Proper protein folding in the endoplasmic reticulum is required for attachment of a glycosylphosphatidylinositol anchor in plants

Endoplasmic reticulum (ER) quality control processes recognize and eliminate misfolded proteins to maintain cellular protein homeostasis and prevent the accumulation of defective proteins in the secretory pathway. Glycosylphosphatidylinositol (GPI)-anchored proteins carry a glycolipid modification, which provides an efficient ER export signal and potentially prevents the entry into ER-associated degradation (ERAD), which is one of the major pathways for clearance of terminally misfolded proteins from the ER. Here, we analyzed the degradation routes of different misfolded glycoproteins carrying a C-terminal GPI-attachment signal peptide in Arabidopsis thaliana. We found that a fusion protein consisting of the misfolded extracellular domain from Arabidopsis STRUBBELIG and the GPI-anchor attachment sequence of COBRA1 was efficiently targeted to hydroxymethylglutaryl reductase degradation protein 1 complex-mediated ERAD without the detectable attachment of a GPI anchor. Non-native variants of the GPI-anchored lipid transfer protein 1 (LTPG1) that lack a severely misfolded domain, on the other hand, are modified with a GPI anchor and targeted to the vacuole for degradation. Impaired processing of the GPI-anchoring signal peptide by mutation of the cleavage site or in a GPI-transamidase-compromised mutant caused ER retention and routed the non-native LTPG1 to ERAD. Collectively, these results indicate that for severely misfolded proteins, ER quality control processes are dominant over ER export. For less severely misfolded proteins, the GPI anchor provides an efficient ER export signal resulting in transport to the vacuole.

Severely misfolded proteins carrying a glycosylphosphatidylinositol (GPI)-anchor attachment sequence undergo a stringent quality control process in the endoplasmic reticulum that prevents GPI anchoring.     

GPI锚定蛋白前体C-端附着信号的疏水性决定其内质网相关蛋白质降解途径

目前,已知超过150种糖基磷脂酰肌醇锚定蛋白(glycosylphosphatidylinositol anchored protein, GPI-anchored protein)在哺乳动物细胞中表达,并参与免疫识别、细胞通讯与信号转导等多种生理过程。当蛋白质无法被GPI修饰时,前体蛋白质通过内质网相关蛋白质降解(endoplasmic reticulum associated degradation, ERAD)途径降解。然而,GPI锚定蛋白ERAD的降解机制尚不清楚。为了探究GPI锚定蛋白前体的ERAD途径的具体机制,本研究敲除人胚胎肾细胞293细胞株(HEK293)的GPI转酰胺酶复合物亚基PIGS基因,进而敲除E3泛素连接酶HRD1和GP78基因,之后随机在PIGS-KO,PIGS-HRD1-KO和PIGS-GP78-KO过表达16种GPI锚定蛋白质(以亲本PIGS-KO细胞株作为对照组),Western印迹结果证明,GPI锚定蛋白前体在细胞株PIGS-HRD1-KO中的蛋白质积累量(I_PHK)和PIGS-GP78-KO中的蛋白质积累量(I_PG...     

Characterization of the glycosylphosphatidylinositol-anchor signal sequence of human Cryptic with a hydrophilic extension

Epidermal Growth Factor-Cripto-1/FRL-1/Cryptic (EGF-CFC) proteins, including human Cripto-1 (hCFC2/hCR-1) and human Cryptic (hCFC1), are membrane-associated Nodal co-receptors, which have critical roles in vertebrate development. Most of the EGF-CFC proteins have been experimentally proven or predicted to be glycosylphosphatidylinositol (GPI)-anchored proteins. However, unlike other EGF-CFC proteins, hCFC1 does not exhibit a typical GPI-signal sequence, containing a 32-amino acid hydrophilic extension in its COOH-terminal end. Here we experimentally demonstrate that the COOH-terminal sequence of hCFC1 functions as a GPI-anchoring signal. Moreover, addition of a hydrophilic epitope tag of 55-amino acids (V5-His) after the GPI signal of hCR-1 interfered with generation of a GPI-anchored form of hCR-1. In contrast, addition of the same epitope tag to the end of GPI signal of hCFC1 did not affect the GPI-attachment of hCFC1. The COOH-terminal signal of hCFC1 could produce two different forms of the protein; a GPI-anchored form and an unprocessed form which was more prone to be secreted into the conditioned medium. The hydrophilic extension of hCFC1 negatively regulates the activity of hCFC1 as a Nodal co-receptor. These results demonstrate the presence of endogenous GPI-signal sequence with a hydrophilic extension, which can generate both GPI-anchored and soluble forms of the protein.     

Yeast ARV1 is required for efficient delivery of an early GPI intermediate to the first mannosyltransferase during GPI assembly and controls lipid flow from the endoplasmic reticulum

Glycosylphosphatidylinositol (GPI), covalently attached to many eukaryotic proteins, not only acts as a membrane anchor but is also thought to be a sorting signal for GPI-anchored proteins that are associated with sphingolipid and sterol-enriched domains. GPI anchors contain a core structure conserved among all species. The core structure is synthesized in two topologically distinct stages on the leaflets of the endoplasmic reticulum (ER). Early GPI intermediates are assembled on the cytoplasmic side of the ER and then are flipped into the ER lumen where a complete GPI precursor is synthesized and transferred to protein. The flipping process is predicted to be mediated by a protein referred as flippase; however, its existence has not been proven. Here we show that yeast Arv1p is an important protein required for the delivery of an early GPI intermediate, GlcN-acylPI, to the first mannosyltransferase of GPI synthesis in the ER lumen. We also provide evidence that ARV1 deletion and mutations in other proteins involved in GPI anchor synthesis affect inositol phosphorylceramide synthesis as well as the intracellular distribution and amounts of sterols, suggesting a role of GPI anchor synthesis in lipid flow from the ER.     

Functional analysis of T-cell mutants defective in the biosynthesis of glycosylphosphatidylinositol anchor. Relative importance of glycosylphosphatidylinositol anchor versus N-linked glycosylation in T-cell activation.

The glycosylphosphatidylinositol (GPI) anchor, potentially capable of generating a number of second messengers, such as diacylglycerol, phosphatidic acid, and inositol phosphate glycan, has been postulated to be involved in signal transduction in various cell types, including T-cells. We have identified a panel of T-cell hybridoma mutants that are defective at various steps of GPI anchor biosynthesis. Since they were derived from a functional T-T hybridoma, we were able to determine the precise role of the GPI anchor in T-cell activation. Two mutants were chosen for this analysis. The first mutant is defective at the first step of GPI anchor biosynthesis, i.e. in the transfer of N-acetylglucosamine to a phosphatidylinositol acceptor. Thus, it cannot form any GPI precursors or GPI-like compounds. Interestingly, this mutant can be activated by antigen, superantigen, and concanavalin A in a manner comparable to the wild-type hybridoma. These data strongly suggest that the GPI anchor, its precursor, or its potential cleavage product, inositol phosphate glycan, is not required for the early phase of T-cell activation. The second mutant is able to synthesize the first two GPI precursors, but is not able to add mannose residues to them due to a deficiency in dolichol-phosphate-mannose (Dol-P-Man) biosynthesis. Unexpectedly, all of the Dol-P-Man mutants are defective in activation by antigen, suprantigen, and concanavalin A despite normal T-cell receptor expression. Here, we show that the activation defect was due to a pleiotropic glycosylation abnormality because Dol-P-Man is required for both GPI anchor and N-linked oligosaccharide biosynthesis. When the yeast Dol-P-Man synthase gene was stably transfected into the mutants, full expression of surface GPI-anchored proteins was restored. However, N-linked glycosylation was either partially or completely corrected in different transfectants. Reconstitution of activation defects correlates well with the status of N-linked glycosylation, but not with the expression of GPI-anchored proteins. These results thus reveal an unexpected role of N-linked glycosylation in T-cell activation.     

Efficient glycosylphosphatidylinositol (GPI) modification of membrane proteins requires a C-terminal anchoring signal of marginal hydrophobicity

Many plasma membrane proteins are anchored to the membrane via a C-terminal glycosylphosphatidylinositol (GPI) moiety. The GPI anchor is attached to the protein in the endoplasmic reticulum by transamidation, a reaction in which a C-terminal GPI-attachment signal is cleaved off concomitantly with addition of the GPI moiety. GPI-attachment signals are poorly conserved on the sequence level but are all composed of a polar segment that includes the GPI-attachment site followed by a hydrophobic segment located at the very C terminus of the protein. Here, we show that efficient GPI modification requires that the hydrophobicity of the C-terminal segment is "marginal": less hydrophobic than type II transmembrane anchors and more hydrophobic than the most hydrophobic segments found in secreted proteins. We further show that the GPI-attachment signal can be modified by the transamidase irrespective of whether it is first released into the lumen of the endoplasmic reticulum or is retained in the endoplasmic reticulum membrane.     

Early events in glycosylphosphatidylinositol anchor addition. substrate proteins associate with the transamidase subunit gpi8p

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.     

Zinc transport via ZNT5-6 and ZNT7 is critical for cell surface glycosylphosphatidylinositol-anchored protein expression

Glycosylphosphatidylinositol (GPI)-anchored proteins play crucial roles in various enzyme activities, cell signaling and adhesion, and immune responses. While the molecular mechanism underlying GPI-anchored protein biosynthesis has been well studied, the role of zinc transport in this process has not yet been elucidated. Zn transporter (ZNT) proteins mobilize cytosolic zinc to the extracellular space and to intracellular compartments. Here, we report that the early secretory pathway ZNTs (ZNT5–ZNT6 heterodimers [ZNT5-6] and ZNT7–ZNT7 homodimers [ZNT7]), which supply zinc to the lumen of the early secretory pathway compartments are essential for GPI-anchored protein expression on the cell surface. We show, using overexpression and gene disruption/re-expression strategies in cultured human cells, that loss of ZNT5-6 and ZNT7 zinc transport functions results in significant reduction in GPI-anchored protein levels similar to that in mutant cells lacking phosphatidylinositol glycan anchor biosynthesis (PIG) genes. Furthermore, medaka fish with disrupted Znt5 and Znt7 genes show touch-insensitive phenotypes similar to zebrafish Pig mutants. These findings provide a previously unappreciated insight into the regulation of GPI-anchored protein expression and protein quality control in the early secretory pathway.     

Identification of a missing link in glycosylphosphatidylinositol anchor biosynthesis in mammalian cells.

A large number of mammalian proteins are anchored to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. Biosynthetic intermediates of the GPI anchor have been identified in mammalian cells. The early GPI precursors are sensitive to phosphatidylinositol (PI)-specific phospholipase C (PLC). However, all of the later GPI precursors, which contain 1 or more mannose residues, are PI-PLC-resistant, suggesting that there is another unidentified precursor. Here, we report the identification of this missing link. This GPI precursor can only be labeled with glucosamine and inositol, and is resistant to PI-PLC but sensitive to GPI-phospholipase D. It accumulates in large quantity only in mutants which are defective in the addition of the first mannose residue to the elongating GPI core. Thus, fatty acylation of glucosaminylphosphatidylinositol, to render it PI-PLC-resistant, is an obligatory step in the biosynthesis of mammalian GPI anchor precursors.     

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.     

Glycosylphosphatidylinositol-anchored proteins are preferentially targeted to the basolateral surface in Fischer rat thyroid epithelial cells

Glycosylphosphatidylinositol (GPI) acts as an apical targeting signal in MDCK cells and other kidney and intestinal cell lines. In striking contrast with these model polarized cell lines, we show here that Fischer rat thyroid (FRT) epithelial cells do not display a preferential apical distribution of GPI-anchored proteins. Six out of nine detectable endogenous GPI-anchored proteins were localized on the basolateral surface, whereas two others were apical and one was not polarized. Transfection of several model GPI proteins, previously shown to be apically targeted in MDCK cells, also led to unexpected results. While the ectodomain of decay accelerating factor (DAF) was apically secreted, 50% of the native, GPI-anchored form, of this protein was basolateral. Addition of a GPI anchor to the ectodomain of Herpes simplex gD-1, secreted without polarity, led to basolateral localization of the fusion protein, gD1-DAF. Targeting experiments demonstrated that gD1-DAF was delivered vectorially from the Golgi apparatus to the basolateral surface. These results indicate that FRT cells have fundamental differences with MDCK cells with regard to the mechanisms for sorting GPI-anchored proteins: GPI is not an apical signal but, rather, it behaves as a basolateral signal. The "mutant" behavior of FRT cells may provide clues to the nature of the mechanisms that sort GPI-anchored proteins in epithelial cells.     

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.     

Genome-Wide Screening of Genes Required for Glycosylphosphatidylinositol Biosynthesis

Glycosylphosphatidylinositol (GPI) is synthesized and transferred to proteins in the endoplasmic reticulum (ER). GPI-anchored proteins are then transported from the ER to the plasma membrane through the Golgi apparatus. To date, at least 17 steps have been identified to be required for the GPI biosynthetic pathway. Here, we aimed to establish a comprehensive screening method to identify genes involved in GPI biosynthesis using mammalian haploid screens. Human haploid cells were mutagenized by the integration of gene trap vectors into the genome. Mutagenized cells were then treated with a bacterial pore-forming toxin, aerolysin, which binds to GPI-anchored proteins for targeting to the cell membrane. Cells that showed low surface expression of CD59, a GPI-anchored protein, were further enriched for. Gene trap insertion sites in the non-selected population and in the enriched population were determined by deep sequencing. This screening enriched 23 gene regions among the 26 known GPI biosynthetic genes, which when mutated are expected to decrease the surface expression of GPI-anchored proteins. Our results indicate that the forward genetic approach using haploid cells is a useful and powerful technique to identify factors involved in phenotypes of interest.     

GPI-anchored GFP signals Ca2+ but is homogeneously distributed on the cell surface

Glycosyl-phosphatidylinositol (GPI)-anchored proteins are unique in that they penetrate only the outer leaflet of the plasma membrane but are still able to mediate intracellular signalling events following antibody-induced ligation. Detergent solubilisation studies suggest that microdomains exist at the cell surface within which are sequestered GPI-linked proteins. Here we report the construction and expression of a fluorescent GPI anchor on the surface of CHO, EL4, and U937 cells by fusing green fluorescent protein (GFP) to the GPI-attachment site of CD59. The resultant GFP-GPI has properties comparable to that of endogenously expressed GPI-anchored molecules as shown by Triton X-114 partitioning. However, sucrose gradient floatation showed that GFP-GPI was only partially resistant to detergent solubilisation. Furthermore confocal scanning laser microscopy revealed a homogeneous distribution of GFP-GPI at the cell surface, which only became clustered following cross-linking of the GPI anchor via an anti-GFP antibody. Surprisingly, GFP-GPI signalled Ca2+ change upon cross-linking demonstrating its signalling competence. Our results suggest that the GPI-anchor itself does not confer a clustered distribution to molecules but that clustering occurs following ligation with antibody, which allows the protein to become Ca2+ signalling competent.     

Dengue virus nonstructural protein 1 is expressed in a glycosyl-phosphatidylinositol-linked form that is capable of signal transduction.

Dengue virus nonstructural protein 1 (NS1) is expressed on the surface of infected cells and is a target of human antibody responses to dengue virus infection. We show here that dengue virus uses the cellular glycosyl-phosphatidylinositol (GPI) linkage pathway to express a GPI-anchored form of NS1 and that GPI anchoring imparts a capacity for signal transduction in response to binding of NS1-specific antibody. This study is the first to identify GPI linkage of a virus-encoded protein. The GPI anchor addition signal for NS1 was identified, by transfection of HeLa cells with dengue cDNA constructs, as a downstream hydrophobic domain in NS2A. GPI linkage of NS1 in both transfected and infected cells was demonstrated by cleavage of NS1 from the surface by PI-specific phospholipase C and by metabolic incorporation of the GPI-specific components ethanolamine and inositol. In common with other GPI-anchored proteins, addition of specific antibody resulted in signal transduction, as evidenced by tyrosine phosphorylation of cellular proteins. Antibody-induced signal transduction by GPI-linked NS1 suggests a mechanism of cellular activation that may contribute to the pathogenesis of human dengue disease. Signal transduction by a GPI-anchored viral antigen interacting with a specific antibody that it induces is a new concept in the pathogenesis of viral disease.     

An internally positioned signal can direct attachment of a glycophospholipid membrane anchor

All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.     

Retrotranslocation of prion proteins from the endoplasmic reticulum by preventing GPI signal transamidation

Neurodegeneration in diseases caused by altered metabolism of mammalian prion protein (PrP) can be averted by reducing PrP expression. To identify novel pathways for PrP down-regulation, we analyzed cells that had adapted to the negative selection pressure of stable overexpression of a disease-causing PrP mutant. A mutant cell line was isolated that selectively and quantitatively routes wild-type and various mutant PrPs for ER retrotranslocation and proteasomal degradation. Biochemical analyses of the mutant cells revealed that a defect in glycosylphosphatidylinositol (GPI) anchor synthesis leads to an unprocessed GPI-anchoring signal sequence that directs both ER retention and efficient retrotranslocation of PrP. An unprocessed GPI signal was sufficient to impart ER retention, but not retrotranslocation, to a heterologous protein, revealing an unexpected role for the mature domain in the metabolism of misprocessed GPI-anchored proteins. Our results provide new insights into the quality control pathways for unprocessed GPI-anchored proteins and identify transamidation of the GPI signal sequence as a step in PrP biosynthesis that is absolutely required for its surface expression. As each GPI signal sequence is unique, these results also identify signal recognition by the GPI-transamidase as a potential step for selective small molecule perturbation of PrP expression.     

GWT1 gene is required for inositol acylation of glycosylphosphatidylinositol anchors in yeast

Glycosylphosphatidylinositol (GPI) is a conserved post-translational modification to anchor cell surface proteins to plasma membrane in all eukaryotes. In yeast, GPI mediates cross-linking of cell wall mannoproteins to beta1,6-glucan. We reported previously that the GWT1 gene product is a target of the novel anti-fungal compound, 1-[4-butylbenzyl]isoquinoline, that inhibits cell wall localization of GPI-anchored mannoproteins in Saccharomyces cerevisiae (Tsukahara, K., Hata, K., Sagane, K., Watanabe, N., Kuromitsu, J., Kai, J., Tsuchiya, M., Ohba, F., Jigami, Y., Yoshimatsu, K., and Nagasu, T. (2003) Mol. Microbiol. 48, 1029-1042). In the present study, to analyze the function of the Gwt1 protein, we isolated temperature-sensitive gwt1 mutants. The gwt1 cells were normal in transport of invertase and carboxypeptidase Y but were delayed in transport of GPI-anchored protein, Gas1p, and were defective in its maturation from the endoplasmic reticulum to the Golgi. The incorporation of inositol into GPI-anchored proteins was reduced in gwt1 mutant, indicating involvement of GWT1 in GPI biosynthesis. We analyzed the early steps of GPI biosynthesis in vitro by using membranes prepared from gwt1 and Deltagwt1 cells. The synthetic activity of GlcN-(acyl)PI from GlcN-PI was defective in these cells, whereas Deltagwt1 cells harboring GWT1 gene restored the activity, indicating that GWT1 is required for acylation of inositol during the GPI synthetic pathway. We further cloned GWT1 homologues in other yeasts, Cryptococcus neoformans and Schizosaccharomyces pombe, and confirmed that the specificity of acyl-CoA in inositol acylation, as reported in studies of endogenous membranes (Franzot, S. P., and Doering, T. L. (1999) Biochem. J. 340, 25-32), is due to the properties of Gwt1p itself and not to other membrane components.