The Role of N-Glycosylation in Transport to the Plasma Membrane and Sorting of the Neuronal Glycine Transporter GLYT2

Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putativeN-glycosylation sites on the transport activity, intracellular t...

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Published inThe Journal of biological chemistry Vol. 276; no. 3; pp. 2168 - 2173
Main Authors Martı́nez-Maza, Rodrigo, Poyatos, Irene, López-Corcuera, Beatriz, Núñez, Enrique, Giménez, Cecilio, Zafra, Francisco, Aragón, Carmen
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 19.01.2001
American Society for Biochemistry and Molecular Biology
Subjects
Amino Acid Transport Systems, Neutral
Animals
Carrier Proteins - chemistry
Carrier Proteins - metabolism
Cell Line
Cell Membrane - metabolism
Cricetinae
Glycine Plasma Membrane Transport Proteins
Glycosylation
Neurons - metabolism
Protein Conformation
Protein Sorting Signals
Protein Transport
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Summary:Glycine transporter GLYT2 is an axonal glycoprotein involved in the removal of glycine from the synaptic cleft. To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putativeN-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in polarized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduced by 35–40% after enzymatic deglycosylation of the transporter reconstituted into liposomes. Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellular loop of GLYT2, produced an inactive protein that was retained in intracellular compartments when transiently transfected in COS cells or in nonpolarized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 localizes in the apical surface as assessed by transport and biotinylation assays. However, a partially unglycosylated mutant (triple mutant) was distributed in a nonpolarized manner in MDCK cells. The apical localization of GLYT2 occurred by a glycolipid rafts independent pathway.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.M006774200