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.

Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway. We found that GPI-anchored proteins could not associate with the detergent-resistant membranes in per1Delta cells. In addition, the mutant cells had a defect in the lipid remodeling from normal phosphatidylinositol (PI) to a C26 fatty acid-containing PI in the GPI anchor. In vitro analysis showed that PER1 is required for the production of lyso-GPI, suggesting that Per1p possesses or regulates the GPI-phospholipase A2 activity. We also found that human PERLD1 is a functional homologue of PER1. Our results demonstrate for the first time that PER1 encodes an evolutionary conserved component of the GPI anchor remodeling pathway, highlighting the close connection between the lipid remodeling of GPI and raft association of GPI-anchored proteins.

Misfolded proteins are recognized in the endoplasmic reticulum (ER), transported back to the cytosol, and degraded by the proteasome. A number of proteins are processed and modified by a glycosylphosphatidylinositol (GPI) anchor in the ER, but the quality control mechanisms of GPI-anchored proteins remain unclear. Here, we report on the quality control mechanism of misfolded GPI-anchored proteins. We have constructed a mutant form of the ␤-1,3-glucanosyltransferase Gas1p (Gas1*p) as a model misfolded GPI-anchored protein. Gas1*p was modified with a GPI anchor but retained in the ER and was degraded rapidly via the proteasome. Disruption of BST1, which encodes GPI inositol deacylase, caused a delay in the degradation of Gas1*p. This delay was because of an effect on the deacylation activity of Bst1p. Disruption of genes involved in GPI-anchored protein concentration and N-glycan processing caused different effects on the degradation of Gas1*p and a soluble misfolded version of carboxypeptidase Y. Furthermore, Gas1*p associated with both Bst1p and BiP/Kar2p, a molecular chaperone, in vivo. Our data suggest that GPI inositol deacylation plays important roles in the quality control and ER-associated degradation of GPI-anchored proteins. INTRODUCTIONCells possess several quality control mechanisms for the maintenance of proper protein folding and function. On synthesis, membrane and secretory proteins are inserted into the lumen of the endoplasmic reticulum (ER), where they are folded and undergo oligomerization. There are a number of chaperones and enzymes in the ER required for proper protein folding (Ellgaard et al., 1999). Before exiting from the ER, proteins are monitored by a quality control system that ensures correct folding. Misfolded proteins that fail to pass the quality control checkpoint are transported back to the cytosol and degraded by an ER-associated degradation (ERAD) mechanism that involves the ubiquitinproteasome pathway (Kopito, 1997;Kostova and Wolf, 2003). N-linked oligosaccharide, one of the major posttranslational modifications in the ER, is trimmed and processed by glucosidase I, glucosidase II, and mannosidase I (Jakob et al., 1998;Helenius and Aebi, 2001). The processing of Nlinked oligosaccharides plays important roles in the quality control of glycoprotein folding in the ER Aebi, 2001, 2004).A number of cell surface proteins are posttranslationally modified in the ER with glycosylphosphatidylinositol (GPI). Mechanisms for quality control and degradation of GPIanchored proteins are important in folding diseases, including prion diseases and transmissible spongiform encephalopathies, which are caused by a conformational modification of prion, a GPI-anchored protein (Prusiner, 1998). There have been several biochemical studies on both the degradation of mutated prions and the quality control of proteins with mutated GPI attachment signals (Field et al., 1994;Oda et al., 1996;Wainwright and Field, 1997;Jin et al., 2000;Ito et al., 2002;Ishida et al., 2003). In contrast, the molecular mech...

The glycosylphosphatidylinositol (GPI)-anchored proteins are subjected to lipid remodeling during their biosynthesis. In the yeast Saccharomyces cerevisiae, the mature GPI-anchored proteins contain mainly ceramide or diacylglycerol with a saturated long-fatty acid, whereas conventional phosphatidylinositol (PI) used for GPI biosynthesis contains an unsaturated fatty acid. Here, we report that S. cerevisiae Cwh43p, whose N-terminal region contains a sequence homologous to mammalian PGAP2, is involved in the remodeling of the lipid moiety of GPI anchors to ceramides. In cwh43 disruptant cells, the PI moiety of the GPI-anchored protein contains a saturated long fatty acid and lyso-PI but not inositolphosphorylceramides, which are the main lipid moieties of GPI-anchored proteins from wild-type cells. Moreover, the C-terminal region of Cwh43p (Cwh43-C), which is not present in PGAP2, is essential for the ability to remodel GPI lipids to ceramides. The N-terminal region of Cwh43p (Cwh43-N) is associated with Cwh43-C, and it enhanced the lipid remodeling to ceramides by Cwh43-C. Our results also indicate that mouse FRAG1 and C130090K23, which are homologous to Cwh43-N and -C, respectively, share these activities.

Background: Serine residues in extensin, a cell wall protein in plants, are glycosylated with O-galactose. Results: Genes encoding the serine O-␣-galactosyltransferase were isolated from Chlamydomonas reinhardtii and plants and were characterized. Conclusion: The serine O-␣-galactosyltransferases belong to a novel glycosyltransferase class existing only in the plant kingdom. Significance: The identification of novel glycosyltransferases contributes to elucidation of how protein glycosylation has evolved in plants.