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

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.

Exit of GPI-anchored proteins from the ER differs in yeast and mammalian cells.

Previous studies have shown that yeast glycosylphosphatidylinositol-anchored proteins (GPI-APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI-APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI-APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER-to-Golgi transport of GPI-APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI-APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI-APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI-APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.

Sorting of GPI-anchored proteins into ER exit sites by p24 proteins is dependent on remodeled GPI.

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a posttranslational modification occurring in the endoplasmic reticulum (ER). After GPI attachment, proteins are transported by coat protein complex II (COPII)-coated vesicles from the ER. Because GPI-anchored proteins (GPI-APs) are localized in the lumen, they cannot interact with cytosolic COPII components directly. Receptors that link GPI-APs to COPII are thought to be involved in efficient packaging of GPI-APs into vesicles; however, mechanisms of GPI-AP sorting are not well understood. Here we describe two remodeling reactions for GPI anchors, mediated by PGAP1 and PGAP5, which were required for sorting of GPI-APs to ER exit sites. The p24 family of proteins recognized the remodeled GPI-APs and sorted them into COPII vesicles. Association of p24 proteins with GPI-APs was pH dependent, which suggests that they bind in the ER and dissociate in post-ER acidic compartments. Our results indicate that p24 complexes act as cargo receptors for correctly remodeled GPI-APs to be sorted into COPII vesicles.