Raft-like membrane domains contain enzymatic activities involved in the synthesis of mammalian glycosylphosphatidylinositol anchor intermediates

The synthesis of the glycosylphosphatidylinositol (GPI) anchor occurs in different compartments within the ER. We have previously shown that GPI anchor intermediates including GlcNAc-PI and GlcN-(acyl)PI are present in Triton insoluble membranes (TIMs), believed to be derived from lipid rafts. The present study was initiated to determine if GPI anchor intermediates move to raft-like domains after their synthesis or if these domains represent another ER compartment for GPI anchor synthesis. We determined that in transfected cells Pig-Ap and Pig-Lp, two proteins involved in the synthesis of GlcNAc-PI and GlcN-PI, respectively, are present in TIMs. In addition, we detected GlcNAc-PI synthase, GlcNAc-PI deacetylase, and GlcN-PI acyltransferase activities in TIMs isolated from untransfected cells. These results lend support to the possibility of additional GPI biosynthetic compartments in the ER and to the notion that GPI anchor intermediates produced in and outside raft-like domains may have a different fate.     

Derivation and characterization of glycoinositol-phospholipid anchor-defective human K562 cell clones.

To aid in studies of human glycoinositol-phospholipid (GPI) anchor pathway biochemistry in normal and affected paroxysmal nocturnal hemoglobinuria cells, GPI anchor-defective human K562 cell lines were derived by negative fluorescent sorting of anti-decay-accelerating factor (DAF) monoclonal antibody-stained cells either following or in the absence of ethylmethylsulfonate pretreatment. The resulting cloned cells showed deficiencies of both DAF and GPI-anchored CD59, some (designated group A) exhibiting total absence and some (designated group B) exhibiting approximately 10% levels of surface expression of the two proteins. In heterologous cell fusions, group A clones complemented defective Thy-1 expression by class A, B, C, E, and I Thy-1-negative lymphoma lines, but not H or D lines, the latter of which is defective in the Thy-1 structural gene. In contrast, group B clones complemented all previously described GPI anchor pathway-defective lymphoma classes. Immunoradiomatic assays of cells and supernatants and 35S biosynthetic labeling showed that group A cells degraded DAF protein while group B cells secreted it but failed to attach a GPI anchor structure. [3H]Man labeling of intact cells and UDP-[3H]GlcNAc and GDP-[3H]Man labeling of broken cell preparations demonstrated that group A cells failed to synthesize GlcNAc- and GlcN-PI (GPI-A and -B) as well as more polar mannolipids, whereas group B cells showed accumulation of GlcNAc-PI with approximately 10-fold diminished levels of GlcN-PI and more polar mannolipids. The failed assembly of GlcNAc-PI in group A cells and the reduced conversion of this intermediate to GlcN-PI in group B cells indicates that the former harbors a defect in UDP-GlcNAc transferase or in assembly of its PI acceptor, while the latter harbors a defect in GlcN-PI deacetylase activity.     

Membrane topology and transient acylation of Toxoplasma gondii glycosylphosphatidylinositols

Using hypotonically permeabilized Toxoplasma gondii tachyzoites, we investigated the topology of the free glycosylphosphatidylinositols (GPIs) within the endoplasmic reticulum (ER) membrane. The morphology and permeability of parasites were checked by electron microscopy and release of a cytosolic protein. The membrane integrity of organelles (ER and rhoptries) was checked by protease protection assays. In initial experiments, GPI biosynthetic intermediates were labeled with UDP-[6-(3)H]GlcNAc in permeabilized parasites, and the transmembrane distribution of the radiolabeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). A new early intermediate with an acyl modification on the inositol was identified, indicating that inositol acylation also occurs in T. gondii. A significant portion of the early GPI intermediates (GlcN-PI and GlcNAc-PI) could be hydrolyzed following PI-PLC treatment, indicating that these glycolipids are predominantly present in the cytoplasmic leaflet of the ER. Permeabilized T. gondii parasites labeled with either GDP-[2-(3)H]mannose or UDP-[6-(3)H]glucose showed that the more mannosylated and side chain (Glc-GalNAc)-modified GPI intermediates are also preferentially localized in the cytoplasmic leaflet of the ER.     

Selective inhibitors of the glycosylphosphatidylinositol biosynthetic pathway of Trypanosoma brucei.

Synthetic analogues of D-GlcNalpha1-6D-myo-inositol-1-HPO(4)-3(sn-1, 2-diacylglycerol) (GlcN-PI), with the 2-position of the inositol residue substituted with an O-octyl ether [D-GlcNalpha1-6D-(2-O-octyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-octyl) PI] or O-hexadecyl ether [D-GlcNalpha1-6D-(2-O-hexadecyl)myo-inositol-1-HPO(4)-3-sn-1, 2-dipalmitoylglycerol; GlcN-(2-O-hexadecyl)PI], were tested as substrates or inhibitors of glycosylphosphatidylinositol (GPI) biosynthetic pathways using cell-free systems of the protozoan parasite Trypanosoma brucei (the causative agent of human African sleeping sickness) and human HeLa cells. Neither these compounds nor their N-acetyl derivatives are substrates or inhibitors of GPI biosynthetic enzymes in the HeLa cell-free system but are potent inhibitors of GPI biosynthesis in the T.brucei cell-free system. GlcN-(2-O-hexadecyl)PI was shown to inhibit the first alpha-mannosyltransferase of the trypanosomal GPI pathway. The N-acetylated derivative GlcNAc-(2-O-octyl)PI is a substrate for the trypanosomal GlcNAc-PI de-N-acetylase and this compound, like GlcN-(2-O-octyl)PI, is processed predominantly to Man(2)GlcN-(2-O-octyl)PI by the T.brucei cell-free system. Both GlcN-(2-O-octyl)PI and GlcNAc(2-O-octyl)PI also inhibit inositol acylation of Man(1-3)GlcN-PI and, consequently, the addition of the ethanolamine phosphate bridge in the T.brucei cell-free system. The data establish these substrate analogues as the first generation of in vitro parasite GPI pathway-specific inhibitors.     

Inositol acylation of a potential glycosyl phosphoinositol anchor precursor from yeast requires acyl coenzyme A.

Glycosyl phosphoinositol (GPI) anchors on proteins can be modified by palmitoylation of their inositol residue, which makes such anchors resistant to cleavage by phosphatidylinositol-specific phospholipase C (PI-PLC) (Roberts, W. L., Myher, J. J., Kuksis, A., Low, M. G., and Rosenberry, T.L. (1988) J. Biol. Chem. 263, 18766-18775). Mannosylated GPI lipids made in trypanosomal and mammalian cells can also be inositol-acylated, indicating that inositol acylation may be a normal step in GPI anchor synthesis. We find that Saccharomyces cerevisiae mutants blocked in dolichyl phosphate mannose synthesis accumulate a lipid that can be radiolabeled in vivo with [3H]myo-inositol, [3H]GlcN, and [3H]palmitic acid. This lipid is resistant to PI-PLC, yet sensitive to mild alkaline hydrolysis, and has been characterized as GlcN-phosphatidylinositol (PI), fatty acylated on its inositol residue. When yeast membranes are incubated with UDP-[14C] GlcNAc, 14C-labeled GlcNAc-PI and GlcN-PI are made. Addition of ATP and CoA, or of palmitoyl-CoA to incubations results in the synthesis of [14C]GlcN-(acyl-inositol)PI. This lipid is also made when membranes are incubated with [1-14C]palmitoyl-CoA and UDP-GlcNAc. We propose that acyl CoA is the donor in inositol acylation of GlcN-PI, and that GlcN-(acyl-inositol)PI is an obligatory intermediate in GPI synthesis.     

Biosynthesis of the glycosyl phosphatidylinositol membrane anchor of the trypanosome variant surface glycoprotein. Origin of the non-acetylated glucosamine

Non-acetylated glucosamine is an unusual structural feature shared by all glycosyl phosphatidylinositol (GPI) lipids, including a variety of membrane anchors, the leishmanial lipophosphoglycan, and a mediator of insulin action. We proposed previously a pathway for biosynthesis of glycolipid A, the precursor of the GPI membrane anchor of the trypanosome variant surface glycoprotein (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800). In this paper we characterize in more detail the initial steps of GPI assembly. The first and committed step in the pathway is the transfer of GlcNAc, from UDP-GlcNAc, to endogenous phosphatidylinositol to form N-acetylglucosaminyl phosphatidylinositol (GlcNAc-PI). The GlcNAc-PI is then efficiently deacetylated to form glucosaminyl phosphatidylinositol (GlcN-PI), the substrate for subsequent reactions en route to glycolipid A.     

Early lipid intermediates in glycosyl-phosphatidylinositol anchor assembly are synthesized in the ER and located in the cytoplasmic leaflet of the ER membrane bilayer

Glycosylated phosphoinositides serve as membrane anchors for numerous eukaryotic cell surface glycoproteins. Recent biochemical and genetic studies indicate that the glycolipids are assembled by sequential addition of components (monosaccharides and phosphoethanolamine) to phosphatidylinositol. The biosynthetic steps are presumed to occur in the ER, but formal proof of this is lacking. We describe experiments designed to establish the subcellular location of the initial steps in glycosyl-phosphatidylinositol (GPI) anchor biosynthesis and to define the transmembrane distribution of early biosynthetic lipid intermediates. The experiments were performed with the thymoma cell line BW5147.3. A subcellular fractionation protocol was used to show that early biosynthetic steps in GPI assembly, i.e., synthesis and deacetylation of N-acetylglucosaminyl phosphatidylinositol, occur in the ER. GPI biosynthetic intermediates were synthesized by incubating the microsomes with UDP-[3H]GlcNAc, and the transmembrane distribution of the labeled lipids was probed with phosphatidylinositol-specific phospholipase C (PI-PLC). Treatment of the radiolabeled microsomes with PI-PLC showed that > 70% of the N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be hydrolyzed, indicating that the two lipids were primarily distributed in the cytoplasmic (outer) leaflet of the microsomes. Similar cleavage results were obtained using Streptolysin O-permeabilized thymoma cells. When permeabilized cells were incubated with UDP-[3H]GlcNAc and treated with PI-PLC, approximately 85% of the radiolabeled N-acetylglucosaminyl phosphatidylinositol and glucosaminyl phosphatidylinositol could be cleaved, indicating that they were accessible to the enzyme. The cumulative data indicate that early GPI intermediates are primarily located in the cytoplasmic leaflet of the ER, and are probably synthesized from PI located in the cytoplasmic leaflet and UDP-GlcNAc synthesized in the cytosol.     

In vitro biosynthesis of glycosylphosphatidylinositol in Aspergillus fumigatus

Glycosylphosphatidylinositol (GPI) represents a mechanism for the attachment of proteins to the plasma membrane found in all eukaryotic cells. GPI biosynthesis has been mainly studied in parasites, yeast, and mammalian cells. Aspergillus fumigatus, a filamentous fungus, produces GPI-anchored molecules, some of them being essential in the construction of the cell wall. An in vitro assay was used to study the GPI biosynthesis in the mycelium form of this organism. In the presence of UDP-GlcNAc and coenzyme A, the cell-free system produces the initial intermediates of the GPI biosynthesis: GlcNAc-PI, GlcN-PI, and GlcN-(acyl)PI. Using GDP-Man, two types of mannosylation are observed. First, one or two mannose residues are added to GlcN-PI. This mannosylation, never described in fungi, does not require dolichol phosphomannoside (Dol-P-Man) as the monosaccharide donor. Second, one to five mannose residues are added to GlcN-(acyl)PI using Dol-P-Man as the mannose donor. The addition of ethanolamine phosphate groups to the first, second, and third mannose residue is also observed. This latter series of GPI intermediates identified in the A. fumigatus cell-free system indicates that GPI biosynthesis in this filamentous fungus is similar to the mammalian or yeast systems. Thus, these biochemical data are in agreement with a comparative genome analysis that shows that all but 3 of the 21 genes described in the Saccharomyces cerevisiae GPI pathways are found in A. fumigatus.     

Defective glycosyl phosphatidylinositol biosynthesis in extracts of three Thy-1 negative lymphoma cell mutants

The glycosyl phosphatidylinositol (GPI) anchors that attach certain proteins to membranes are preassembled by sequential addition of glycan components to phosphatidylinositol (PI) before being transferred to nascent polypeptide. A cell-free system consisting of trypanosome membranes has been reported to catalyze GPI biosynthesis (Masterson, W. J., Doering, T. L., Hart, G. W., and Englund, P. T. (1989) Cell 56, 793-800; Menon, A. K., Schwarz, R. T., Mayor, S., and Cross, G. A. M. (1990) J. Biol. Chem. 265, 9033-9042). We now describe conditions for studying the initial steps of GPI biosynthesis in extracts of murine lymphoma cells. Two chloroform-soluble products, tentatively identified as [6-3H]GlcNAc-PI and [6-3H]GlcN-PI were generated during incubations of EL4 cell lysates with UDP-[6-3H]GlcNAc. The involvement of PI in the reaction was established by the sensitivity of the products to hydrolysis by PI-specific phospholipase C and the finding that the addition of exogenous PI to the incubation stimulated the reaction. The minor, more polar product was sensitive to nitrous acid cleavage and was converted to the major product, as judged by TLC, after treatment with acetic anhydride. The glycolipids generated in lymphoma extracts appeared to be the same as the products produced in parallel incubations with trypanosome membranes. Analysis of available lymphoma mutants deficient in Thy-1 surface expression revealed that extracts of the class A, C, and H mutants are completely defective in synthesizing GlcNAc-PI and GlcN-PI.     

A water-soluble analogue of glucosaminylphosphatidylinositol distinguishes two activities that palmitoylate inositol on GPI anchors

2-Palmitoylation of the inositol residue occurs during biosynthesis of glycosylphosphatidylinositol (GPI) anchors, but the enzymology of this step has been enigmatic. With endogenously synthesized glucosamine-PI (GlcN-PI; a GPI intermediate), a CoA-dependent palmitoyl-CoA-independent acyltransfer activity (AT-1) has been reported in rodent preparations. In contrast, a palmitoyl-CoA-dependent GlcN-PI acyltransferase activity (AT-2) was reported in both rodent and yeast preparations with a novel water-soluble dioctanoyl GlcN-PI analogue, GlcN-PI(C8). We report that AT-1, as well as AT-2, can be detected in rodent microsomes with GlcN-PI(C8), thus demonstrating the coexistence of these activities in a single membrane preparation and the general utility of GlcN-PI(C8) for studying the GPI pathway. Unexpectedly, AT-2 was peripherally associated with microsomes, a property atypical for GPI biosynthetic enzymes.     

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 approximately 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.     

Biosynthesis of glycolipid precursors for glycosylphosphatidylinositol membrane anchors in a Toxoplasma gondii cell-free system.

Toxoplasmosis, a disease that affects humans and a wide variety of mammals is caused by Toxoplasma gondii, the obligate intracellular coccidian protozoan parasite. Most T. gondii research has focused on the rapidly growing invasive form, the tachyzoite, which expresses five major surface proteins attached to the parasite membrane by glycosylphosphatidylinositol (GPI) anchors. We have recently reported the purification and partial characterization of candidate precursor glycolipids (GPIs) from metabolically labeled parasites and have presented evidence that these GPIs have a linear glycan backbone sequence indistinguishable from the GPI core glycan of the major tachyzoite surface protein, P30. In this report, we describe a cell-free system derived from tachyzoite membranes which is capable of catalyzing GPI biosynthesis. Incubation of the membrane preparations with radioactive sugar nucleotides (GDP-[3H]mannose or UDP-[3H]GlcNAc) resulted in incorporation of radiolabeled into numerous glycolipids. By using a combination of chemical/enzymatic tests and chromatographic analysis, a series of incompletely glycosylated lipid species and mature GPIs have been identified. We have also established the involvement of Dol-P-mannose in the synthesis of T. gondii GPIs by demonstrating that the incorporation of [3H]mannose into the mannosylated GPIs is stimulated by dolichylphosphate and inhibited by amphomycin. In addition, increasing the concentration of nonradioactive GDP mannose resulted in a loss of radiolabel from the first easily detectable GPI precursor, GlcN-PI, and a concomittant appearance of the radio-activity into mannosylated glycolipids. Altogether, our data suggest that the GPI core glycan in T. gondii is assembled via sequential glycosylation of phosphatidylinositol, as proposed for the biosynthesis of GPIs in Trypanosoma brucei. In contrast to T. brucei, preliminary experiments indicate that the core glycan of some GPIs synthesized by the T. gondii cell-free system is modified by N-acetylgalactosamine similar to the situation for mammalian Thy-1.     

The role of inositol acylation and inositol deacylation in the Toxoplasma gondii glycosylphosphatidylinositol biosynthetic pathway

Toxoplasma gondii is a ubiquitous parasitic protozoan that invades nucleated cells in a process thought to be in part due to several surface glycosylphosphatidylinositol (GPI)-anchored proteins, like the major surface antigen SAG1 (P30), which dominates the plasma membrane. The serine protease inhibitors phenylmethylsulfonyl fluoride and diisopropyl fluoride were found to have a profound effect on the T. gondii GPI biosynthetic pathway, leading to the observation and characterization of novel inositol-acylated mannosylated GPI intermediates. This inositol acylation is acyl-CoA-dependent and takes place before mannosylation, but uniquely for this class of inositol-acyltransferase, it is inhibited by phenylmethylsulfonyl fluoride. The subsequent inositol deacylation of fully mannosylated GPI intermediates is inhibited by both phenylmethylsulfonyl fluoride and diisopropyl fluoride. The use of these serine protease inhibitors allows observations as to the timing of inositol acylation and subsequent inositol deacylation of the GPI intermediates. Inositol acylation of the non-mannosylated GPI intermediate D-GlcNalpha1-6-D-myo-inositol-1-HPO4-sn-lipid precedes mannosylation. Inositol deacylation of the fully mannosylated GPI intermediate allows further processing, i.e. addition of GalNAc side chain to the first mannose. Characterization of the phosphatidylinositol moieties present on both free GPIs and GPI-anchored proteins shows the presence of a diacylglycerol lipid, whose sn-2 position contains almost exclusively an C18:1 acyl chain. The data presented here identify key novel inositol-acylated mannosylated intermediates, allowing the formulation of an updated T. gondii GPI biosynthetic pathway along with identification of the putative genes involved.     

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.     

Pig-n, a mammalian homologue of yeast Mcd4p, is involved in transferring phosphoethanolamine to the first mannose of the glycosylphosphatidylinositol

Many cell surface proteins are anchored to the membrane via a glycosylphosphatidylinositol (GPI) moiety, which is attached to the C terminus of the proteins. The core of the GPI anchor is conserved in all eukaryotes but is modified by various side chains. We cloned a mouse phosphatidylinositol glycan-class N (Pig-n) gene that encodes a 931amino acid protein expressed in the endoplasmic reticulum, which is homologous to yeast Mcd4p. We disrupted the gene in F9 embryonal carcinoma cells. In the Pig-n knockout cells, the first mannose in the GPI precursors was not modified by phosphoethanolamine. Nevertheless, further biosynthetic steps continued with the addition of the third mannose and the terminal phosphoethanolamine. The surface expression of Thy-1 was only partially affected, indicating that modification of the first mannose by phosphoethanolamine is not essential for attachment of GPI anchors in mammalian cells. An inhibitor of GPI biosynthesis, YW3548/BE49385A, inhibited transfer of phosphoethanolamine to the first mannose in mammalian cells but only slightly affected the surface expression of GPI-anchored proteins. Biosynthesis of GPI in the Pig-n knockout cells was not affected by YW3548/BE49385A, and yeast overexpressing MCD4 was highly resistant to YW3548/BE49385A, suggesting that Pig-n and Mcd4p are targets of this drug.     

Phosphatidylethanolamine is the donor of the terminal phosphoethanolamine group in trypanosome glycosylphosphatidylinositols.

A variety of eukaryotic cell surface proteins, including the variant surface glycoproteins of African trypanosomes, rely on a covalently attached lipid, glycosylphosphatidylinositol (GPI), for membrane attachment. GPI anchors are synthesized in the endoplasmic reticulum by stepwise glycosylation of phosphatidylinositol (via UDP-GlcNAc and dolichol-P-mannose) followed by the addition of phosphoethanolamine. The experiments described in this paper are aimed at identifying the biosynthetic origin of the terminal phosphoethanolamine group. We show that trypanosome GPIs can be labelled via CDP-[3H]ethanolamine or [beta-32P]CDP-ethanolamine in a cell-free system, indicating that phosphoethanolamine is acquired en bloc. In pulse-chase experiments with CDP-[3H]ethanolamine we show that the GPI phosphoethanolamine is not derived directly from CDP-ethanolamine, but instead from a relatively stable metabolite, such as phosphatidylethanolamine (PE), generated from CDP-ethanolamine in the cell-free system. To test the possibility that PE is the immediate donor of the GPI phosphoethanolamine moiety, we describe metabolic labelling experiments with [3H]serine and show that GPIs can be labelled in the absence of detectable radiolabelled CDP-ethanolamine, presumably via [3H]PE generated from [3H]phosphatidylserine (PS). The data support the proposal that the terminal phosphoethanolamine group in trypanosome GPIs is derived from PE.     

The GPI anchor of cell-surface proteins is synthesized on the cytoplasmic face of the endoplasmic reticulum

Glycosylphosphatidylinositol (GPI) membrane protein anchors are synthesized from sugar nucleotides and phospholipids in the ER and transferred to newly synthesized proteins destined for the cell surface. The topology of GPI synthesis in the ER was investigated using sealed trypanosome microsomes and the membrane-impermeant probes phosphatidylinositol-specific phospholipase C, Con A, and proteinase K. All the GPI biosynthetic intermediates examined were found to be located on the external face of the microsomal vesicles suggesting that the principal steps of GPI assembly occur in the cytoplasmic leaflet of the ER. Protease protection experiments showed that newly GPI-modified trypanosome variant surface glycoprotein was primarily oriented towards the ER lumen, consistent with eventual expression at the cell surface. The unusual topographical arrangement of the GPI assembly pathway suggests that a biosynthetic intermediate, possibly the phosphoethanolamine-containing anchor precursor, must be translocated across the ER membrane bilayer in the process of constructing a GPI anchor.     

Differences between the trypanosomal and human GlcNAc-PI de-N-acetylases of glycosylphosphatidylinositol membrane anchor biosynthesis

De-N-acetylation of N-acetylglucosaminyl-phosphatidylino-sitol (GlcNAc-PI) is the second step of glycosylphosphatidylino-sitol (GPI) membrane anchor biosynthesis in eukaryotes. This step is a prerequisite for the subsequent processing of glucosaminyl-phosphatidylinositol (GlcN-PI) that leads to mature GPI membrane anchor precursors, which are transferred to certain proteins in the endoplasmic reticulum. In this article, we used a direct de-N-acetylase assay, based on the release of [14C]acetate from synthetic GlcN[14C]Ac-PI and analogues thereof, and an indirect assay, based on the mannosylation of GlcNAc-PI analogues, to study the substrate specificities of the GlcNAc-PI de-N-acetylase activities of African trypanosomes and human (HeLa) cells. The HeLa enzyme was found to be more fastidious than the trypanosomal enzyme such that, unlike the trypanosomal enzyme, it was unable to act on a GlcNAc-PI analogue containing 2-O-octyl-d- myo -inositol or on the GlcNAc-PI diastereoisomer containing l- myo -inositol (GlcNAc-P(l)I). These results suggest thatselective inhibition of the trypanosomal de-N-acetylase may be possible and that this enzyme should be considered as a possible therapeutic target. The lack of strict stereospecificity of the trypanosomal de-N-acetylase for the d- myo -inositol component was also seen for the trypanosomal GPI alpha-manno-syltransferases when GlcNAc-P(l)I was added to the trypanosome cell-free system, but not when GlcN-P(l)I was used. In an attempt to rationalize these data, we modeled the structure and dynamics of d-GlcNAcalpha1-6d- myo -inositol-1-HPO4-( sn )-3-glycerol and its diastereoisomer d-GlcNAcalpha1-6l- myo -inositol-1-HPO4-( sn )-3-glycerol. These studies indicate that the latter compound visits two energy minima, one of which resembles the low-energy conformer of former compound. Thus, it is conceivable that the trypanosomal de-N-acetylase acts on GlcNAc-P(l)I when it occupies a GlcNAc-PI-likeconformation and that GlcN-P(l)I emerging from the de-N-acetylase may be channeled to the alpha-mannosyltransferases in this conformation.     

MCD4 encodes a conserved endoplasmic reticulum membrane protein essential for glycosylphosphatidylinositol anchor synthesis in yeast

Glycosylphosphatidylinositol (GPI)-anchored proteins are cell surface-localized proteins that serve many important cellular functions. The pathway mediating synthesis and attachment of the GPI anchor to these proteins in eukaryotic cells is complex, highly conserved, and plays a critical role in the proper targeting, transport, and function of all GPI-anchored protein family members. In this article, we demonstrate that MCD4, an essential gene that was initially identified in a genetic screen to isolate Saccharomyces cerevisiae mutants defective for bud emergence, encodes a previously unidentified component of the GPI anchor synthesis pathway. Mcd4p is a multimembrane-spanning protein that localizes to the endoplasmic reticulum (ER) and contains a large NH2-terminal ER lumenal domain. We have also cloned the human MCD4 gene and found that Mcd4p is both highly conserved throughout eukaryotes and has two yeast homologues. Mcd4p's lumenal domain contains three conserved motifs found in mammalian phosphodiesterases and nucleotide pyrophosphases; notably, the temperature-conditional MCD4 allele used for our studies (mcd4-174) harbors a single amino acid change in motif 2. The mcd4-174 mutant (1) is defective in ER-to-Golgi transport of GPI-anchored proteins (i.e., Gas1p) while other proteins (i.e., CPY) are unaffected; (2) secretes and releases (potentially up-regulated cell wall) proteins into the medium, suggesting a defect in cell wall integrity; and (3) exhibits marked morphological defects, most notably the accumulation of distorted, ER- and vesicle-like membranes. mcd4-174 cells synthesize all classes of inositolphosphoceramides, indicating that the GPI protein transport block is not due to deficient ceramide synthesis. However, mcd4-174 cells have a severe defect in incorporation of [3H]inositol into proteins and accumulate several previously uncharacterized [3H]inositol-labeled lipids whose properties are consistent with their being GPI precursors. Together, these studies demonstrate that MCD4 encodes a new, conserved component of the GPI anchor synthesis pathway and highlight the intimate connections between GPI anchoring, bud emergence, cell wall function, and feedback mechanisms likely to be involved in regulating each of these essential processes. A putative role for Mcd4p as participating in the modification of GPI anchors with side chain phosphoethanolamine is also discussed.