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 carbohydr ate moiety on GLYT2 function, we analyzed the effect of the disruption of t he putative N-glycosylation sites on the transport activity, intracellular traffic in COS cells, and asymmetrical distribution of this protein in pola rized Madin-Darby canine kidney (MDCK) cells. Transport activity was reduce d by 35-40% after enzymatic deglycosylation of the transporter reconstitute d into liposomes, Site-directed mutagenesis of the four glycosylation sites (Asn-345, Asn-355, Asn-360, and Asn-366), located in the large extracellul ar loop of GLYT2, produced an inactive protein that was retained in intrace llular compartments when transiently transfected in COS cells or in nonpola rized MDCK cells. When expressed in polarized MDCK cells, wild type GLYT2 l ocalizes in the apical surface as assessed by transport and biotinylation a ssays. However, a partially unglycosylated mutant (triple mutant) was distr ibuted in a nonpolarized manner in MDCK cells. The apical localization of G LYT2 occurred by a glycolipid rafts independent pathway.