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

Abstract

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.

Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc

“…The conserved cysteine residue involved in the intersubunit disulfide bridge is located between TMD3 and TMD4 (11). Structure-function studies (12,13) suggest that the structures of the light subunit of HATs should be similar to those of remote bacterial amino acid/polyamine/ organocation (APC) transporters (∼20% amino acid sequence identities), which present the . In contrast, there is no structural information about the interaction between the two HAT subunits.…”

The Structural and Functional Units of Heteromeric Amino Acid Transporters

“…After 6 h of incubation, the cells were washed twice with phosphate-buffered saline, trypsinized, and reseeded on a 6-well plate containing one coverslip (22 (22,25). The effect of pCMB and MTS reagents (Toronto Research Chemicals, Inc.) was assayed as described (3,22). 1 mM pCMB and 2.5 mM MTSEA inhibited the activity of wild-type xCT (22) and wildtype b 0,ϩ AT, respectively.…”

“…The effect of pCMB and MTS reagents (Toronto Research Chemicals, Inc.) was assayed as described (3,22). 1 mM pCMB and 2.5 mM MTSEA inhibited the activity of wild-type xCT (22) and wildtype b 0,ϩ AT, respectively. Reconstitution of wild type b 0,ϩ AT and the C321S mutant into liposomes and uptake measurements in the reconstituted system were performed as described (21).…”

“…This medium was incubated with plasmids encoding the CFP and YFP fusion proteins (1 g each) for 30 min, and the mixture was added to the cells grown in culture medium. After 6 h of incubation, the cells were washed twice with phosphate-buffered saline, trypsinized, and reseeded on a 6-well plate containing one coverslip (22 (22,25). The effect of pCMB and MTS reagents (Toronto Research Chemicals, Inc.) was assayed as described (3,22).…”

“…Despite the important roles attributed to HAT, few studies have addressed the structure-function relationship of these transporters; (i) most HAT are obligate antiporters with a 1:1 stoichiometry (20), (ii) light subunits appear to be necessary and sufficient for transport activity, as demonstrated for b 0,ϩ AT (21); (iii) in using xCT as a model for the light subunits, we showed a membrane topology with 12 transmembrane domains and revealed that the residues His-110 and Cys-327 are crucial for function (3,22). In non-reducing SDS-PAGE conditions 4F2hc-associated heterodimers run mainly as a 125-kDa band (23), whereas rBAT and b 0,ϩ AT run as a band of 130 kDa (the disulfide-linked b 0,ϩ AT-rBAT) and an additional band of 250 kDa (4).…”