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 affinity glycine transporter GLYT1, we have investigated the effect of the glycosylation inhibitor tunicamycin as well as the effect of the disruption of the putative glycosylation sites by site-directed mutagenesis. SDS-polyacrylamide gel electrophoresis of proteins from GLYT1-transfected 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 transport activity and a decrease in the intensity of the 80-100-kDa band, whereas the 57-kDa band decreases in size to yield a 47-kDa protein corresponding to the unglycosylated form of the transporter. Simultaneous mutation of Asn-169, Asn-172, Asn-182, and Asn-188 to Gln also produces the 47-kDa form of the protein, indicating that there are no additional sites for N-glycosylation. Progressive mutation of the potential glycosylation sites produces a progressive decrease in transport activity 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 demonstrated by enzymatic deglycosylation of the transporter. Analysis of surface proteins by biotinylation and by immunofluorescence demonstrates that a significant portion of the unglycosylated GLYT1 mutant remains in the intracellular compartment. This suggests that the carbohydrate moiety of glycine transporter GLYT1 is necessary for the proper trafficking of the protein to the plasma membrane.
Olivares
L
L
Centro de Biología Molecular Severo Ochoa, Facultad de Ciencias, Universidad Autónoma de Madrid, Spain.
Aragón
C
C
Giménez
C
C
Zafra
F
F
D026921:Amino Acid Transport Systems, Neutral; D000818:Animals; D001483:Base Sequence; D002352:Carrier Proteins / Q000737:chemistry* / Q000502:physiology; D005998:Glycine / Q000378:metabolism*; D050487:Glycine Plasma Membrane Transport Proteins; D006031:Glycosylation; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011817:Rabbits; D013329:Structure-Activity Relationship
D026921:Amino Acid Transport Systems, Neutral; D002352:Carrier Proteins; D050487:Glycine Plasma Membrane Transport Proteins; D005998:Glycine