Transportador de aminoácidos heteromérico xCT: identificación, caracterización funcional y topología
ENGLISH ABSTRACT: The family of heteromeric amino acid transporters has the particular characteristic of being composed of two proteins: a light subunit (LSHAT) and a heavy subunit (HSHAT) linked by a disulfide bridge. Some of its members are associated with hereditary aminoacidurias. Characterisation of a new human LSHAT, responsible for transport system xc-, is reported here. Human xCT associates with 4F2 heavy chain and induces cystine/glutamate exchange with sodium independence and 1:1 molar ratio. Topology studies using immunodetection and substituted cysteine accessibility method reveal a model compatible with 12 transmembrane domains and intracellular N and C-terminus. The results obtained in the intracellular loop between 2 and 3 transmembrane domain (IL2-3), with two residues of extracellular accessibility (110 and 112) flanked by residues of intracellular accessibility (the 102, 109 and 116) in a rank of 15 amino acids, resembles the structure of a reentrant loop with apex in position H110. The fact that these structures are usually associated with the substrate pathway through the transporter, lead us to further studies with residue H110. In summary, the results on that position show that: i) biotinylation of H110C is blocked by its substrate and the non-transportable inhibitor 4-S-carboxiphenyl-glycine (4-S-CPG); (ii) the inactivation caused by MTSES in the transport of mutant H110C is also protected by each substrate with a IC50 similar to the Km; (iii) this protection is independent of temperature, and therefore it does not imply great conformational changes; and (iv) although mutants H110C and H110D did not alter the Km of the transporter nor their specificity of substrate, the substitution by a lysine inactivates the function of xCT totally. Therefore, we conclude that residue H110 resides near the binding site/translocation pathway of the substrates in the transporter xCT. These results can be found in the following publications: J Biol Chem (2004) vol. 279, 31228-36, J Biol Chem (2004) vol. 279, 11214-21, y Pflugers Arch. (2001) vol. 442 (2) 286-296.
Advisor:Palacín Prieto, Manuel
School:Universitat de Barcelona
Source Type:Master's Thesis
Keywords:bioquímica i biologia molecular divisió iii
Date of Publication:10/21/2004