title: Detergent-induced stabilization and improved 3D map of the human heteromeric amino acid transporter 4F2hc-LAT2.
abstract: Human heteromeric amino acid transporters (HATs) are membrane protein complexes that facilitate the transport of specific amino acids across cell membranes. Loss of function or overexpression of these transporters is implicated in several human diseases such as renal aminoacidurias and cancer. HATs are composed of two subunits, a heavy and a light subunit, that are covalently connected by a disulphide bridge. Light subunits catalyse amino acid transport and consist of twelve transmembrane α-helix domains. Heavy subunits are type II membrane N-glycoproteins with a large extracellular domain and are involved in the trafficking of the complex to the plasma membrane. Structural information on HATs is scarce because of the difficulty in heterologous overexpression. Recently, we had a major breakthrough with the overexpression of a recombinant HAT, 4F2hc-LAT2, in the methylotrophic yeast Pichia pastoris. Microgram amounts of purified protein made possible the reconstruction of the first 3D map of a human HAT by negative-stain transmission electron microscopy. Here we report the important stabilization of purified human 4F2hc-LAT2 using a combination of two detergents, i.e., n-dodecyl-β-D-maltopyranoside and lauryl maltose neopentyl glycol, and cholesteryl hemisuccinate. The superior quality and stability of purified 4F2hc-LAT2 allowed the measurement of substrate binding by scintillation proximity assay. In addition, an improved 3D map of this HAT could be obtained. The detergent-induced stabilization of the purified human 4F2hc-LAT2 complex presented here paves the way towards its crystallization and structure determination at high-resolution, and thus the elucidation of the working mechanism of this important protein complex at the molecular level.
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Marcel
authors.initials: M
authors.lastname: Meury
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Costa
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Harder
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Mirko
authors.initials: M
authors.lastname: Stauffer
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Jeckelmann
authors.affiliation: Institute of Biotechnology, School of Life Sciences and Facility Management Zürich University of Applied Sciences (ZHAW), Campus Grüntal, Wädenswil, Switzerland.
authors.forename: Béla
authors.initials: B
authors.lastname: Brühlmann
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Barcelona, Spain.
authors.forename: Albert
authors.initials: A
authors.lastname: Rosell
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Hüseyin
authors.initials: H
authors.lastname: Ilgü
authors.affiliation: Institute of Biotechnology, School of Life Sciences and Facility Management Zürich University of Applied Sciences (ZHAW), Campus Grüntal, Wädenswil, Switzerland.
authors.forename: Karin
authors.initials: K
authors.lastname: Kovar
authors.affiliation: Institute for Research in Biomedicine (IRB Barcelona), Barcelona, Spain; Centro de Investigación Biomédica en Red de Enfermedades Raras, Barcelona, Spain; Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, Barcelona, Spain.
authors.forename: Manuel
authors.initials: M
authors.lastname: Palacín
authors.affiliation: Institute of Biochemistry and Molecular Medicine, and Swiss National Centre of Competence in Research (NCCR) TransCure, University of Bern, Bern, Switzerland.
authors.forename: Dimitrios
authors.initials: D
authors.lastname: Fotiadis
mesh_terms: D027182:Amino Acid Transport System y+ / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D000596:Amino Acids / Q000737:chemistry / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D027282:Fusion Regulatory Protein 1, Heavy Chain / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D027301:Fusion Regulatory Protein 1, Light Chains / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008320:Maltose / Q000031:analogs & derivatives / Q000494:pharmacology; D046912:Multiprotein Complexes / Q000737:chemistry / Q000302:isolation & purification; D010843:Pichia; D055550:Protein Stability / Q000187:drug effects; D011994:Recombinant Proteins / Q000187:drug effects / Q000302:isolation & purification*
chemical_list: D027182:Amino Acid Transport System y+; D026905:Amino Acid Transport Systems; D000596:Amino Acids; D003902:Detergents; D027282:Fusion Regulatory Protein 1, Heavy Chain; D027301:Fusion Regulatory Protein 1, Light Chains; D046912:Multiprotein Complexes; D011994:Recombinant Proteins; C420646:SLC7A8 protein, human; C411362:dodecyl maltopyranoside; D008320:Maltose