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 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.
Martínez-Maza
R
R
Centro de Biologia Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain.
Poyatos
I
I
López-Corcuera
B
B
N úñez
E
E
Giménez
C
C
Zafra
F
F
Aragón
C
C
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D002352:Carrier Proteins / Q000737:chemistry / Q000378:metabolism*; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D006224:Cricetinae; D050487:Glycine Plasma Membrane Transport Proteins; D006031:Glycosylation; D009474:Neurons / Q000378:metabolism*; D011487:Protein Conformation; D021382:Protein Sorting Signals; D021381:Protein Transport
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D021382:Protein Sorting Signals