Fatty Acid Remodeling of GPI-anchored Proteins Is Required for Their Raft Association

Abstract

Whereas most of the cellular phosphatidylinositol (PI) contain unsaturated fatty chains and are excluded from rafts, GPI-anchored proteins (APs) unusually contain two saturated fatty chains in their PI moiety, and they are typically found within lipid rafts. However, the origin of the saturated chains and whether they are essential for raft association are unclear. Here, we report that GPI-APs, with two saturated fatty chains, are generated from those bearing an unsaturated chain by fatty acid remodeling that occurs most likely in the Golgi and requires post-GPI-attachment to proteins (PGAP)2 and PGAP3. The surface GPI-APs isolated from the PGAP2 and -3 double-mutant Chinese hamster ovary (CHO) cells had unsaturated chains, such as oleic, arachidonic, and docosatetraenoic acids in the sn-2 position, whereas those from wild-type CHO cells had exclusively stearic acid, a saturated chain, indicating that the sn-2 chain is exchanged to a saturated chain. We then assessed the association of GPI-APs with lipid rafts. Recovery of unremodeled GPI-APs from the double-mutant cells in the detergent-resistant membrane fraction was very low, indicating that GPI-APs become competent to be incorporated into lipid rafts by PGAP3-and PGAP2-mediated fatty acid remodeling. We also show that the remodeling requires the preceding PGAP1-mediated deacylation from inositol of GPI-APs in the endoplasmic reticulum.

Potential Roles of GPI-Anchor Remodeling in Protein Trafficking and Raft Association in Mammalian Cells

“…Inositol deacylation by PGAP1 is required for the efficient transport of GPI-anchored proteins from the ER to the Golgi, as described in Section D. In yeast, the reaction is also needed for the efficient degradation of misfolded GPIanchored proteins (38). In addition, inositol deacylation is indispensable for the following fatty acid remodeling described in Section C-4 (39). Fatty acid remodeling is not actually performed in human erythrocytes, which express inositol-acylated GPI-anchored proteins.…”

“…Mammalian PGAP3 (yeast Per1p) is a multiple membrane-spanning protein required for GPI phospholipase A 2 activity during fatty acid remodeling (39,53). Recently, it was reported that PGAP3 together with alkaline ceramidase form a superfamily of putative transmembrane hydrolases named CREST (54).…”

“…Since the late 1980s, it has been suggested that the association of GPI-anchored proteins with DRMs or lipid rafts is important for the regulation of signal transduction, apical transport and endocytosis (86)(87)(88). Fatty acid remodeling of GPI-anchored proteins is critical for their DRM association (39,53). Mammalian PGAP3 (yeast Per1p) removes the unsaturated fatty acid from the sn-2 position in the GPI lipid during the fatty acid remodeling reaction.…”

Special issue of “Organization and functions of lipid membrane microdomains”

“…The ceramide structure of each glycosphingolipid is highly variable (7), whereas GPI-anchored proteins contain C18 and/or C16 fatty acid chains. In particular, the C18:0 fatty acid chain at the sn-2 position of GPIanchored proteins is critical for their association with membrane microdomains (8), while the presence of a 24:0 or C24:1 fatty acid chain in glycosphingolipids has been demonstrated to be necessary for their functional connection with Src family members in membrane microdomains (6,9). In contrast, GPI-anchored receptor clusters transiently recruit Src family kinases, leading to their temporary immobilization and consequent activation (10,11).…”

Characterizing the interactions between GPI-anchored alkaline phosphatases and membrane domains by AFM

“…Furthermore, recent data strongly suggest they could also play a determinant role in mammalian cells in the distribution of GPI proteins between subset of microdomains [72]. Fatty acid remodeling of the GPI anchors, required for their association with membrane ordered domains [45,65], could be part of the process leading to the synthesis of these specific anchors. In accordance with this view, the differen- In this experiment, the amount of BIAP incorporated in the bilayer was much lower than in Fig.…”