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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.
Volume 270,
Number 16,
Issue of April 21, pp. 9437-9442, 1995
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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