Membrane topology of system xc- light subunit reveals a re-entrant loop with substrate-restricted accessibility.
Heteromeric amino acid transporters are composed of a heavy and a light subunit linked by a disulfide bridge. 4F2hc/xCT elicits sodium-independent exchange of anionic L-cysteine and L-glutamate (system x(c)(-)). Based on the accessibility of single cysteines to 3-(N-maleimidylpropionyl)biocytin, we propose a topological model for xCT of 12 transmembrane domains with the N and C termini located inside the cell. This location of N and C termini was confirmed by immunofluorescence. Studies of biotinylation and accessibility to sulfhydryl reagents revealed a re-entrant loop within intracellular loops 2 and 3. Residues His(110) and Thr(112), facing outside, are located at the apex of the re-entrant loop. Biotinylation of H110C was blocked by xCT substrates, by the nontransportable inhibitor (S)-4-carboxyphenylglycine, and by the impermeable reagent (2-sulfonatoethyl) methanethiosulfonate, which produced an inactivation of H110C that was protected by L-glutamate and L-cysteine with an IC(50) similar to the K(m). Protection was temperatureindependent. The data indicate that His(110) may lie close to the substrate binding/permeation pathway of xCT. The membrane topology of xCT could serve as a model for other light subunits of heteromeric amino acid transporters.
Gasol
Emma
E
Department of Biochemistry and Molecular Biology, Faculty of Biology and Barcelona Science Park, University of Barcelona, Spain.
Jiménez-Vidal
Maite
M
Chillarón
Josep
J
Zorzano
Antonio
A
Palacín
Manuel
M
D027182:Amino Acid Transport System y+ / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D026905:Amino Acid Transport Systems / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D001710:Biotin / Q000737:chemistry; D002462:Cell Membrane / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D027182:Amino Acid Transport System y+; D026905:Amino Acid Transport Systems; D021122:Protein Subunits; D011994:Recombinant Proteins; C118895:SLC7A11 protein, human; D001710:Biotin; D003545:Cysteine