Reika Watanabe

The first step of glycosylphosphatidylinositol biosynthesis is mediated by a complex of PIG-A, PIG-H, PIG-C and GPI1.

Biosynthesis of glycosylphosphatidylinositol (GPI) is initiated by transfer of N‐acetylglucosamine (GlcNAc) from UDP‐GlcNAc to phosphatidylinositol (PI). This chemically simple step is genetically complex because three genes are required in both mammals and yeast. Mammalian PIG‐A and PIG‐C are homologous to yeast GPI3 and GPI2, respectively; however, mammalian PIG‐H is not homologous to yeast GPI1. Here, we report cloning of a human homolog of GPI1 (hGPI1) and demonstrate that four mammalian gene products form a protein complex in the endoplasmic reticulum membrane. PIG‐L, which is involved in the second step in GPI synthesis, GlcNAc‐PI de‐N‐acetylation, did not associate with the isolated complex. The protein complex had GPI–GlcNAc transferase (GPI–GnT) activity in vitro, but did not mediate the second reaction. Bovine PI was utilized ∼100‐fold more efficiently than soybean PI as a substrate, and lyso PI was a very inefficient substrate. These results suggest that GPI–GnT recognizes the fatty acyl chains of PI. The unusually complex organization of GPI–GnT may be relevant to selective usage of PI and/or regulation.

PIG-M transfers the first mannose to glycosylphosphatidylinositol on the lumenal side of the ER.

Glycosylphosphatidylinositol (GPI) acts as a membrane anchor of many cell surface proteins. Its structure and biosynthetic pathway are generally conserved among eukaryotic organisms, with a number of differences. In particular, mammalian and protozoan mannosyltransferases needed for addition of the first mannose (GPI‐MT‐I) have different substrate specificities and are targets of species‐ specific inhibitors of GPI biosynthesis. GPI‐MT‐I, however, has not been molecularly characterized. Characterization of GPI‐MT‐I would also help to clarify the topology of GPI biosynthesis. Here, we report a human cell line defective in GPI‐MT‐I and the gene responsible, PIG‐M. PIG‐M encodes a new type of mannosyltransferase of 423 amino acids, bearing multiple transmembrane domains. PIG‐M has a functionally important DXD motif, a characteristic of many glycosyltransferases, within a domain facing the lumen of the endoplasmic reticulum (ER), indicating that transfer of the first mannose to GPI occurs on the lumenal side of the ER membrane.

Concentration of GPI‐Anchored Proteins upon ER Exit in Yeast

Previous biochemical work has revealed two parallel routes of exit from the endoplasmic reticulum (ER) in the yeast Saccharomyces cerevisiae, one seemingly specific for glycosyl‐phosphatidylinositol (GPI)‐anchored proteins. Using the coat protein II (COPII) mutant sec31‐1, we visualized ER exit sites (ERES) and identified three distinct ERES populations in vivo. One contains glycosylated pro‐α‐factor, the second contains the GPI‐anchored proteins Cwp2p, Ccw14p and Tos6p and the third is enriched with the hexose transporter, Hxt1p. Concentration of GPI‐anchored proteins prior to budding requires anchor remodeling, and Hxt1p incorporation into ERES requires the COPII components Sec12p and Sec16p. Additionally, we have found that GPI‐anchored protein ER exit is controlled by the p24 family member Emp24p, whereas ER export of most transmembrane proteins requires the Cornichon homologue Erv14p.

Initial enzyme for glycosylphosphatidylinositol biosynthesis requires PIG-P and is regulated by DPM2

Glycosylphosphatidylinositols (GPIs) are attached to the C‐termini of many proteins, thereby acting as membrane anchors. Biosynthesis of GPI is initiated by GPI‐N‐acetylglucosaminyltransferase (GPI‐GnT), which transfers N‐acetylglucosamine from UDP‐ N‐acetylglucosamine to phosphatidylinositol. GPI‐GnT is a uniquely complex glycosyltransferase, consisting of at least four proteins, PIG‐A, PIG‐H, PIG‐C and GPI1. Here, we report that GPI‐GnT requires another component, termed PIG‐P, and that DPM2, which regulates dolichol‐phosphate‐mannose synthase, also regulates GPI‐GnT. PIG‐P, a 134‐amino acid protein having two hydrophobic domains, associates with PIG‐A and GPI1. PIG‐P is essential for GPI‐GnT since a cell lacking PIG‐P is GPI‐anchor negative. DPM2, but not two other components of dolichol‐phosphate‐mannose synthase, associates with GPI‐GnT through interactions with PIG‐A, PIG‐C and GPI1. Lec15 cell, a null mutant of DPM2, synthesizes early GPI intermediates, indicating that DPM2 is not essential for GPI‐GnT; however, the enzyme activity is enhanced 3‐fold in the presence of DPM2. These results reveal new essential and regulatory components of GPI‐GnT and imply co‐regulation of GPI‐GnT and the dolichol‐phosphate‐mannose synthase that generates a mannosyl donor for GPI.

Expression cloning of PIG-L, a candidate N-acetylglucosaminyl-phosphatidylinositol deacetylase.

Many eukaryotic cell surface proteins are bound to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Several genes involved in GPI anchor biosynthesis have been cloned using complementation of mutant mammalian cell lines and yeasts that are defective in its biosynthesis pathway. However, the gene involved in the second step of this pathway, in whichN-acetylglucosaminyl-phosphatidylinositol (GlcNAc-PI) isN-deacetylated to form glucosaminyl (GlcN)-PI, has not been cloned. In this study, we established a GPI anchor-deficient mutant of Chinese hamster ovary (CHO) cells defective in the second step. Complementation analysis with the known GPI anchor mutant cells demonstrated that it belonged to the same complementation group as the CHO cell mutant G9PLAP.85. Using the new mutant, we cloned a rat gene termed PIG-L (forphosphatidylinositol glycan classL) that is involved in this step. PIG-L encodes a 252-amino acid, endoplasmic reticulum membrane protein, most of which is in the cytoplasmic side. This orientation of PIG-L protein is consistent with the notion that the second step of GPI anchor biosynthesis occurs on the cytoplasmic side of the endoplasmic reticulum.

DPM2 regulates biosynthesis of dolichol phosphate‐mannose in mammalian cells: correct subcellular localization and stabilization of DPM1, and binding of dolichol phosphate

Biosynthesis of glycosylphosphatidylinositol and N‐glycan precursor is dependent upon a mannosyl donor, dolichol phosphate‐mannose (DPM). The Thy‐1negative class E mutant of mouse lymphoma and Lec15 mutant Chinese hamster ovary (CHO) cells are incapable of DPM synthesis. The class E mutant is defective in the DPM1 gene which encodes a mammalian homologue of Saccharomyces cerevisiae Dpm1p that is a DPM synthase, whereas Lec15 is a different mutant, indicating that mammalian DPM1 is not sufficient for DPM synthesis. Here we report expression cloning of a new gene, DPM2, which is defective in Lec15 cells. DPM2, an 84 amino acid membrane protein expressed in the endoplasmic reticulum (ER), makes a complex with DPM1 that is essential for the ER localization and stable expression of DPM1. Moreover, DPM2 enhances binding of dolichol phosphate, a substrate of DPM synthase. Mammalian DPM1 is catalytic because a fusion protein of DPM1 that was stably expressed in the ER synthesized DPM without DPM2. Therefore, biosynthesis of DPM in mammalian cells is regulated by DPM2.

Sphingolipids Are Required for the Stable Membrane Association of Glycosylphosphatidylinositol-anchored Proteins in Yeast

Ongoing sphingolipid synthesis is specifically required in vivo for the endoplasmic reticulum (ER) to Golgi transport of glycosylphosphatidylinositol (GPI)-anchored proteins. However, the sphingolipid intermediates that are required for transport nor their role(s) have been identified. Using stereoisomers of dihydrosphingosine, together with specific inhibitors and a mutant defective for sphingolipid synthesis, we now show that ceramides and/or inositol sphingolipids are indispensable for GPI-anchored protein transport. Furthermore, in the absence of sphingolipid synthesis, a significant fraction of GPI-anchored proteins is no longer associated tightly with the ER membrane. The loose membrane association is neither because of the lack of a GPI-anchor nor because of prolonged ER retention of GPI-anchored proteins. These results indicate that ceramides and/or inositol sphingolipids are required to stabilize the association of GPI-anchored proteins with membranes. They could act either by direct involvement as membrane components or as substrates for the remodeling of GPI lipid moieties.

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

Two functions of the p24 complex are described: one connects GPI-anchored proteins to COPII proteins at ER exit sites to facilitate their incorporation into ER-derived vesicles, and the other serves in quality control of GPI-anchored proteins to retrieve unremodeled GPI-anchored proteins from the Golgi back to the ER.

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

p24 complexes act as cargo receptors for sorting GPI-anchored proteins into COPII vesicles.

Requirement of PIG-F and PIG-O for Transferring Phosphoethanolamine to the Third Mannose in Glycosylphosphatidylinositol

Many eukaryotic proteins are anchored by glycosylphosphatidylinositol (GPI) to the cell surface membrane. The GPI anchor is linked to proteins by an amide bond formed between the carboxyl terminus and phosphoethanolamine attached to the third mannose. Here, we report the roles of two mammalian genes involved in transfer of phosphoethanolamine to the third mannose in GPI. We cloned a mouse gene termed Pig-o that encodes a 1101-amino acid PIG-O protein bearing regions conserved in various phosphodiesterases.Pig-o knockout F9 embryonal carcinoma cells expressed very little GPI-anchored proteins and accumulated the same major GPI intermediate as the mouse class F mutant cell, which is defective in transferring phosphoethanolamine to the third mannose due to mutantPig-f gene. PIG-O and PIG-F proteins associate with each other, and the stability of PIG-O was dependent upon PIG-F. However, the class F cell is completely deficient in the surface expression of GPI-anchored proteins. A minor GPI intermediate seen inPig-o knockout but not class F cells had more than three mannoses with phosphoethanolamines on the first and third mannoses, suggesting that this GPI may account for the low expression of GPI-anchored proteins. Therefore, mammalian cells have redundant activities in transferring phosphoethanolamine to the third mannose, both of which require PIG-F.