Biotinylation of single cysteine mutants of the glutamate transporter GLT-1 from rat brain reveals its unusual topology.
In the central nervous system, (Na+ + K+)-coupled glutamate transporters restrict the neurotoxicity of this transmitter and limit the duration of synaptic excitation at some synapses. The various isotransporters exhibit a particularly high homology in an extended hydrophobic domain of ill-defined topology that contains several determinants involved in ion and transmitter binding. Here, we describe the determination of the membrane topology of the cloned astroglial glutamate transporter GLT-1. A series of functional transporters containing single cysteines was engineered. Their topological disposition was determined by using a biotinylated sulfhydryl reagent. The glutamate transporter has eight transmembrane domains long enough to span the membrane as et heiices. Strikingly, between the seventh and eighth domains, a structure reminiscent of a pore loop and an outward-facing hydrophobic linker are positioned.
Grunewald
M
M
Department of Biochemistry, Hadassah Medical School, The Hebrew University, Jerusalem, Israel.
Bendahan
A
A
Kanner
B I
BI
D018528:ATP-Binding Cassette Transporters / Q000737:chemistry* / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D027322:Amino Acid Transport System X-AG; D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D001692:Biological Transport; D019921:Biotinylation; D001921:Brain / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine; D006031:Glycosylation; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000737:chemistry; D017354:Point Mutation; D011487:Protein Conformation; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism
D018528:ATP-Binding Cassette Transporters; D027322:Amino Acid Transport System X-AG; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003545:Cysteine