Diagnosis of paroxysmal nocturnal hemoglobinuria by fluorescent clostridium septicum alpha toxin

Paroxysmal nocturnal hemoglobinuria (PNH), a hematopoietic stem cell disorder, is caused by the loss of glycosylphosphatidylinositol (GPI)-anchored proteins on the cell membrane. PNH can be simply diagnosed by flow cytometry using monoclonal antibodies against GPI-anchored proteins or fluorescent-tagged aerolysin, a bacterial toxin that binds GPI anchored proteins. Clostridium septicum alpha toxin is homologous to aerolysin and specifically binds GPI-anchored proteins. Previously, we found that an alpha toxin m45 mutant with two amino acid changes, S189C/S238C, lost cytotoxicity but still possessed binding activity for GPI-anchored proteins. To use this mutant toxin as a diagnostic probe in flow cytometry, we constructed the EGFP-AT(m45) expression vector, comprising a S189C/S238C alpha toxin mutant with EGFP and His tags at the N and C termini, respectively. The recombinant EGFP-AT(m45) was easily purified using single-step affinity chromatography against His tag from Escherichia coli. EGFP-AT(m45) bound to CHO and HeLa cells in a similar manner to monoclonal antibodies against GPI-anchored proteins or aerolysin. In whole blood from a PNH patient, GPI-deficient granulocytes could be differentiated by EGFP-AT(m45) using the same procedure as that employed with commercially available monoclonal antibodies. Therefore, nontoxic EGFP-conjugated C. septicum alpha toxin could be used clinically for PNH diagnosis.     

Use of Clostridium septicum alpha toxins for isolation of various glycosylphosphatidylinositol-deficient cells

In eukaryotic cells, various proteins are anchored to the plasma membrane through glycosylphosphatidylinositol (GPI). To study the biosynthetic pathways and modifications of GPI, various mutant cells have been isolated from the cells of Chinese hamster ovaries (CHO) supplemented with several exogenous genes involved in GPI biosynthesis using aerolysin, a toxin secreted from gram-negative bacterium Aeromonas hydrophila. Alpha toxin from Gram-positive bacterium Clostridium septicum is homologous to large lobes (LL) of aerolysin, binds GPI-anchored proteins and possesses a cell-destroying mechanism similar to aerolysin. Here, to determine whether alpha toxins can be used as an isolation tool of GPI-mutants, like aerolysin, CHO cells stably transfected with several exogenous genes involved in GPI biosynthesis were chemically mutagenized and cultured in a medium containing alpha toxins. We isolated six mutants highly resistant to alpha toxins and deficient in GPI biosynthesis. By genetic complementation, we determined that one mutant cell was defective of the second subunit of dolichol phosphate mannose synthase (DPM2) and other five cells were of a putative catalytic subunit of inositol acyltransferase (PIG-W). Therefore, C. septicum alpha toxins are a useful screening probe for the isolation of various GPI-mutant cells.     

Affected paroxysmal nocturnal hemoglobinuria T lymphocytes harbor a common defect in assembly of N-acetyl-D-glucosamine inositol phospholipid corresponding to that in class A Thy-1- murine lymphoma mutants.

Deficient expression of glycoinositol phospholipid (GPI) anchored proteins in affected paroxysmal nocturnal hemoglobinuria (PNH) cells has been traced to a defect in GPI anchor assembly. In a previous study (Schubert, J., Schmidt, R. E., and Medof, M. E. (1993) J. Biol. Chem., in press) we characterized the biosynthesis of putative Man-containing GPI anchor precursors in normal peripheral blood lymphocytes and investigated assembly of these intracellular GPI intermediates in CD48- affected and CD48+ unaffected T and natural killer cell lines of PNH patients. We found that affected T cells from five patients exhibited a uniform defect in which dolichol-phosphoryl-Man was synthesized but no GPI mannolipids were expressed. In this study, membranes of patients' affected T cells were labeled with UDP-[3H]GlcNAc to evaluate earlier steps in GPI synthesis, and intact cells were fused to Thy-1- murine lymphoma mutants harboring different defects in early GPI assembly to test for the presence of corresponding or complementary lesions. In all cases, affected cell membranes failed to assemble GlcNAc-inositol phospholipid, the initial precursor of GPI anchor structures, and the intact cells failed to complement class A mutants while complementing other classes. Affected polymorphonuclear leukocytes from three additional patients of different origin were then labeled with [3H]Man and the labeling patterns found to correspond to those obtained with the T lymphocytes. Taken together the data indicate that the genetic lesion in PNH cells resides in a DNA element which: 1) encodes a product required for the synthesis of GlcNAc-inositol phospholipid, 2) corresponds to that altered in class A Thy-1- murine lymphoma mutants, and 3) is commonly affected in different patients.     

Cross-talk between caveolae and glycosylphosphatidylinositol-rich domains.

Most mammalian cells have in their plasma membrane at least two types of lipid microdomains, non-invaginated lipid rafts and caveolae. Glycosylphosphatidylinositol (GPI)-anchored proteins constitute a class of proteins that are enriched in rafts but not caveolae at steady state. We have analyzed the effects of abolishing GPI biosynthesis on rafts, caveolae, and cholesterol levels. GPI-deficient cells were obtained by screening for resistance to the pore-forming toxin aerolysin, which uses this class of proteins as receptors. Despite the absence of GPI-anchored proteins, mutant cells still contained lipid rafts, indicating that GPI-anchored proteins are not crucial structural elements of these domains. Interestingly, the caveolae-specific membrane proteins, caveolin-1 and 2, were up-regulated in GPI-deficient cells, in contrast to flotillin-1 and GM1, which were expressed at normal levels. Additionally, the number of surface caveolae was increased. This effect was specific since recovery of GPI biosynthesis by gene recomplementation restored caveolin expression and the number of surface caveolae to wild type levels. The inverse correlation between the expression of GPI-anchored proteins and caveolin-1 was confirmed by the observation that overexpression of caveolin-1 in wild type cells led to a decrease in the expression of GPI-anchored proteins. In cells lacking caveolae, the absence of GPI-anchored proteins caused an increase in cholesterol levels, suggesting a possible role of GPI-anchored proteins in cholesterol homeostasis, which in some cells, such as Chinese hamster ovary cells, can be compensated by caveolin up-regulation.     

Analysis of receptor binding by the channel-forming toxin aerolysin using surface plasmon resonance.

Aerolysin is a channel-forming bacterial toxin that binds to glycosylphosphatidylinositol (GPI) anchors on host cell-surface structures. The nature of the receptors and the location of the receptor-binding sites on the toxin molecule were investigated using surface plasmon resonance. Aerolysin bound to the GPI-anchored proteins Thy-1, variant surface glycoprotein, and contactin with similar rate constants and affinities. Enzymatic removal of N-linked sugars from Thy-1 did not affect toxin binding, indicating that these sugars are not involved in the high affinity interaction with aerolysin. Aerolysin is a bilobal protein, and both lobes were shown to be required for optimal binding. The large lobe by itself bound Thy-1 with an affinity that was at least 10-fold weaker than that of the whole toxin, whereas the small lobe bound the GPI-anchored protein at least 1000-fold more weakly than the intact toxin. Mutation analyses provided further evidence that both lobes were involved in GPI anchor binding, with certain single amino acid substitutions in either domain leading to reductions in affinity of as much as 100-fold. A variant with single amino acid substitutions in both lobes of the protein was completely unable to bind the receptor. The membrane protein glycophorin, which is heavily glycosylated but not GPI-anchored, bound weakly to immobilized proaerolysin, suggesting that interactions with cell-surface carbohydrate structures other than GPI anchors may partially mediate toxin binding to host cells.     

Flow cytometric analysis of CD55 and CD59 expression on blood cells in paroxysmal nocturnal haemoglobinuria

PNH is a rare clonal disorder of hematopoietic stem cells, therefore all blood cells lineages are involved. The main feature is an increased sensitivity of erythrocytes to complement-mediated cell lysis due to deficiency of membrane-bound GPI (glycosylphosphatidylinositol)-anchored proteins which normally function as inhibitors of reactive hemolysis. In the present study, we performed flow cytometric analysis using monoclonal antibodies against CD55 and CD59 for the detection of PNH-type clone in the blood of 50 patients (28 females and 22 males, age range 7-67 yrs). In one patient only we found a large population (95%) of granulocytes with decreased expression of both CD55 and CD59 molecules (type I PNH) and in two others with partial loss of CD55 expression (type II PNH). The expression was determined chiefly on granulocytes which in the control group showed reliable and high expression of CD55 and CD59.     

Clostridium septicum alpha toxin uses glycosylphosphatidylinositol-anchored protein receptors.

The alpha toxin produced by Clostridium septicum is a channel-forming protein that is an important contributor to the virulence of the organism. Chinese hamster ovary (CHO) cells are sensitive to low concentrations of the toxin, indicating that they contain toxin receptors. Using retroviral mutagenesis, a mutant CHO line (BAG15) was generated that is resistant to alpha toxin. FACS analysis showed that the mutant cells have lost the ability to bind the toxin, indicating that they lack an alpha toxin receptor. The mutant cells are also resistant to aerolysin, a channel-forming protein secreted by Aeromonas spp., which is structurally and functionally related to alpha toxin and which is known to bind to glycosylphosphatidylinositol (GPI)-anchored proteins, such as Thy-1. We obtained evidence that the BAG15 cells lack N-acetylglucosaminyl-phosphatidylinositol deacetylase-L, needed for the second step in GPI anchor biosynthesis. Several lymphocyte cell lines lacking GPI-anchored proteins were also shown to be less sensitive to alpha toxin. On the other hand, the sensitivity of CHO cells to alpha toxin was increased when the cells were transfected with the GPI-anchored folate receptor. We conclude that alpha toxin, like aerolysin, binds to GPI-anchored protein receptors. Evidence is also presented that the two toxins bind to different subsets of GPI-anchored proteins.     

The receptor and transporter for internalization of Clostridium botulinum type C progenitor toxin into HT-29 cells

Orally ingested botulinum toxin enters the circulatory system and eventually reaches the peripheral nerves, where it elicits a response of neurological dysfunction. In this study, we report the important findings concerning the mechanism of Clostridium botulinum type C progenitor toxin (C16S) endocytic mechanism. C16S toxin bound to high molecular weight proteins on the surface of human colon carcinoma HT-29 cells and was internalized, but not if the cells were pretreated with neuraminidase. Benzyl-GalNAc which inhibited O-glycosylation of glycoproteins also interfered in the toxin's ability to bind the cell surface. On the other hand, the toxin was internalized in spite of pretreatment of the cells with PPMP, an inhibitor of ganglioside synthesis. These results suggest that the glycoproteins, like mucin, fulfill the important roles of receptor and transporter of C16S toxin.     

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.     

Asn183 in alpha5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin

The proposed toxicity mechanism of the Bacillus thuringiensis Cry insecticidal proteins involves membrane penetration and lytic pore formation of the alpha4-alpha5 hairpins in the target larval midgut cell membranes. In this study, alanine substitutions of selected polar residues (Tyr(178), Gln(180), Asn(183), Asn(185), and Asn(195)) in the hydrophobic helix-alpha5 of the Cry4Ba mosquito-larvicidal protein were initially conducted via PCR-based directed mutagenesis. Upon IPTG induction, all the 130-kDa mutant protoxins were highly expressed in Escherichia coli as cytoplasmic inclusions, with yields similar to the wild-type protoxin. When E. coli cells expressing each mutant toxin were tested against Stegomyia aegypti mosquito larvae, the larvicidal activity of the N183A mutant was almost completely abolished whereas the four other mutants showed only a small reduction in toxicity. Additionally, replacements of this critical residue with various amino acids revealed that the uncharged polar residue at position 183 in alpha5 is crucial for larvicidal activity. Further characterisation of the N183K bio-inactive mutant revealed that the 65-kDa activated toxin was unable to form oligomers in lipid vesicles and its ability to induce the release of entrapped calcein from liposomes was much weaker than that of the wild-type toxin. These results suggest that the highly conserved Asn(183) located in the middle of the transmembrane alpha5 of Cry4Ba plays a crucial role in toxicity and toxin oligomerisation in the lipid membranes.     

Analysis of tryptophan‐rich region in Clostridium septicum alpha‐toxin involved with binding to glycosylphosphatidylinositol‐anchored proteins

Clostridium septicum alpha‐toxin has a unique tryptophan‐rich region (³⁰²NGYSEWDWKWV³¹²) that consists of 11 amino acid residues near the C‐terminus. Using mutant toxins, the contribution of individual amino acids in the tryptophan‐rich region to cytotoxicity and binding to glycosylphosphatidylinositol (GPI)‐anchored proteins was examined. For retention of maximum cytotoxic activity, W307 and W311 are essential residues and residue 309 has to be hydrophobic and possess an aromatic side chain, such as tryptophan or phenylalanine. When residue 308, which lies between tryptophans (W307 and W309) is changed from an acidic to a basic amino acid, the cytotoxic activity of the mutant is reduced to less than that of the wild type. It was shown by a toxin overlay assay that the cytotoxic activity of each mutant toxin correlates closely with affinity to GPI‐anchored proteins. These findings indicate that the WDW_W sequence in the tryptophan‐rich region plays an important role in the cytotoxic mechanism of alpha‐toxin, especially in the binding to GPI‐anchored proteins as cell receptors.     

Identification of six complementation classes involved in the biosynthesis of glycosylphosphatidylinositol anchors in Saccharomyces cerevisiae

Glycosylphosphatidylinositol (GPI)-anchored membrane proteins are synthesized by the posttranslational attachment of a preformed glycolipid to newly made glycoproteins. alpha-Agglutinin is a GPI- anchored glycoprotein that gets expressed at the cell surface of MAT alpha cells after induction with type a mating factor. Mutants affecting the biosynthesis of GPI anchors were obtained by selecting for the absence of alpha-agglutinin from the cell wall after induction with a-factor at 37 degrees C. 10 recessive mutants were grouped into 6 complementation classes, gpi4 to gpi9. Mutants are considered to be deficient in the biosynthesis of GPI anchors, since each mutant accumulates an abnormal, incomplete GPI glycolipid containing either zero, two, or four mannoses. One mutant accumulates a complete precursor glycolipid, suggesting that it might be deficient in the transfer of complete precursor lipids to proteins. When labeled with [2- 3H]inositol, mutants accumulate reduced amounts of radiolabeled GPI- anchored proteins, and the export of the GPI-anchored Gas1p out of the ER is severely delayed in several mutant strains. On the other hand, invertase and acid phosphatase are secreted by all but one mutant. All mutants show an increased sensitivity to calcofluor white and hygromycin B. This suggests that GPI-anchored proteins are required for the integrity of the yeast cell wall.     

Isolation of Novel Animal Cell Lines Defective in Glycerolipid Biosynthesis Reveals Mutations in Glucose-6-phosphate Isomerase

Glycerolipids are structural components for membranes and serve in energy storage. We describe here the use of a photodynamic selection technique to generate a population of Chinese hamster ovary cells that display a global deficiency in glycerolipid biosynthesis. One isolate from this population, GroD1, displayed a profound reduction in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triglycerides but presented high levels of phosphatidic acid and normal levels of phosphatidylinositol synthesis. This was accompanied by a reduction in phosphatidate phosphatase 1 (PAP1) activity. Expression cloning and sequencing of the cDNA obtained from GroD1 revealed a point mutation, Gly-189 → Glu, in glucose-6-phosphate isomerase (GPI), a glycolytic enzyme involved in an inherited disorder that results in anemia and neuromuscular symptoms in humans. GPI activity was reduced by 87% in GroD1. No significant differences were found in DNA synthesis, protein synthesis, and ATP levels, whereas glycerol 3-phosphate levels were increased in the mutant. Expression of wild-type hamster GPI restored GPI activity, glycerolipid biosynthesis, and PAP1 activity in GroD1. Two additional, independently isolated GPI-deficient mutants displayed similar phenotypes with respect to PAP1 activity and glycerolipid biosynthesis. These findings uncover a novel relationship between GPI, involved in carbohydrate metabolism, and PAP1, a lipogenic enzyme. These results may also help to explain neuromuscular symptoms associated with inherited GPI deficiency.     

Biochemical background of paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder characterized by paroxysms of intravascular hemolysis. A considerable part of erythrocytes in patient blood is susceptible to autologous complement activation because of the deficiency of CD59, which is a glycosylphosphatidylinositol (GPI)-anchored protein and inhibits the formation of the membrane attack complex (MAC) of complement. The deficiency of CD59 is derived from the inability of GPI-anchor synthesis. Although more than 10 proteins are involved in the GPI-anchor synthesis, the mutation of only one protein, PIG-A, causes the defect in about 200 patients with PNH who have been analyzed. The reason why only PIG-A causes the deficiency of GPI anchor is due to the location of its gene on X chromosome. The clonal stem cell mutated with PIG-A gene in the bone marrow loses the capability of the synthesis of GPI-anchor. The mutation of PIG-A gene alone, however, seems to be insufficient to account for the survival of the PIG-A-deficient cells in the bone marrow. Thus, a fraction of the mutant stem cells probably gain a survival advantage by some additional changes, either additional mutations or changes in immunological circumstances. The release of the surviving cells into blood stream results in a clinical syndrome with PNH.     

GPI anchor biosynthesis in yeast: phosphoethanolamine is attached to the alpha1,4-linked mannose of the complete precursor glycophospholipid

Cells synthesize the GPI anchor carbohydrate core by successively adding N-acetylglucosamine, three mannoses, and phosphoethanolamine (EtN-P) onto phosphatidylinositol, thus forming the complete GPI precursor lipid which is then added to proteins. Previously, we isolated a GPI deficient yeast mutant accumulating a GPI intermediate containing only two mannoses, suggesting that it has difficulty in adding the third, alpha1,2-linked Man of GPI anchors. The mutant thus displays a similar phenotype as the mammalian mutant cell line S1A-b having a mutation in the PIG-B gene. The yeast mutant, herein named gpi10-1 , contains a mutation in YGL142C, a yeast homolog of the human PIG-B. YGL142C predicts a highly hydrophobic integral membrane protein which by sequence is related to ALG9, a yeast gene required for adding Man in alpha1,2 linkage to N-glycans. Whereas gpi10-1 cells grow at a normal rate and make normal amounts of GPI proteins, the microsomes of gpi10-1 are completely unable to add the third Man in an in vitro assay. Further analysis of the GPI intermediate accumulating in gpi10 shows it to have the structure Manalpha1-6(EtN-P-)Manalpha1-4GlcNalpha1- 6(acyl) Inositol-P-lipid. The presence of EtN-P on the alpha1,4-linked Man of GPI anchors is typical of mammalian and a few other organisms but had not been observed in yeast GPI proteins. This additional EtN-P is not only found in the abnormal GPI intermediate of gpi10-1 but is equally present on the complete GPI precursor lipid of wild type cells. Thus, GPI biosynthesis in yeast and mammals proceeds similarly and differs from the pathway described for Trypanosoma brucei in several aspects.      

Requirement of N-glycan on GPI-anchored proteins for efficient binding of aerolysin but not Clostridium septicum alpha-toxin

Aerolysin of the Gram-negative bacterium Aeromonas hydrophila consists of small (SL) and large (LL) lobes. The alpha-toxin of Gram-positive Clostridium septicum has a single lobe homologous to LL. These toxins bind to glycosylphosphatidylinositol (GPI)-anchored proteins and generate pores in the cell's plasma membrane. We isolated CHO cells resistant to aerolysin, with the aim of obtaining GPI biosynthesis mutants. One mutant unexpectedly expressed GPI-anchored proteins, but nevertheless bound aerolysin poorly and was 10-fold less sensitive than wild-type cells. A cDNA of N-acetylglucosamine transferase I (GnTI) restored the binding of aerolysin to this mutant. Therefore, N-glycan is involved in the binding. Removal of mannoses by alpha-mannosidase II was important for the binding of aerolysin. In contrast, the alpha-toxin killed GnTI-deficient and wild-type CHO cells equally, indicating that its binding to GPI-anchored proteins is independent of N-glycan. Because SL bound to wild-type but not to GnTI-deficient cells, and because a hybrid toxin consisting of SL and the alpha-toxin killed wild-type cells 10-fold more efficiently than GnTI- deficient cells, SL with its binding site for N-glycan contributes to the high binding affinity of aerolysin.     

Discovery and development of the complement inhibitor eculizumab for the treatment of paroxysmal nocturnal hemoglobinuria

The complement system provides critical immunoprotective and immunoregulatory functions but uncontrolled complement activation can lead to severe pathology. In the rare hemolytic disease paroxysmal nocturnal hemoglobinuria (PNH), somatic mutations result in a deficiency of glycosylphosphatidylinositol-linked surface proteins, including the terminal complement inhibitor CD59, on hematopoietic stem cells. In a dysfunctional bone marrow background, these mutated progenitor blood cells expand and populate the periphery. Deficiency of CD59 on PNH red blood cells results in chronic complement-mediated intravascular hemolysis, a process central to the morbidity and mortality of PNH. A recently developed, humanized monoclonal antibody directed against complement component C5, eculizumab (Soliris; Alexion Pharmaceuticals Inc., Cheshire, CT, USA), blocks the proinflammatory and cytolytic effects of terminal complement activation. The recent approval of eculizumab as a first-in-class complement inhibitor for the treatment of PNH validates the concept of complement inhibition as an effective therapy and provides rationale for investigation of other indications in which complement plays a role.     

Biosynthesis of glycosylphosphatidylinositols in mammals and unicellular microbes.

Membrane anchoring of cell surface proteins via glycosylphosphatidylinositol (GPI) occurs in all eukaryotic organisms. In addition, GPI-related glycophospholipids are important constituents of the glycan coat of certain protozoa. Defects in GPI biosynthesis can retard, if not abolish growth of these organisms. In humans, a defect in GPI biosynthesis can cause paroxysmal nocturnal hemoglobinuria (PNH), a severe acquired bone marrow disorder. Here, we review advances in the characterization of GPI biosynthesis in parasitic protozoa, yeast and mammalian cells. The GPI core structure as well as the major steps in its biosynthesis are conserved throughout evolution. However, there are significant biosynthetic differences between mammals and microbes. First indications are that these differences could be exploited as targets in the design of novel pharmacotherapeutics that selectively inhibit GPI biosynthesis in unicellular microbes.     

Mutations of GS alpha designed to alter the reactivity of the protein with bacterial toxins. Substitutions at ARG187 result in loss of GTPase activity

We have introduced two types of mutations into cDNAs that encode the alpha subunit of Gs, the guanine nucleotide-binding regulatory protein that stimulates adenylyl cyclase. The arginine residue (Arg187) that is the presumed site of ADP-ribosylation of Gs alpha by cholera toxin has been changed to Ala, Glu, or Lys. The rate constant for hydrolysis of GTP by all of these mutants is reduced approximately 100-fold compared with the wild-type protein. As predicted from this change, these proteins activate adenylyl cyclase constitutively in the presence of GTP. Despite these substitutions, cholera toxin still catalyzes the incorporation of 0.2-0.3 mol of ADP-ribose/mol of mutant alpha subunit. The sequence near the carboxyl terminus of Gs alpha was altered to resemble those in Gi alpha polypeptides, which are substrates for pertussis toxin. Despite this change, the mutant protein is a poor substrate for pertussis toxin. Although this protein has unaltered rates of GDP dissociation and GTP hydrolysis, its ability to activate adenylyl cyclase in the presence of GTP is enhanced by 3-fold when compared with the wild-type protein but only when these assays are performed after reconstitution of Gs alpha into cyc- (Gs alpha-deficient) S49 cell membranes.     

YLL031c belongs to a novel family of membrane proteins involved in the transfer of ethanolaminephosphate onto the core structure of glycosylphosphatidylinositol anchors in yeast

MCD4 and GPI7 are important for the addition of glycosylphosphatidylinositol (GPI) anchors to proteins in the yeast Saccharomyces cerevisiae. Mutations in these genes lead to a reduction of GPI anchoring and cell wall fragility. Gpi7 mutants accumulate a GPI lipid intermediate of the structure Manalpha1-2[NH(2)-(CH(2))(2)-PO(4)-->]Manalpha1-2Manalpha 1-6[NH(2)-(C H(2))(2)-PO(4)-->]Manalpha1-4GlcNalpha1-6[acyl-->]inositol-P O(4)-lipi d, which, in comparison with the complete GPI precursor lipid CP2, lacks an HF-sensitive side chain on the alpha1-6-linked mannose. In contrast, mcd4-174 accumulates only minor amounts of abnormal GPI intermediates. Here we investigate whether YLL031c, an open reading frame predicting a further homologue of GPI7 and MCD4, plays any role in GPI anchoring. YLL031c is an essential gene. Its depletion results in a reduction of GPI anchor addition to GPI proteins as well as to cell wall fragility. YLL031c-depleted cells accumulate GPI intermediates with the structures Manalpha1-2Manalpha1-2Manalpha1-6[NH(2)-(CH(2))(2)-PO( 4)-->]Manalpha1 -4GlcNalpha1-6[acyl-->]inositol-PO(4)-lipid and Manalpha1-2Manalpha1-2Manalpha1-6Manalpha1-4G lcNalpha1-6[acyl-->]inos itol-PO(4)-lipid. Subcellular localization studies of a tagged version of YLL031c suggest that this protein is mainly in the ER, in contrast to Gpi7p, which is found at the cell surface. The data are compatible with the idea that YLL031c transfers the ethanolaminephosphate to the inner alpha1-2-linked mannose, i.e. the group that links the GPI lipid anchor to proteins, whereas Mcd4p and Gpi7p transfer ethanolaminephosphate onto the alpha1-4- and alpha1-6-linked mannoses of the GPI anchor, respectively.