Analysis of the transmembrane topology and membrane assembly of the GAT-1 gamma-aminobutyric acid transporter.

The Journal of Biological Chemistry
J A Clark

Abstract

The transmembrane topology of the Na+- and Cl--dependent gamma-aminobutyric acid transporter GAT-1 has been studied using protein chimeras in Xenopus oocytes. A series of COOH-terminal truncations was generated to which a prolactin epitope was fused. Following expression of transporter-prolactin chimeras in Xenopus oocytes, the transmembrane orientation of each chimera was determined by testing for protease sensitivity in an oocyte membrane preparation. Data from protease protection assays with GAT-1-prolactin chimeras has shown that residues in the loops connecting hydrophobic domain (HD)3 and HD4 and HD7 and HD8 are accessible to protease in the cytoplasm and suggest the presence of pore loop structures which extend into the membrane from the extracellular face. Such pore loop structures may be involved in the formation of the substrate-binding pocket. Studies presented herein confirm that the NH2 and COOH termini are cytosolic and hydrophobic domains span the membrane in a manner consistent with the predicted hydropathy model for Na+- and Cl--dependent transporters. These data also provide insight into GAT-1 transmembrane assembly and suggest that a complex series of topogenic sequences directs this process. A potential paus...Continue Reading

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Citations

Nov 13, 2007·The Journal of Membrane Biology·Albert L GonzalesSepehr Eskandari
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