The H,K-ATPase beta subunit as a model to study the role of N-glycosylation in membrane trafficking and apical sorting.
The role of N-glycosylation in trafficking of an apical membrane protein, the gastric H,K-ATPase beta subunit linked to yellow fluorescent protein, was analyzed in polarized LLC-PK1 cells by confocal microscopy and surface-specific biotinylation. Deletion of the N-glycosylation sites at N1, N3, N5, and N7 but not at N2, N4, and N6 significantly slowed endoplasmic reticulum-to-Golgi trafficking, impaired apical sorting, and enhanced endocytosis from the apical membrane, resulting in decreased apical expression. Golgi mannosidase inhibition to prevent carbohydrate chain branching and elongation resulted in faster internalization and degradation of the beta subunit, indicating that terminal glycosylation is important for stabilization of the protein in the apical membrane and protection of internalized protein from targeting to the degradation pathway. The decrease in the apical content of the beta subunit was less with mannosidase inhibition compared with that found in the N1, N3, N5, and N7 site mutants, suggesting that the core region sugars are more important than the terminal sugars for apical sorting.
Vagin
Olga
O
Department of Physiology, School of Medicine, UCLA, Los Angeles, California, USA.
Turdikulova
Shahlo
S
Sachs
George
G
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D019921:Biotinylation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004705:Endocytosis; D004721:Endoplasmic Reticulum / Q000378:metabolism; D017353:Gene Deletion; D006026:Glycoside Hydrolases / Q000378:metabolism; D006031:Glycosylation; D006056:Golgi Apparatus / Q000378:metabolism; D018374:LLC-PK1 Cells; D008164:Luminescent Proteins / Q000737:chemistry; D018613:Microscopy, Confocal; D009154:Mutation; D009284:Naphthols / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry*; D021381:Protein Transport; D011817:Rabbits; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*; D013270:Stomach / Q000201:enzymology; D017026:Swainsonine / Q000494:pharmacology; D014162:Transfection
D001426:Bacterial Proteins; D018076:DNA, Complementary; D008164:Luminescent Proteins; D009284:Naphthols; D010455:Peptides; D021122:Protein Subunits; C054562:yellow fluorescent protein, Bacteria; C038630:1-(N-dimethyl)amino-7-naphthol; D006026:Glycoside Hydrolases; D000254:Sodium-Potassium-Exchanging ATPase; D017026:Swainsonine