Membrane topology of the high-affinity L-glutamate transporter (GLAST-1) of the central nervous system.
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.
Wahle
S
S
Institute of Biochemistry I, Medical Faculty, University of Cologne, Germany.
Stoffel
W
W
D000595:Amino Acid Sequence; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002352:Carrier Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D002462:Cell Membrane / Q000737:chemistry; D002490:Central Nervous System / Q000737:chemistry* / Q000378:metabolism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry; D005260:Female; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism; D006031:Glycosylation; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014981:Xenopus
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D006023:Glycoproteins; D011993:Recombinant Fusion Proteins