Apical sorting of lysoGPI-anchored proteins occurs independent of association with detergent-resistant membranes but dependent on their N-glycosylation

Mol Biol Cell. 2013 Jun;24(12):2021-33. doi: 10.1091/mbc.E13-03-0160. Epub 2013 Apr 24.

Abstract

Most glycosylphosphatidylinositol-anchored proteins (GPI-APs) are located at the apical surface of epithelial cells. The apical delivery of GPI-APs is believed to result from their association with lipid rafts. We find that overexpression of C-terminally tagged PGAP3 caused predominant production of lysoGPI-APs, an intermediate precursor in the GPI lipid remodeling process in Madin-Darby canine kidney cells. In these cells, produced lysoGPI-APs are not incorporated into detergent-resistant membranes (DRMs) but still are delivered apically, suggesting that GPI-AP association with DRMs is not necessary for apical targeting. In contrast, apical transport of both fully remodeled and lyso forms of GPI-APs is dependent on N-glycosylation, confirming a general role of N-glycans in apical protein transport. We also find that depletion of cholesterol causes apical-to-basolateral retargeting not only of fully remodeled GPI-APs, but also of lysoGPI-APs, as well as endogenous soluble and transmembrane proteins that would normally be targeted to the apical membrane. These findings confirm the essential role for cholesterol in the apical protein targeting and further demonstrate that the mechanism of cholesterol-dependent apical sorting is not related to DRM association of GPI-APs.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Blotting, Western
  • Carboxylic Ester Hydrolases / genetics
  • Carboxylic Ester Hydrolases / metabolism
  • Cell Line
  • Cell Membrane / metabolism*
  • Cholesterol / metabolism
  • Detergents / pharmacology
  • Dogs
  • Glycosylation
  • Glycosylphosphatidylinositols / metabolism*
  • Green Fluorescent Proteins / genetics
  • Green Fluorescent Proteins / metabolism
  • Humans
  • Membrane Microdomains / metabolism
  • Membrane Proteins / genetics
  • Membrane Proteins / metabolism*
  • Microscopy, Fluorescence
  • Protein Binding
  • Protein Transport / drug effects
  • RNA Interference
  • Receptors, Cell Surface / genetics
  • Receptors, Cell Surface / metabolism

Substances

  • Detergents
  • Glycosylphosphatidylinositols
  • Membrane Proteins
  • Receptors, Cell Surface
  • Green Fluorescent Proteins
  • Cholesterol
  • Carboxylic Ester Hydrolases
  • PGAP3 protein, human