THE ROLE OF N-GLYCOSYLATION IN THE TARGETING AND ACTIVITY OF THE GLYT1 GLYCINE TRANSPORTER

To elucidate the role of N-glycosylation in the function of the high a ffinity glycine transporter GLYT1, we have investigated the effect of the glycosylation inhibitor tunicamycin as well as the effect of the d isruption of the putative glycosylation sites by site-directed mutagen esis, SDS-polyacrylamide gel electrophoresis of proteins from GLYT1-tr ansfected COS cells reveals a major band of 80-100 kDa and a minor one of 57 kDa, Treatment with tunicamycin produces a 40% inhibition in tr ansport activity and a decrease in the intensity of the 80-100-kDa ban d, whereas the 57-kDa band decreases in size to yield a 47-kDa protein corresponding to the unglycosylated form of the transporter, Simultan eous mutation of Asn-169, Asn-172, Asn-182, and Asn-188 to Gln also pr oduces the 47-kDa form of the protein, indicating that there are no ad ditional sites for N-glycosylation, Progressive mutation of the potent ial glycosylation sites produces a progressive decrease in transport a ctivity and in size of the protein, indicating that the four putative glycosylation sites are actually glycosylated. N-Glycosylation of the GLYT1 is not indispensable for the transport activity itself, as demon strated by enzymatic deglycosylation of the transporter, Analysis of s urface proteins by biotinylation and by immunofluorescence demonstrate s that a significant portion of the unglycosylated GLYT1 mutant remain s in the intracellular compartment, This suggests that the carbohydrat e moiety of glycine transporter GLYT1 is necessary for the proper traf ficking of the protein to the plasma membrane.