Stable cell surface expression of GPI-anchored proteins, but not intracellular transport, depends on their fatty acid structure

Abstract

The importance of two saturated fatty acids within the phosphatidylinositol of glycosylphosphatidylinositol-anchored proteins (GPI-APs) was examined. The lipid remodeling reaction that replaces unsaturated fatty acid with saturated fatty acid in the GPI lipid moiety was not required for efficient Golgi-to-plasma membrane transport, endocytosis via GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway and further sorting to the recycling endosome. In contrast, more than 60% of unremodeled GPI-APs carrying an unsaturated fatty acid were specifically released from cell surface during cholesterol extraction, highlighting the essential role of remodeling for stable membrane association under membrane lipid perturbation. Glycosylphosphatidylinositol-anchored proteins (GPI-APs) are a class of lipid anchored proteins expressed on the cell surface of eukaryotes. The potential interaction of GPI-APs with ordered lipid domains enriched in cholesterol and sphingolipids has been proposed to function in the intracellular transport of these lipid anchored proteins. Here, we examined the biological importance of two saturated fatty acids present in the phosphatidylinositol moiety of GPI-APs. These fatty acids are introduced by the action of lipid remodeling enzymes and required for the GPI-AP association within ordered lipid domains. We found that the fatty acid remodeling is not required for either efficient Golgi-to-plasma membrane transport or selective endocytosis via GPI-enriched early endosomal compartment (GEEC)/ clathrin-independent carrier (CLIC) pathway, whereas cholesterol depletion significantly affects both pathways independent of their fatty acid structure. Therefore, the mechanism of cholesterol dependence does not appear to be related to the interaction with ordered lipid domains mediated by two saturated fatty acids. Furthermore, cholesterol extraction drastically releases the unremodeled GPI-APs carrying an unsaturated fatty acid from the cell surface, but not remodeled GPI-APs carrying two saturated fatty acids. This underscores the essential role of lipid remodeling to ensure a stable membrane association of GPI-APs particularly under potential membrane lipid perturbation.