PIG-S and PIG-T, essential for GPI anchor attachment to proteins, form a complex with GAA1 and GPI8

Many eukaryotic cell surface proteins are anchored to the plasma membrane via glycosylphosphatidylinositol (GPI). The GPI transamidase mediates GPI anchoring in the endoplasmic reticulum, by replacing a protein's C-terminal GPI attachment signal peptide with a pre-assembled GPI. During this transamidation reaction, the GPI transamidase forms a carbonyl intermediate with a substrate protein. It was known that the GPI transamidase is a complex containing GAA1 and GPI8. Here, we report two new components of this enzyme: PIG-S and PIG-T. To determine roles for PIG-S and PIG-T, we disrupted these genes in mouse F9 cells by homologous recombination. PIG-S and PIG-T knockout cells were defective in transfer of GPI to proteins, particularly in formation of the carbonyl intermediates. We also demonstrate that PIG-S and PIG-T form a protein complex with GAA1 and GPI8, and that PIG-T maintains the complex by stabilizing the expression of GAA1 and GPI8. Saccharomyces cerevisiae Gpi16p (YHR188C) and Gpi17p (YDR434W) are orthologues of PIG-T and PIG-S, respectively.     

Gaa1p and gpi8p are components of a glycosylphosphatidylinositol (GPI) transamidase that mediates attachment of GPI to proteins

Many eukaryotic cell surface proteins are anchored to the membrane via glycosylphosphatidylinositol (GPI). The GPI is attached to proteins that have a GPI attachment signal peptide at the carboxyl terminus. The GPI attachment signal peptide is replaced by a preassembled GPI in the endoplasmic reticulum by a transamidation reaction through the formation of a carbonyl intermediate. GPI transamidase is a key enzyme of this posttranslational modification. Here we report that Gaa1p and Gpi8p are components of a GPI transamidase. To determine a role of Gaa1p we disrupted a GAA1/GPAA1 gene in mouse F9 cells by homologous recombination. GAA1 knockout cells were defective in the formation of carbonyl intermediates between precursor proteins and transamidase as determined by an in vitro GPI-anchoring assay. We also show that cysteine and histidine residues of Gpi8p, which are conserved in members of a cysteine protease family, are essential for generation of a carbonyl intermediate. This result suggests that Gpi8p is a catalytic component that cleaves the GPI attachment signal peptide. Moreover, Gaa1p and Gpi8p are associated with each other. Therefore, Gaa1p and Gpi8p constitute a GPI transamidase and cooperate in generating a carbonyl intermediate, a prerequisite for GPI attachment.     

New mutant Chinese hamster ovary cell representing an unknown gene for attachment of glycosylphosphatidylinositol to proteins

Aerolysin, a secreted bacterial toxin from Aeromonas hydrophila, binds to glycosylphosphatidylinositol (GPI)-anchored protein and kills the cells by forming pores. Both GPI and N-glycan moieties of GPI-anchored proteins are involved in efficient binding of aerolysin. We isolated various Chinese hamster ovary (CHO) mutant cells resistant to aerolysin. Among them, CHOPA41.3 mutant cells showed several-fold decreased expression of GPI-anchored proteins. After transfection of N-acetylglucosamine transferase I (GnT1) cDNA, aerolysin was efficiently bound to the cells, indicating that the resistance against aerolysin in this cells was mainly ascribed to the defect of N-glycan maturation. CHOPA41.3 cells also accumulated GPI intermediates lacking ethanolamine phosphate modification on the first mannose. After stable transfection of PIG-N cDNA encoding GPI-ethanolamine phosphate transferase1, a profile of accumulated GPI intermediates became similar to that of GPI transamidase mutant cells. It indicated, therefore, that CHOPA41.3 cells are defective in GnT1, ethanolamine phosphate modification of the first mannose, and attachment of GPI to proteins. The GPI accumulation in CHOPA41.3 cells carrying PIG-N cDNA was not normalized after transfection with cDNAs of all known components in GPI transamidase complex. Microsomes from CHOPA41.3 cells had normal GPI transamidase activity. Taken together, there is an unknown gene required for efficient attachment of GPI to proteins.     

Transamidase subunit GAA1/GPAA1 is a M28 family metallo-peptide-synthetase that catalyzes the peptide bond formation between the substrate protein’s omega-site and the GPI lipid anchor’s phosphoethanolamine

The transamidase subunit GAA1/GPAA1 is predicted to be the enzyme that catalyzes the attachment of the glycosylphosphatidyl (GPI) lipid anchor to the carbonyl intermediate of the substrate protein at the ω-site. Its ~300-amino acid residue lumenal domain is a M28 family metallo-peptide-synthetase with an α/β hydrolase fold, including a central 8-strand β-sheet and a single metal (most likely zinc) ion coordinated by 3 conserved polar residues. Phosphoethanolamine is used as an adaptor to make the non-peptide GPI lipid anchor look chemically similar to the N terminus of a peptide.     

Human PIG-U and yeast Cdc91p are the fifth subunit of GPI transamidase that attaches GPI-anchors to proteins

Many eukaryotic proteins are anchored to the cell surface via glycosylphosphatidylinositol (GPI), which is posttranslationally attached to the carboxyl-terminus by GPI transamidase. The mammalian GPI transamidase is a complex of at least four subunits, GPI8, GAA1, PIG-S, and PIG-T. Here, we report Chinese hamster ovary cells representing a new complementation group of GPI-anchored protein-deficient mutants, class U. The class U cells accumulated mature and immature GPI and did not have in vitro GPI transamidase activity. We cloned the gene responsible, termed PIG-U, that encoded a 435-amino-acid hydrophobic protein. The GPI transamidase complex affinity-purified from cells expressing epitope-tagged-GPI8 contained PIG-U and four other known components. Cells lacking PIG-U formed complexes of the four other components normally but had no ability to cleave the GPI attachment signal peptide. Saccharomyces cerevisiae Cdc91p, with 28% amino acid identity to PIG-U, partially restored GPI-anchored proteins on the surface of class U cells. PIG-U and Cdc91p have a functionally important short region with similarity to a region conserved in long-chain fatty acid elongases. Taken together, PIG-U and the yeast orthologue Cdc91p are the fifth component of GPI transamidase that may be involved in the recognition of either the GPI attachment signal or the lipid portion of GPI.     

Cloning of murine glycosyl phosphatidylinositol anchor attachment protein, GPAA1

Glycosyl phosphatidylinositols (GPIs) are usedto anchor many proteins to the cell surface membrane and are utilizedin all eukaryotic cells. GPI anchoring units are attached to proteins via a transamidase reaction mediated by a GPI transamidase complex. Weisolated one of the components of this complex,mGPAA1 (murine GPI anchor attachment), by the signalsequence trap method. mGPAA1 cDNA is about 2 kb in lengthand encodes a putative 621 amino acid protein. The mGPAA1gene has 12 small exons and 11 small introns. mGPAA1 mRNA isubiquitously expressed in mammalian cells, and in situ hybridizationanalysis revealed that it is abundant in the choroid plexus, skeletalmuscle, osteoblasts of rib, and occipital bone in mouse embryos. Itsexpression levels and transamidation efficiency decreased withdifferentiation of embryonic stem cells. The 3T3 cell lines expressingantisense mGPAA1 failed to express GPI-anchored proteins onthe cell surface membrane.

     

Two subunits of glycosylphosphatidylinositol transamidase,GPI8 and PIG-T,form a functionally important intermolecular disulfide bridge

Many eukaryotic proteins are tethered to the plasma membrane via glycosylphosphatidylinositol (GPI). GPI transamidase is localized in the endoplasmic reticulum and mediates post-translational transfer of preformed GPI to proteins bearing a carboxyl-terminal GPI attachment signal. Mammalian GPI transamidase is a multimeric complex consisting of at least five subunits. Here we report that two subunits of mammalian GPI transamidase, GPI8 and PIG-T, form a functionally important disulfide bond between conserved cysteine residues. GPI8 and PIG-T mutants in which relevant cysteines were replaced with serines were unable to fully restore the surface expression of GPI-anchored proteins upon transfection into their respective mutant cells. Microsomal membranes of these transfectants had markedly decreased activities in an in vitro transamidase assay. The formation of this disulfide bond is not essential but required for full transamidase activity. Antibodies against GPI8 and PIG-T revealed that endogenous as well as exogenous proteins formed a disulfide bond. Furthermore trypanosome GPI8 forms a similar intermolecular disulfide bond via its conserved cysteine residue, suggesting that the trypanosome GPI transamidase is also a multimeric complex likely containing the orthologue of PIG-T. We also demonstrate that an inactive human GPI transamidase complex that consists of non-functional GPI8 and four other components was co-purified with the proform of substrate proteins, indicating that these five components are sufficient to hold the substrate proteins.     

TbGPI16 is an essential component of GPI transamidase in Trypanosoma brucei

Glycosylphosphatidylinositol (GPI) is widely used by eukaryotic cell surface proteins for membrane attachment. De novo synthesized GPI precursors are attached to proteins post-translationally by the enzyme complex, GPI transamidase. TbGPI16, a component of the trypanosome transamidase, shares similarity with human PIG-T. Here, we show that TbGPI16 is the orthologue of PIG-T and an essential component of GPI transamidase by creating a TbGPI16 knockout. TbGPI16 forms a disulfide-linked complex with TbGPI8. A cysteine to serine mutant of TbGPI16 was unable to fully restore the surface expression of GPI-anchored proteins upon transfection into the knockout cells, indicating that its disulfide linkage with TbGPI8 is important for the full transamidase activity.     

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.     

Yeast Gpi8p is essential for GPI anchor attachment onto proteins.

Glycosylphosphatidylinositol (GPI) anchors are added onto newly synthesized proteins in the ER. Thereby a putative transamidase removes a C-terminal peptide and attaches the truncated protein to the free amino group of the preformed GPI. The yeast mutant gpi8-1 is deficient in this addition of GPIs to proteins. GPI8 encodes for an essential 47 kDa type I membrane glycoprotein residing on the luminal side of the ER membrane. GPI8 shows significant homology to a novel family of vacuolar plant endopeptidases one of which is supposed to catalyse a transamidation step in the maturation of concanavalin A and acts as a transamidase in vitro. Humans have a gene which is highly homologous to GPI8 and can functionally replace it.     

Glycosylphosphatidylinositol lipid anchoring of plant proteins. Sensitive prediction from sequence- and genome-wide studies for Arabidopsis and rice

Posttranslational glycosylphosphatidylinositol (GPI) lipid anchoring is common not only for animal and fungal but also for plant proteins. The attachment of the GPI moiety to the carboxyl-terminus after proteolytic cleavage of a C-terminal propeptide is performed by the transamidase complex. Its four known subunits also have obvious full-length orthologs in the Arabidopsis and rice (Oryza sativa) genomes; thus, the mechanism of substrate protein processing appears similar for all eukaryotes. A learning set of plant proteins (substrates for the transamidase complex) has been collected both from the literature and plant sequence databases. We find that the plant GPI lipid anchor motif differs in minor aspects from the animal signal (e.g. the plant hydrophobic tail region can contain a higher fraction of aromatic residues). We have developed the "big-Pi plant" program for prediction of compatibility of query protein C-termini with the plant GPI lipid anchor motif requirements. Validation tests show that the sensitivity for transamidase targets is approximately 94%, and the rate of false positive prediction is about 0.1%. Thus, the big-Pi predictor can be applied as unsupervised genome annotation and target selection tool. The program is also suited for the design of modified protein constructs to test their GPI lipid anchoring capacity. The big-Pi plant predictor Web server and lists of potential plant precursor proteins in Swiss-Prot, SPTrEMBL, Arabidopsis, and rice proteomes are available at http://mendel.imp.univie.ac.at/gpi/plants/gpi_plants.html. Arabidopsis and rice protein hits have been functionally classified. Several GPI lipid-anchored arabinogalactan-related proteins have been identified in rice.     

Analysis of the signal for attachment of a glycophospholipid membrane anchor

The COOH terminus of decay accelerating factor (DAF) contains a signal that directs attachment of a glycophospholipid (GPI) membrane anchor. To define this signal we deleted portions of the DAF COOH terminus and expressed the mutant cDNAs it CV1 origin-deficient SV-40 cells. Our results show that the COOH-terminal hydrophobic domain (17 residues) is absolutely required for GPI anchor attachment. However, when fused to the COOH terminus of a secreted protein this hydrophobic domain is insufficient to direct attachment of a GPI anchor. Additional specific information located within the adjacent 20 residues appears to be necessary. We speculate that by analogy with signal sequences for membrane translocation, GPI anchor attachment requires both a COOH-terminal hydrophobic domain (the GPI signal) as well as a suitable cleavage/attachment site located NH2 terminal to the signal.     

Leishmania mexicana mutants lacking glycosylphosphatidylinositol (GPI):protein transamidase provide insights into the biosynthesis and functions of GPI-anchored proteins

The major surface proteins of the parasitic protozoon Leishmania mexicana are anchored to the plasma membrane by glycosylphosphatidylinositol (GPI) anchors. We have cloned the L. mexicana GPI8 gene that encodes the catalytic component of the GPI:protein transamidase complex that adds GPI anchors to nascent cell surface proteins in the endoplasmic reticulum. Mutants lacking GPI8 (DeltaGPI8) do not express detectable levels of GPI-anchored proteins and accumulate two putative protein-anchor precursors. However, the synthesis and cellular levels of other non-protein-linked GPIs, including lipophosphoglycan and a major class of free GPIs, are not affected in the DeltaGPI8 mutant. Significantly, the DeltaGPI8 mutant displays normal growth in liquid culture, is capable of differentiating into replicating amastigotes within macrophages in vitro, and is infective to mice. These data suggest that GPI-anchored surface proteins are not essential to L. mexicana for its entry into and survival within mammalian host cells in vitro or in vivo and provide further support for the notion that free GPIs are essential for parasite growth.     

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

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

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

Post-translational GPI lipid anchor modification of proteins in kingdoms of life: analysis of protein sequence data from complete genomes

To investigate the occurrence of glycosylphosphatidylinositol (GPI) lipid anchor modification in various taxonomic ranges, potential substrate proteins have been searched for in completely sequenced genomes. We applied the big-pi predictor for the recognition of propeptide cleavage and anchor attachment sites with a new, generalized analytical form of the extreme-value distribution for evaluating false-positive prediction rates. (i) We find that GPI modification is present among lower and higher Eukaryota (approximately 0.5% of all proteins) but it seems absent in all eubacterial and three archaeobacterial species studied. Four other archaean genomes appear to encode such a fraction of substrate proteins (in the range of eukaryots) that they cannot be explained as false-positive predictions. This result supports the possible existence of GPI anchor modification in an archaean subgroup. (ii) The frequency of GPI-modified proteins on various chromosomes of a given eukaryotic species is different. (iii) Lists of potentially GPI-modified proteins in complete genomes with their predicted cleavage sites are available at http://mendel.imp.univie.ac.at/gpi/gpi_genomes.html. (iv) Orthologues of known transamidase subunits have been found only for EUKARYA: Inconsistencies in domain structure among homologues some of which may indicate sequencing errors are described. We present a refined model of the transamidase complex.     

PER1 is required for GPI-phospholipase A2 activity and involved in lipid remodeling of GPI-anchored proteins

Glycosylphoshatidylinositol (GPI) anchors are remodeled during their transport to the cell surface. Newly synthesized proteins are transferred to a GPI anchor, consisting of diacylglycerol with conventional C16 and C18 fatty acids, whereas the lipid moiety in mature GPI-anchored proteins is exchanged to either diacylglycerol containing a C26:0 fatty acid in the sn-2 position or ceramide in Saccharomyces cerevisiae. Here, we report on PER1, a gene encoding a protein that is required for the GPI remodeling pathway. We found that GPI-anchored proteins could not associate with the detergent-resistant membranes in per1Delta cells. In addition, the mutant cells had a defect in the lipid remodeling from normal phosphatidylinositol (PI) to a C26 fatty acid-containing PI in the GPI anchor. In vitro analysis showed that PER1 is required for the production of lyso-GPI, suggesting that Per1p possesses or regulates the GPI-phospholipase A2 activity. We also found that human PERLD1 is a functional homologue of PER1. Our results demonstrate for the first time that PER1 encodes an evolutionary conserved component of the GPI anchor remodeling pathway, highlighting the close connection between the lipid remodeling of GPI and raft association of GPI-anchored proteins.     

Comparative efficiencies of C‐terminal signals of native glycophosphatidylinositol (GPI)‐anchored proproteins in conferring GPI‐anchoring

Every protein fated to receive the glycophosphatidylinositol (GPI) anchor post‐translational modification has a C‐terminal GPI‐anchor attachment signal sequence. This signal peptide varies with respect to length, content, and hydrophobicity. With the exception of predictions based on an upstream amino acid triplet termed ω→ω + 2 which designates the site of GPI uptake, there is no information on how the efficiencies of different native signal sequences compare in the transamidation reaction that catalyzes the substitution of the GPI anchor for the C‐terminal peptide. In this study we utilized the placental alkaline phosphatase (PLAP) minigene, miniPLAP, and replaced its native 3′ end‐sequence encoding ω‐2 to the C‐terminus with the corresponding C‐terminal sequences of nine other human GPI‐anchored proteins. The resulting chimeras then were fed into an in vitro processing microsomal system where the cleavages leading to mature product from the nascent preproprotein could be followed by resolution on an SDS–PAGE system after immunoprecipitation. The results showed that the native signal of each protein differed markedly with respect to transamidation efficiency, with the signals of three proteins out‐performing the others in GPI‐anchor addition and those of two proteins being poorer substrates for the GPI transamidase. The data additionally indicated that the hierarchical order of efficiency of transamidation did not depend solely on the combination of permissible residues at ω→ω + 2. J. Cell. Biochem. 84: 68–83, 2002. © 2001 Wiley‐Liss, Inc.     

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.     

Processing and trafficking of Leishmania mexicana GP63. Analysis using GP18 mutants deficient in glycosylphosphatidylinositol protein anchoring

GPI8 is a clan CD, family C13 cysteine protease and the catalytic core of the GPI-protein transamidase complex. In Leishmania mexicana, GPI8 is nonessential, and Deltagpi8 mutants lack the GPI-anchored metalloprotease GP63, which is the major surface protein of promastigotes. We have identified the active site histidine and cysteine residues of leishmanial GPI8 and generated Deltagpi8 lines expressing modified GPI8 proteins. This has allowed us to study the processing and trafficking of GP63 in wild type and Deltagpi8 mutants. We show using pulse-chase labeling that in Deltagpi8 non-GPI-anchored GP63 was glycosylated and secreted without further processing from the cell with a t(12) of 120 min. This secretion was prevented by growth of cells in the presence of tunicamycin, indicating that glycosylation is necessary for secretion of non-GPI-anchored proteins. In contrast, in wild type cells the majority of GP63 was rapidly glycosylated, GPI-anchored, and trafficked to the surface with defined processing intermediate forms. Tunicamycin inhibited glycosylation but did not prevent GPI anchor addition or trafficking. These results show that GPI-anchored and unanchored GP63 are trafficked via different pathways. In addition, the balance between GPI anchor addition and secretion of GP63 in Leishmania can vary depending on the activity of the GPI-protein transamidase, which has implications for the host-parasite interaction.