title: Identification of cysteine residues in human cationic amino acid transporter hCAT-2A that are targets for inhibition by N-ethylmaleimide.
abstract: In most cells, cationic amino acids such as l-arginine, l-lysine, and l-ornithine are transported by cationic (CAT) and y(+)L (y(+)LAT) amino acid transporters. In human erythrocytes, the cysteine-modifying agent N-ethylmaleimide (NEM) has been shown to inhibit system y(+) (most likely CAT-1), but not system y(+)L (Devés, R., Angelo, S., and Chávez, P. (1993) J. Physiol. 468, 753-766). We thus wondered if sensitivity to NEM distinguishes generally all CAT and y(+)LAT isoforms. Transport assays in Xenopus laevis oocytes established that indeed all human CATs (including the low affinity hCAT-2A), but neither y(+)LAT isoform, are inhibited by NEM. hCAT-2A inhibition was not due to reduced transporter expression in the plasma membrane, indicating that NEM reduces the intrinsic transporter activity. Individual mutation of each of the seven cysteine residues conserved in all CAT isoforms did not lead to NEM insensitivity of hCAT-2A. However, a cysteine-less mutant was no longer inhibited by NEM, suggesting that inhibition occurs through modification of more than one cysteine in hCAT-2A. Indeed, also the double mutant C33A/C273A was insensitive to NEM inhibition, whereas reintroduction of a cysteine at either position 33 or 273 in the cysteine-less mutant led to NEM sensitivity. We thus identified Cys-33 and Cys-273 in hCAT-2A as the targets of NEM inhibition. In addition, all proteins with Cys-33 mutations showed a pronounced reduction in transport activity, suggesting that, surprisingly, this residue, located in the cytoplasmic N terminus, is important for transporter function.
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany.
authors.forename: Sarah R
authors.initials: SR
authors.lastname: Beyer
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany.
authors.forename: Robert T
authors.initials: RT
authors.lastname: Mallmann
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany.
authors.forename: Isabel
authors.initials: I
authors.lastname: Jaenecke
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany.
authors.forename: Alice
authors.initials: A
authors.lastname: Habermeier
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany.
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Boissel
authors.affiliation: From the Department of Pharmacology, University Medical Center, Johannes Gutenberg University, 55131 Mainz, Germany. Electronic address: closs@uni-mainz.de.
authors.forename: Ellen I
authors.initials: EI
authors.lastname: Closs
mesh_terms: D019943:Amino Acid Substitution; D026923:Amino Acid Transport Systems, Basic / Q000037:antagonists & inhibitors* / Q000737:chemistry / Q000378:metabolism*; D000818:Animals; D003545:Cysteine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology*; D005033:Ethylmaleimide / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D020125:Mutation, Missense; D009865:Oocytes; D017434:Protein Structure, Tertiary; D014982:Xenopus laevis
chemical_list: D026923:Amino Acid Transport Systems, Basic; D004791:Enzyme Inhibitors; C414177:SLC7A2 protein, human; D003545:Cysteine; D005033:Ethylmaleimide
keywords: Amino Acid Transport; Mutagenesis Site-specific; N-Ethylmaleimide (NEM); Plasma Membrane; Solute Carrier Family 7; Sulfhydryl; System y+; System y+L; Xenopus; l-Arginine Transport
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