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

Beatriz López-Corcuera,Rodrigo Martı́nez-Maza,Cecilio Giménez,Francisco Zafra,Irene Poyatos,Carmen Aragón,Enrique Núñez

doi:10.1074/jbc.m006774200

Abstract

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 putative N-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.

Highlights

To terminate synaptic transmission in the central nervous system, neurotransmitters are inactivated by either enzymatic degradation or active transport into neuronal and/or glial cells [1, 2]
To elucidate the role of the carbohydrate moiety on GLYT2 function, we analyzed the effect of the disruption of the putative N-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
The specific mechanisms of such a nonuniform distribution have been studied in experimental systems of polarized cultured cells, especially in the epithelial MDCK cell line, since somatodendritic neuronal proteins use to be localized in the basolateral domain, whereas axonal proteins are in the apical one [22]

Summary

EXPERIMENTAL PROCEDURES

Materials—Dulbecco’s modified Eagle’s medium and G418 were from Life Technologies, Inc. Glycine Transport Assay—Transport assays in transfected COS-7 cells were performed at 37 °C in HEPES-buffered saline (150 mM NaCl, 10 mM HEPES-Tris, pH 7.4, 1 mM Ca2Cl, 2.5 mM KCl, 2.5 mM MgSO4, 10 mM glucose) as described previously [26]. Vectorial Glycine Uptake Assay—Parental and transfected MDCK cells were plated at 50% confluence on Transwell tissue culture inserts (6.5 mm diameter, 0.4 ␮m filter pore size, Costar Co., Cambridge, MA) and grown for 5–7 days. Glycosidase Treatment—Wild type GLYT2 protein transiently transfected in COS cells was solubilized and reconstituted into liposomes as described above. After three more washes with PBS/Ca/Mg, filters were excised, and cells were lysed with 1 ml of lysis buffer (150 mM NaCl, 5 mM EDTA, 50 mM HEPES-Tris, 0.25% deoxycholate, 1% Triton X-100, 0.1% SDS, pH 7.4) for 30 min. Protein Concentration—Protein concentration was determined by the method of Bradford [32]

RESULTS AND DISCUSSION

Targeting and Sorting of a Glycine Transporter

To determine whether the impaired transport of the mutants