. Rat SN2 exhibits 63% identity with rat SN1. It also shows considerable sequence identity (50–56%) with the members of the amino acid transporter A subfamily. In the rat, SN2 mRNA is most abundant in the liver but is detectable in the brain, lung, stomach, kidney, testis, and spleen. When expressed in Xenopus laevis oocytes and in mammalian cells, rat SN2 mediates Na+-dependent transport of several neutral amino acids, including glycine, asparagine, alanine, serine, glutamine, and histidine. The transport process is electrogenic, Li+tolerant, and pH sensitive. The transport mechanism involves the influx of Na+ and amino acids coupled to the efflux of H+, resulting in intracellular alkalization. Proline, α-(methylamino)isobutyric acid, and anionic and cationic amino acids are not recognized by rat SN2.

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

International Journal of Molecular Sciences ◽

10.3390/ijms20040931 ◽

2019 ◽

Vol 20 (4) ◽

pp. 931 ◽

Cited By ~ 9

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.

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Characterization of two Mg2+transporters in sealed plasma membrane vesicles from rat liver

AJP Cell Physiology ◽

10.1152/ajpcell.1998.275.4.c995 ◽

1998 ◽

Vol 275 (4) ◽

pp. C995-C1008 ◽

Cited By ~ 49

Author(s):

Christie Cefaratti ◽

Andrea Romani ◽

Antonio Scarpa

Keyword(s):

Plasma Membrane ◽

Rat Liver ◽

Intracellular Signaling ◽

Mammalian Cells ◽

Plasma Membranes ◽

Membrane Vesicles ◽

Channel Blockers ◽

Transport Mechanisms ◽

Plasma Membrane Vesicles ◽

New Information

The plasma membrane of mammalian cells possesses rapid Mg2+ transport mechanisms. The identity of Mg2+ transporters is unknown, and so are their properties. In this study, Mg2+ transporters were characterized using a biochemically and morphologically standardized preparation of sealed rat liver plasma membranes (LPM) whose intravesicular content could be set and controlled. The system has the advantages that it is not regulated by intracellular signaling machinery and that the intravesicular ion milieu can be designed. The results indicate that 1) LPM retain trapped intravesicular total Mg2+with negligible leak; 2) the addition of Na+ or Ca2+ induces a concentration- and temperature-dependent efflux corresponding to 30–50% of the intravesicular Mg2+; 3) the rate of flux is very rapid (137.6 and 86.8 nmol total Mg2+ ⋅ μm−2 ⋅ min−1after Na+ and Ca2+ addition, respectively); 4) coaddition of maximal concentrations of Na+ and Ca2+ induces an additive Mg2+ efflux; 5) both Na+- and Ca2+-stimulated Mg2+ effluxes are inhibited by amiloride, imipramine, or quinidine but not by vanadate or Ca2+ channel blockers; 6) extracellular Na+ or Ca2+ can stimulate Mg2+ efflux in the absence of Mg2+ gradients; and 7) Mg2+ uptake occurs in LPM loaded with Na+ but not with Ca2+, thus indicating that Na+/Mg2+but not Ca2+/Mg2+exchange is reversible. These data are consistent with the operation of two distinct Mg2+ transport mechanisms and provide new information on rates of Mg2+ transport, specificity of the cotransported ions, and reversibility of the transport.

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