Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Jeckelmann JM

PROVIDER: S-EPMC6413005 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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

Jeckelmann Jean-Marc JM Fotiadis Dimitrios D

International journal of molecular sciences 20190221 4

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 ...[more]

PMID: 30795505

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Project description:The cationic amino acid arginine, due to its positive charge, is usually accumulated in the cytosol. Nevertheless, arginine has to be released by a number of cell types, e.g. kidney cells, which supply other organs with this amino acid, or the endothelial cells of the blood-brain barrier which release arginine into the brain. Arginine release in mammalian cells can be mediated by two different transporters, y(+)LAT1 and y(+)LAT2. For insertion into the plasma membrane, these transporters have to be associated with the type-II membrane glycoprotein 4F2hc [Torrents, Estevez, Pineda, Fernandez, Lloberas, Shi, Zorzano and Palacin (1998) J. Biol. Chem. 273, 32437-32445]. The present study elucidates the function and distribution of y(+)LAT2. In contrast to y(+)LAT1, which is expressed mainly in kidney epithelial cells, lung and leucocytes, y(+)LAT2 has a wider tissue distribution, including brain, heart, testis, kidney, small intestine and parotis. When co-expressed with 4F2hc in Xenopus laevis oocytes, y(+)LAT2 mediated uptake of arginine, leucine and glutamine. Arginine uptake was inhibited strongly by lysine, glutamate, leucine, glutamine, methionine and histidine. Mutual inhibition was observed when leucine or glutamine was used as substrate. Inhibition of arginine uptake by neutral amino acids depended on the presence of Na(+), which is a hallmark of y(+)LAT-type transporters. Although arginine transport was inhibited strongly by glutamate, this anionic amino acid was only weakly transported by 4F2hc/y(+)LAT2. Amino acid transport via 4F2hc/y(+)LAT2 followed an antiport mechanism similar to the other members of this new family. Only preloaded arginine could be released in exchange for extracellular amino acids, whereas marginal release of glutamine or leucine was observed under identical conditions. These results indicated that arginine has the highest affinity for the intracellular binding site and that arginine release may be the main physiological function of this transporter.

Dataset Information

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

Publications

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

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