Plasma membrane delivery of the gastric H,K-ATPase: the role of beta-subunit glycosylation.
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 (Asn103), which is not conserved among the beta-subunits from different species, is not critical for plasma delivery of the protein.
Vagin
O
O
Division of Nephrology, David Geffen School of Medicine, University of California, Los Angeles Center for Health, 90095, USA. olgav@ucla.edu
Denevich
S
S
Sachs
G
G
D001426:Bacterial Proteins / Q000235:genetics; D001665:Binding Sites; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D006031:Glycosylation; D017506:H(+)-K(+)-Exchanging ATPase / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013270:Stomach / Q000201:enzymology*
D001426:Bacterial Proteins; D007527:Isoenzymes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria; D017506:H(+)-K(+)-Exchanging ATPase