1. Plasma membrane delivery of the gastric H,K-ATPase: the role of [beta]-subunit glycosylation
- Author
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Vagin, O., Denevich, S., and Sachs, G.
- Subjects
Glycosylation -- Physiological aspects ,Biological sciences - Abstract
The factors determining trafficking of the gastric H,K-ATPase to the apical membrane remain elusive. To identify such determinants in the gastric H,K-ATPase, fusion proteins of yellow fluorescent protein (YFP) and the gastric H,K-ATPase [beta]-subunit (YFP-[beta]) and cyan fluorescent protein (CFP) and the gastric H,K-ATPase [alpha]-subunit (CFP-[alpha]) were expressed in HEK-293 cells. Then plasma membrane delivery of wild-type CFP-[alpha], wild-type YFP-[beta], and YFP-[beta] mutants lacking one or two of the seven [beta]-subunit glycosylation sites was determined using confocal microscopy and surface biotinylation. Expression of the wild-type YFP-[beta] resulted in the plasma membrane localization of the protein, whereas the expressed CFP-[alpha] was retained intracellularly. When coexpressed, both CFP-[alpha] and YFP-[beta] were delivered to the plasma membrane. Removing each of the seven glycosylation sites, except the second one, from the extracellular loop of YFP-[beta] prevented plasma membrane delivery of the protein. Only the mutant lacking the second glycosylation site (Asn103Gln) was localized both intracellularly and on the plasma membrane. A double mutant lacking the first (Asn99Gln) and the second (Asn103Gln) glycosylation sites displayed intracellular accumulation of the protein. Therefore, six of the seven glycosylation sites in the [beta]-subunit are essential for the plasma membrane delivery of the [beta]-subunit of the gastric H,K-ATPase, whereas the second glycosylation site (Asnl03), which is not conserved among the [beta]-subunits from different species, is not critical for plasma delivery of the protein. sorting and trafficking; apical signals; plasma membrane targeting
- Published
- 2003