Saccharomyces cerevisiae CWH43 is involved in the remodeling of the lipid moiety of GPI anchors to ceramides

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.     

Multiple functions of inositolphosphorylceramides in the formation and intracellular transport of glycosylphosphatidylinositol-anchored proteins in yeast

The mature sphingolipids of yeast consist of IPCs (inositolphosphorylceramides) and glycosylated derivatives thereof. Beyond being an abundant membrane constituent in the organelles of the secretory pathway, IPCs are also used to constitute the lipid moiety of the majority of GPI (glycosylphosphatidylinositol) proteins, while a minority of GPI proteins contain PI (phosphatidylinositol). Thus all GPI anchor lipids (as well as free IPCs) typically contain C26 fatty acids. However, the primary GPI lipid that isadded to newly synthesized proteins in the endoplasmic reticulum consists of a PI with conventional C16 and C18 fatty acids. A new class of enzymes is required to replace the fatty acid in sn-2 by a C26 fatty acid. Cells lacking this activity make normal amounts of GPI proteins but accumulate GPI anchors containing lyso-PI. As a consequence, the endoplasmic reticulum to Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. The GPI anchor containing C26 in sn-2 can further be remodelled by the exchange of diacylglycerol for ceramide. This process is also dependent on the presence of specific phosphorylethanolamine side-chains on the GPI anchor.     

Lipid remodeling of GPI-anchored proteins and its function

Many proteins are attached to the cell surface via a conserved post-translational modification, the glycosylphosphatidylinositol (GPI) anchor. GPI-anchored proteins are functionally diverse, but one of their most striking features is their association with lipid microdomains, which consist mainly of sphingolipids and sterols. GPI-anchored proteins modulate various biological functions when they are incorporated into these specialized domains. The biosynthesis of GPI and its attachment to proteins occurs in the endoplasmic reticulum. The lipid moieties of GPI-anchored proteins are further modified during their transport to the cell surface, and these remodeling processes are essential for the association of proteins with lipid microdomains. Recently, several genes required for GPI lipid remodeling have been identified in yeast and mammalian cells. In this review, we describe the pathways for lipid remodeling of GPI-anchored proteins in yeast and mammalian cells, and discuss how lipid remodeling affects the association of GPI-anchored proteins with microdomains in cellular events.     

CWH43 is required for the introduction of ceramides into GPI anchors in Saccharomyces cerevisiae

After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, the fatty acid in sn-2 of the diacylglycerol moiety can be replaced by a C26:0 fatty acid by a deacylation-reacylation cycle catalysed by Per1p and Gup1p. Furthermore the diacylglycerol moiety of the yeast GPI anchor can also be replaced by ceramides. CWH43 of yeast is homologous to PGAP2, a gene that recently was implicated in a similar deacylation reacylation cycle of GPI proteins in mammalian cells, where PGAP2 is required for the reacylation of monoradylglycerol-type GPI anchors. Here we show that mutants lacking CWH43 are unable to synthesize ceramide-containing GPI anchors, while the replacement of C18 by C26 fatty acids on the primary diacylglycerol anchor by Per1p and Gup1p is still intact. CWH43 contains the COG3568 metal hydrolase motif, which is found in many eukaryotic and prokaryotic enzymes. The conserved His 802 residue of this motif was identified as being essential for ceramide remodelling. Ceramide remodelling is not required for the normal integration of GPI proteins into the cell wall. All remodelling reactions are dependent on prior removal of the inositol-linked fatty acid by Bst1p.     

Glycosylphosphatidylinositol anchors regulate glycosphingolipid levels

Glycosylphosphatidylinositol (GPI) anchor biosynthesis takes place in the endoplasmic reticulum (ER). After protein attachment, the GPI anchor is transported to the Golgi where it undergoes fatty acid remodeling. The ER exit of GPI-anchored proteins is controlled by glycan remodeling and p24 complexes act as cargo receptors for GPI anchor sorting into COPII vesicles. In this study, we have characterized the lipid profile of mammalian cell lines that have a defect in GPI anchor biosynthesis. Depending on which step of GPI anchor biosynthesis the cells were defective, we observed sphingolipid changes predominantly for very long chain monoglycosylated ceramides (HexCer). We found that the structure of the GPI anchor plays an important role in the control of HexCer levels. GPI anchor-deficient cells that generate short truncated GPI anchor intermediates showed a decrease in very long chain HexCer levels. Cells that synthesize GPI anchors but have a defect in GPI anchor remodeling in the ER have a general increase in HexCer levels. GPI-transamidase-deficient cells that produce no GPI-anchored proteins but generate complete free GPI anchors had unchanged levels of HexCer. In contrast, sphingomyelin levels were mostly unaffected. We therefore propose a model in which the transport of very long chain ceramide from the ER to Golgi is regulated by the transport of GPI anchor molecules.     

Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae.

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins of Saccharomyces cerevisiae exist with two types of lipid moiety--diacylglycerol or ceramide--both of which contain 26:0 fatty acids. To understand at which stage of biosynthesis these long-chain fatty acids become incorporated into diacylglycerol anchors, we compared the phosphatidylinositol moieties isolated from myo-[2-(3)H]inositol-labelled protein anchors and from GPI intermediates. There is no evidence for the presence of long-chain fatty acids in any intermediate of GPI biosynthesis. However, GPI-anchored proteins contain either the phosphatidylinositol moiety characteristic of the precursor lipids or a version with a long-chain fatty acid in the sn-2 position of glycerol. The introduction of long-chain fatty acids into sn-2 occurs in the endoplasmic reticulum (ER) and is independent of the sn-2-specific acyltransferase SLC1. Analysis of ceramide anchors revealed the presence of two types of ceramide, one added in the ER and another more polar molecule which is found only on proteins which have reached the mid Golgi. In summary, the lipid of GPI-anchored proteins can be exchanged by at least three different remodelling pathways: (i) remodelling from diacylglycerol to ceramide in the ER as proposed previously; (ii) remodelling from diacylglycerol to a more hydrophobic diacylglycerol with a long-chain fatty acid in sn-2 in the ER; and (iii) remodelling to a more polar ceramide in the Golgi.     

Enhanced response of T lymphocytes from Pgap3 knockout mouse: Insight into roles of fatty acid remodeling of GPI anchored proteins

Glycosylphosphatidylinositol (GPI) is a complex glycolipid that serves as a membrane anchor for many cell-surface proteins, such as Thy-1 and CD48. GPI-anchored proteins (GPI-APs) play important roles in many biological processes, such as signal transduction and cell-cell interaction, through their association with lipid rafts. Fatty acid remodeling of GPI-APs in the Golgi apparatus is required for their efficient association with lipid rafts, i.e., the unsaturated fatty acid at the sn-2 position of the PI moiety is exchanged for the saturated fatty acid by PGAP2 and PGAP3. To investigate the immunological role of the fatty acid remodeling of GPI-APs, we generated a Pgap3 knockout mouse. In this mouse, GPI-APs are expressed on the cell surface without fatty acid remodeling, and fail to associate with lipid rafts. Male Pgap3 knockout mice were born alive at a ratio lower than expected from Mendel's law, whereas the number of female mice followed Mendel's law. All mice exhibited growth retardation and abnormal reflexes such as limb grasping. We focused T cell function in these mice and found that T cell development in the absence of Pgap3 was normal. However, the response of T cells was enhanced in Pgap3 knockout mice in both in vitro and in vivo studies, including alloreactive response, antigen-specific immune response, and experimental autoimmune encephalomyelitis. Cross-linking of Thy-1 in wild-type cells inhibited the signal transduced by the T cell receptor (TCR), whereas cross-linking of Thy-1 in Pgap3 knockout cells enhanced the TCR signal. These results suggest that GPI-APs localized in lipid rafts may modulate signaling through the TCR.     

AtPGAP1 functions as a GPI inositol-deacylase required for efficient transport of GPI-anchored proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) play an important role in a variety of plant biological processes including growth, stress response, morphogenesis, signaling, and cell wall biosynthesis. The GPI anchor contains a lipid-linked glycan backbone that is synthesized in the endoplasmic reticulum (ER) where it is subsequently transferred to the C-terminus of proteins containing a GPI signal peptide by a GPI transamidase. Once the GPI anchor is attached to the protein, the glycan and lipid moieties are remodeled. In mammals and yeast, this remodeling is required for GPI-APs to be included in Coat Protein II-coated vesicles for their ER export and subsequent transport to the cell surface. The first reaction of lipid remodeling is the removal of the acyl chain from the inositol group by Bst1p (yeast) and Post-GPI Attachment to Proteins Inositol Deacylase 1 (PGAP1, mammals). In this work, we have used a loss-of-function approach to study the role of PGAP1/Bst1 like genes in plants. We have found that Arabidopsis (Arabidopsis thaliana) PGAP1 localizes to the ER and likely functions as the GPI inositol-deacylase that cleaves the acyl chain from the inositol ring of the GPI anchor. In addition, we show that PGAP1 function is required for efficient ER export and transport to the cell surface of GPI-APs.

The inositol deacylase AtPGAP1 mediates the first step of glycosylphosphatidylinositol (GPI) anchor-lipid remodeling and is required for efficient transport of GPI-anchored proteins     

Defective lipid remodeling of GPI anchors in peroxisomal disorders, Zellweger syndrome, and rhizomelic chondrodysplasia punctata

Many cell surface proteins in mammalian cells are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The predominant form of mammalian GPI contains 1-alkyl-2-acyl phosphatidylinositol (PI), which is generated by lipid remodeling from diacyl PI. The conversion of diacyl PI to 1-alkyl-2-acyl PI occurs in the ER at the third intermediate in the GPI biosynthetic pathway. This lipid remodeling requires the alkyl-phospholipid biosynthetic pathway in peroxisome. Indeed, cells defective in dihydroxyacetone phosphate acyltransferase (DHAP-AT) or alkyl-DHAP synthase express only the diacyl form of GPI-anchored proteins. A defect in the alkyl-phospholipid biosynthetic pathway causes a peroxisomal disorder, rhizomelic chondrodysplasia punctata (RCDP), and defective biogenesis of peroxisomes causes Zellweger syndrome, both of which are lethal genetic diseases with multiple clinical phenotypes such as psychomotor defects, mental retardation, and skeletal abnormalities. Here, we report that GPI lipid remodeling is defective in cells from patients with Zellweger syndrome having mutations in the peroxisomal biogenesis factors PEX5, PEX16, and PEX19 and in cells from patients with RCDP types 1, 2, and 3 caused by mutations in PEX7, DHAP-AT, and alkyl-DHAP synthase, respectively. Absence of the 1-alkyl-2-acyl form of GPI-anchored proteins might account for some of the complex phenotypes of these two major peroxisomal disorders.     

The yeast p24 complex regulates GPI-anchored protein transport and quality control by monitoring anchor remodeling

Glycosylphosphatidylinositol (GPI)-anchored proteins are secretory proteins that are attached to the cell surface of eukaryotic cells by a glycolipid moiety. Once GPI anchoring has occurred in the lumen of the endoplasmic reticulum (ER), the structure of the lipid part on the GPI anchor undergoes a remodeling process prior to ER exit. In this study, we provide evidence suggesting that the yeast p24 complex, through binding specifically to GPI-anchored proteins in an anchor-dependent manner, plays a dual role in their selective trafficking. First, the p24 complex promotes efficient ER exit of remodeled GPI-anchored proteins after concentration by connecting them with the COPII coat and thus facilitates their incorporation into vesicles. Second, it retrieves escaped, unremodeled GPI-anchored proteins from the Golgi to the ER in COPI vesicles. Therefore the p24 complex, by sensing the status of the GPI anchor, regulates GPI-anchored protein intracellular transport and coordinates this with correct anchor remodeling.     

Glycosylphosphatidylinositol-anchored fungal polysaccharide in Aspergillus fumigatus

Galactomannan is a characteristic polysaccharide of the human filamentous fungal pathogen Aspergillus fumigatus that can be used to diagnose invasive aspergillosis. In this study, we report the isolation of a galactomannan fraction associated to membrane preparations from A. fumigatus mycelium by a lipid anchor. Specific chemical and enzymatic degradations and mass spectrometry analysis showed that the lipid anchor is a glycosylphosphatidylinositol (GPI). The lipid part is an inositol phosphoceramide containing mainly C18-phytosphingosine and monohydroxylated lignoceric acid (2OH-C(24:0) fatty acid). GPI glycan is a tetramannose structure linked to a glucosamine residue: Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN. The galactomannan polymer is linked to the GPI structure through the mannan chain. The GPI structure is a type 1, closely related to the one previously described for the GPI-anchored proteins of A. fumigatus. This is the first time that a fungal polysaccharide is shown to be GPI-anchored.     

Determination and physiological roles of the glycosylphosphatidylinositol lipid remodelling pathway in yeast

In the yeast Saccharomyces cerevisiae, glycosylphosphatidylinositol (GPI)‐anchored proteins play important roles in cell wall biogenesis/assembly and the formation of lipid microdomains. The lipid moieties of mature GPI‐anchored proteins in yeast typically contain either ceramide moieties or diacylglycerol. Recent studies have identified that the GPI phospholipase A₂ Per1p and O‐acyltransferase Gup1p play essential roles in diacylglycerol‐type lipid remodelling of GPI‐anchored proteins, while Cwh43p is involved in the remodelling of lipid moieties to ceramide. It has been generally proposed that phosphatidylinositol with diacylglycerol containing a C26 saturated fatty acid, which is generated by the sequential activity of Per1p and Gup1p, is converted to inositolphosphorylceramide by Cwh43p. In this report, we constructed double‐mutant strains defective in lipid remodelling and investigated their growth phenotypes and the lipid moieties of GPI‐anchored proteins. Based on our analyses of single‐ and double‐mutants of proteins involved in lipid remodelling, we demonstrate that an alternative pathway, in which lyso‐phosphatidylinositol generated by Per1p is used as a substrate for Cwh43p, is involved in the remodelling of GPI lipid moieties to ceramide when the normal sequential pathway is inhibited. In addition, mass spectrometric analysis of lipid species of Flag‐tagged Gas1p revealed that Gas1p contains ceramide moieties in its GPI anchor.     

GUP1 of Saccharomyces cerevisiae encodes an O-acyltransferase involved in remodeling of the GPI anchor

The anchors of mature glycosylphosphatidylinositol (GPI)-anchored proteins of Saccharomyces cerevisiae contain either ceramide or diacylglycerol with a C26:0 fatty acid in the sn2 position. The primary GPI lipid added to newly synthesized proteins in the ER consists of diacylglycerol with conventional C16 and C18 fatty acids. Here we show that GUP1 is essential for the synthesis of the C26:0-containing diacylglycerol anchors. Gup1p is an ER membrane protein with multiple membrane-spanning domains harboring a motif that is characteristic of membrane-bound O-acyl-transferases (MBOAT). Gup1Delta cells make normal amounts of GPI proteins but most mature GPI anchors contain lyso-phosphatidylinositol, and others possess phosphatidylinositol with conventional C16 and C18 fatty acids. The incorporation of the normal ceramides into the anchors is also disturbed. As a consequence, the ER-to-Golgi transport of the GPI protein Gas1p is slow, and mature Gas1p is lost from the plasma membrane into the medium. Gup1Delta cells have fragile cell walls and a defect in bipolar bud site selection. GUP1 function depends on the active site histidine of the MBOAT motif. GUP1 is highly conserved among fungi and protozoa and the gup1Delta phenotype is partially corrected by GUP1 homologues of Aspergillus fumigatus and Trypanosoma cruzi.     

Structures of the glycosylphosphatidylinositol membrane anchors from Aspergillus fumigatus membrane proteins

Glycosylphosphatidylinositol (GPI)-anchored proteins have been identified in all eukaryotes. In fungi, structural and biosynthetic studies of GPIs have been restricted to the yeast Saccharomyces cerevisiae. In this article, four GPI-anchored proteins were purified from a membrane preparation of the human filamentous fungal pathogen Aspergillus fumigatus. Using new methodology applied to western blot protein bands, the GPI structures were characterized by ES-MS, fluorescence labeling, HPLC, and specific enzymatic digestions. The phosphatidylinositol moiety of the A. fumigatus GPI membrane anchors was shown to be an inositol-phosphoceramide containing mainly phytosphingosine and monohydroxylated C24:0 fatty acid. In constrast to yeast, only ceramide was found in the GPI anchor structures of A. fumigatus, even for Gel1p, a homolog of Gas1p in S. cerevisiae that contains diacylglycerol. The A. fumigatus GPI glycan moiety is mainly a linear pentomannose structure linked to a glucosamine residue: Manalpha1-3Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4GlcN.     

Proteomic identification of glycosylphosphatidylinositol anchor-dependent membrane proteins elevated in breast carcinoma

The glycosylphosphatidylinositol (GPI) anchor is a lipid and glycan modification added to the C terminus of certain proteins in the endoplasmic reticulum by the activity of a multiple subunit enzyme complex known as the GPI transamidase (GPIT). Several subunits of GPIT have increased expression levels in breast carcinoma. In an effort to identify GPI-anchored proteins and understand the possible role of these proteins in breast cancer progression, we employed a combination of strategies. First, alpha toxin from Clostridium septicum was used to capture GPI-anchored proteins from human breast cancer tissues, cells, and serum for proteomic analysis. We also expressed short interfering RNAs targeting the expression of the GPAA1 and PIGT subunits of GPIT in breast cancer cell lines to identify proteins in which membrane localization is dependent on GPI anchor addition. Comparative membrane proteomics using nano-ESI-RPLC-MS/MS led to the discovery of several new potential diagnostic and therapeutic targets for breast cancer. Furthermore, we provide evidence that increased levels of GPI anchor addition in malignant breast epithelial cells promotes the dedifferentiation of malignant breast epithelial cells in part by increasing the levels of cell surface markers associated with mesenchymal stem cells.     

Changes in molecular species profiles of glycosylphosphatidylinositol anchor precursors in early stages of biosynthesis

Glycosylphosphatidylinositol (GPI) anchor is a major lipidation in posttranslational modification. GPI anchor precursors are biosynthesized from endogenous phosphatidylinositols (PIs) and attached to proteins in the endoplasmic reticulum. Endogenous PIs are characterized by domination of diacyl species and the presence of polyunsaturated fatty acyl chain, such as 18:0-20:4, at the sn-2 position. In contrast, the features of mammalian glycosylphosphatidylinositol-anchored proteins (GPI-APs) are domination of alkyl/acyl PI species and the presence of saturated fatty acyl chains at the sn-2 position, the latter being consistent with association with lipid rafts. Recent studies showed that saturated fatty acyl chain at sn-2 is introduced by fatty acid remodeling that occurs in GPI-APs. To gain insight into the former feature, we analyzed the molecular species of several different GPI precursors derived from various mammalian mutant cell lines. Here, we show that the PI species profile greatly changed in the precursor glucosamine (GlcN)-acyl-PI and became very similar to that of GPI-APs before fatty acid remodeling. They had alkyl (or alkenyl)/acyl types with unsaturated acyl chain as the major PI species. Therefore, a specific feature of the PI moieties of mature GPI-APs, domination of alkyl (or alkenyl)/acyl type species over diacyl types, is established at the stage of GlcN-acyl-PI.     

Structural remodeling of GPI anchors during biosynthesis and after attachment to proteins

Glycosylphosphatidylinositol (GPI) anchoring of proteins is a conserved post-translational modification in eukaryotes. In mammalian cells, approximately 150 proteins on the plasma membrane are attached to the cell surface by GPI anchors, which confer specific properties on proteins, such as association with membrane microdomains. The structures of lipid and glycan moieties on GPI anchors are remodeled during biosynthesis and after attachment to proteins. The remodeling processes are critical for transport and microdomain-association of GPI-anchored proteins. Here, we describe the structural remodeling of GPI anchors and genes required for the processes in mammals, yeast, and trypanosomes.     

Two different types of lipid moieties are present in glycophosphoinositol-anchored membrane proteins of Saccharomyces cerevisiae.

Numerous glycoproteins of Saccharomyces cerevisiae are anchored in the lipid bilayer by a glycophosphatidylinositol (GPI) anchor. Mild alkaline hydrolysis reveals that the lipid components of these anchors are heterogeneous in that both base-sensitive and base-resistant lipid moieties can be found on most proteins. The relative abundance of base-resistant lipid moieties is different for different proteins. Strong alkaline or acid hydrolysis of the mild base-resistant lipid component liberates C18-phytosphingosine indicating the presence of a ceramide. Two lines of evidence suggest that proteins are first attached to a base-sensitive GPI anchor, the lipid moiety of which subsequently gets exchanged for a base-resistant ceramide: (i) an early glycolipid intermediate of GPI biosynthesis only contains base-sensitive lipid moieties; (ii) after a pulse with [3H]myo-inositol the relative abundance of base-sensitive GPI anchors decreases significantly during chase. This decrease does not take place if GPI-anchored proteins are retained in the ER.     

Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI- anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.     

C26-CoA-dependent ceramide synthesis of Saccharomyces cerevisiae is operated by Lag1p and Lac1p

Lag1p and Lac1p are two highly homologous membrane proteins of the endoplasmic reticulum (ER). When both genes are deleted, cells cannot transport glycosylphosphatidylinositol (GPI)-anchored proteins from the ER to the Golgi at a normal rate. Here we show that microsomes or detergent extracts from lag1lac1 double mutants lack an activity transferring C26 fatty acids from C26-coenzyme A onto dihydrosphingosine or phytosphingosine. As a consequence, in intact cells, the normal ceramides and inositolphosphorylceramides are drastically reduced. lag1lac1 cells compensate for the lack of normal sphingolipids by making increased amounts of C26 fatty acids, which become incorporated into glycerophospholipids. They also contain 20- to 25-fold more free long chain bases than wild type and accumulate very large amounts of abnormally polar ceramides. They make small amounts of abnormal mild base-resistant inositolphospholipids. The lipid remodelling of GPI-anchored proteins is severely compromised in lag1lac1 double mutants since only few and mostly abnormal ceramides are incorporated into the GPI anchors. The participation of Lag1p and Lac1p in ceramide synthesis may explain their role in determining longevity.