Localization of N-glycosylation sites and functional role of the carbohydrate units of GLAST-1, a cloned rat brain L-glutamate/L-aspartate transporter.
The L-glutamate transporter GLAST-1 belongs to the newly discovered family of Na(+)-dependent, high-affinity glutamate transporters, which are involved in the regulation of synaptic excitatory neurotransmitter concentration in mammalian brain. The members of this family have a similar topological organisation with at least six transmembrane helices (TMHs) and two putative N-glycosylation sites located in the extracellular loop connecting TMH 3 and TMH 4. Besides these two conserved N-glycosylation motifs at Asn206 and Asn216, GLAST-1 possesses an additional one at Asn35. The putative N-glycosylation consensus motifs (Asn-Xaa-Ser/Thr) were deleted by replacement of Asn206 and/or Asn216 by Thr using site-directed mutagenesis (mutants N206T, N216T and N206,216T). The cDNAs encoding wild-type GLAST-1 and the three glycosylation-defective transport proteins were expressed in the Xenopus laevis oocyte system. Immunoprecipitation of the [35S]methionine-labeled and glycopeptidase-F-treated transporter molecules indicates that GLAST-1 is glycosylated at Asn206 and Asn216, whereas Asn35 remains unglycosylated. To assess a possible functional role of the two glycosylation sites wild-type and glycosylation-deficient GLAST-1 were expressed in Xenopus oocytes and characterized functionally by using the whole-cell voltage-clamp technique. The results prove that N-glycosylation has no impact on the transport activity of GLAST-1.
Conradt
M
M
Institute of Biochemistry, Medical Faculty, University of Cologne, Germany.
Storck
T
T
Stoffel
W
W
D027322:Amino Acid Transport System X-AG; D000818:Animals; D001216:Asparagine; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D011233:Precipitin Tests; D051381:Rats; D014982:Xenopus laevis
D027322:Amino Acid Transport System X-AG; D002352:Carrier Proteins; D017931:DNA Primers; D006023:Glycoproteins; D001216:Asparagine