Thiol modification of cysteine 327 in the eighth transmembrane domain of the light subunit xCT of the heteromeric cystine/glutamate antiporter suggests close proximity to the substrate binding site/permeation pathway.
We measured sensitivity to thiol modification of the heteromeric glutamate/cystine transporter 4F2hc-xCT expressed in Xenopus oocytes. p-Chloromercuribenzoate (pCMB) and p-chloromercuribenzenesulfonate (pCMBS) rapidly blocked transport activity. Cys(327), located in the middle of the eighth transmembrane domain of the light subunit (xCT), was found to be the main target of inactivation. Cysteine, an impermeant reducing reagent, reversed pCMB and pCMBS effects only when applied from the extracellular medium. l-Glutamate and l-cystine, but not l-arginine, protected from the inactivation with an IC(50) similar to the K(m). Protection was not temperature-dependent, suggesting that it did not depend on large substrate-induced conformational changes. Mutation of Cys(327) to Ala and Ser slightly modified the K(m) and a C327L mutant abolished transport function without compromising transporter expression at the plasma membrane. The results indicate that Cys(327) is a functionally important residue accessible to the aqueous extracellular environment and is structurally linked to the permeation pathway and/or the substrate binding site.
Jiménez-Vidal
Maite
M
Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, E-08028 Barcelona, Spain.
Gasol
Emma
E
Zorzano
Antonio
A
Nunes
Virginia
V
Palacín
Manuel
M
Chillarón
Josep
J
D000595:Amino Acid Sequence; D027182:Amino Acid Transport System y+; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism*; D019281:Dimerization; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017669:Mercury Compounds / Q000494:pharmacology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D014981:Xenopus
D027182:Amino Acid Transport System y+; D002352:Carrier Proteins; D008565:Membrane Proteins; D017669:Mercury Compounds; C118895:SLC7A11 protein, human; D013438:Sulfhydryl Compounds; D003545:Cysteine