The H,K-ATPase β Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting

Olga Vagin,George Sachs,Shahlo Turdikulova

doi:10.1074/jbc.m405453200

Olga Vagin, George Sachs + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m405453200

Copy DOI

Abstract

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.

Highlights

The H,K-ATPase ␤ Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting*
In the mature complextype glycoprotein, the core region sugars are more important for the apical sorting event in trans-Golgi network (TGN)/endosomes compared with terminal sugars, whereas the terminal sugars anchor the protein on the apical membrane and protect the protein from targeting to the degradation pathway after endocytosis
Removal of all the sugars from apical Yellow Fluorescent Protein (YFP)-␤ by PNGase F in LLC-PK1 cells resulted in internalization of the protein from the apical membrane and rapid degradation (Fig. 9), showing that carbohydrates are crucial for stabilization of both newly synthesized and recycling proteins

Summary

THE JOURNAL OF BIOLOGICAL CHEMISTRY

Vol 279, No 37, Issue of September 10, pp. 39026 –39034, 2004 Printed in U.S.A. The H,K-ATPase ␤ Subunit as a Model to Study the Role of N-Glycosylation in Membrane Trafficking and Apical Sorting*. After coupling the core to the asparagine of the specific amino acid NXS or NXT motif, the N-glycosylation site, the terminal glucose residues are trimmed by ER glucosidases Various chaperones such as calnexin bind to sugar chains as quality control elements at this and perhaps later stages. We determined the effect of either deletion of individual N-glycosylation sites or treatment of the cells with N-glycan-trimming inhibitors on the steady-state distribution of the H,K-ATPase ␤ subunit between the ER, Golgi, and apical and basolateral membranes as well as on endocytosis efficiency and degradation rate in polarized LLC-PK1 cells by quantifying the immature and mature forms of the protein that differ in their molecular masses and by using surface-specific biotinylation and confocal microscopy. In the mature complextype glycoprotein, the core region sugars are more important for the apical sorting event in TGN/endosomes compared with terminal sugars, whereas the terminal sugars anchor the protein on the apical membrane and protect the protein from targeting to the degradation pathway after endocytosis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION