Membrane topology of the high-affinity L-glutamate transporter (GLAST- 1) of the central nervous system

doi:10.1083/jcb.135.6.1867

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Membrane topology of the high-affinity L-glutamate transporter (GLAST- 1) of the central nervous system

S Wahle and W Stoffel
Institute of Biochemistry I, Medical Faculty, University of Cologne, Germany.

The membrane topology of the high affinity, Na(+)-coupled L-glutamate/L- aspartate transporter (GLAST-1) of the central nervous system has been determined. Truncated GLAST-1 cDNA constructs encoding protein fragments with an increasing number of hydrophobic regions were fused to a cDNA encoding a reporter peptide with two N-glycosylation sites. The respective cRNA chimeras were translated in vitro and in vivo in Xenopus oocytes. Posttranslational N-glycosylation of the two reporter consensus sites monitors the number, size, and orientation of membrane- spanning domains. The results of our experiments suggest a novel 10- transmembrane domain topology of GLAST-1, a representative of the L- glutamate neurotransmitter transporter family, with its NH2 and COOH termini on the cytoplasmic side, six NH2-terminal hydrophobic transmembrane alpha-helices, and four COOH-terminal short hydrophobic domains spanning the bilayer predicted as beta-sheets.
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