Deficiencies in the endoplasmic reticulum (ER)-membrane protein Gab1p perturb transfer of glycosylphosphatidylinositol to proteins and cause perinuclear ER-associated actin bar formation

The essential GAB1 gene, which encodes an endoplasmic reticulum (ER)-membrane protein, was identified in a screen for mutants defective in cellular morphogenesis. A temperature-sensitive gab1 mutant accumulates complete glycosylphosphatidylinositol (GPI) precursors, and its temperature sensitivity is suppressed differentially by overexpression of different subunits of the GPI transamidase, from strong suppression by Gpi8p and Gpi17p, to weak suppression by Gaa1p, and to no suppression by Gpi16p. In addition, both Gab1p and Gpi17p localize to the ER and are in the same protein complex in vivo. These findings suggest that Gab1p is a subunit of the GPI transamidase with distinct relationships to other subunits in the same complex. We also show that depletion of Gab1p or Gpi8p, but not Gpi17p, Gpi16p, or Gaa1p causes accumulation of cofilin-decorated actin bars that are closely associated with the perinuclear ER, which highlights a functional interaction between the ER network and the actin cytoskeleton.     

The GPI transamidase complex of Saccharomyces cerevisiae contains Gaa1p, Gpi8p, and Gpi16p

Gpi8p and Gaa1p are essential components of the GPI transamidase that adds glycosylphosphatidylinositols (GPIs) to newly synthesized proteins. After solubilization in 1.5% digitonin and separation by blue native PAGE, Gpi8p is found in 430-650-kDa protein complexes. These complexes can be affinity purified and are shown to consist of Gaa1p, Gpi8p, and Gpi16p (YHR188c). Gpi16p is an essential N-glycosylated transmembrane glycoprotein. Its bulk resides on the lumenal side of the ER, and it has a single C-terminal transmembrane domain and a small C-terminal, cytosolic extension with an ER retrieval motif. Depletion of Gpi16p results in the accumulation of the complete GPI lipid CP2 and of unprocessed GPI precursor proteins. Gpi8p and Gpi16p are unstable if either of them is removed by depletion. Similarly, when Gpi8p is overexpressed, it largely remains outside the 430-650-kDa transamidase complex and is unstable. Overexpression of Gpi8p cannot compensate for the lack of Gpi16p. Homologues of Gpi16p are found in all eucaryotes. The transamidase complex is not associated with the Sec61p complex and oligosaccharyltransferase complex required for ER insertion and N-glycosylation of GPI proteins, respectively. When GPI precursor proteins or GPI lipids are depleted, the transamidase complex remains intact.     

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.     

A conserved proline in the last transmembrane segment of Gaa1 is required for glycosylphosphatidylinositol (GPI) recognition by GPI transamidase

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized as precursor proteins that are processed in the endoplasmic reticulum by GPI transamidase (GPIT). Human GPIT is a multisubunit membrane-bound protein complex consisting of Gaa1, Gpi8, phosphatidylinositol glycan (PIG)-S, PIG-T, and PIG-U. The enzyme recognizes a C-terminal signal sequence in the proprotein and replaces it with a preformed GPI lipid. The nature of the functional interaction of the GPIT subunits with each other and with the proprotein and GPI substrates is largely unknown. We recently analyzed the GPIT subunit Gaa1, a polytopic protein with seven transmembrane (TM) spans, to identify sequence determinants in the protein that are required for its interaction with other subunits and for function (Vainauskas, S., Maeda, Y., Kurniawan, H., Kinoshita, T., and Menon, A. K. (2002) J. Biol. Chem. 277, 30535-30542). We showed that elimination of the C-terminal TM segment of Gaa1 allows the protein to interact with Gpi8, PIG-S, and PIG-T but renders the resulting GPIT complex nonfunctional. We now show that GPIT complexes containing C-terminally truncated Gaa1 possess a full complement of subunits and are able to interact with a proprotein substrate but cannot co-immunoprecipitate GPI. We go on to show that mutation of a conserved proline residue centrally located within the C-terminal TM span of Gaa1 is sufficient to abrogate the ability of the resulting GPIT complex to co-immunoprecipitate GPI. We suggest that the putative dynamic hinge created by the proline residue provides a structural basis for the interaction of GPI with GPIT.     

Endoplasmic reticulum proteins involved in glycosylphosphatidylinositol-anchor attachment: photocrosslinking studies in a cell-free system.

Assembly of glycosylphosphatidylinositol (GPtdIns)-anchored proteins requires translocation of the nascent polypeptide chain across the endoplasmic reticulum (ER) membrane and replacement of the C-terminal signal sequence with a GPtdIns moiety. The anchoring reaction is carried out by an ER enzyme, GPtdIns transamidase. Genetic studies with yeast indicate that the transamidase consists of a dynamic complex of at least two subunits, Gaa1p and Gpi8p. To study the GPtdIns-anchoring reaction, we used a small reporter protein that becomes GPtdIns-anchored when the corresponding mRNA is translated in the presence of microsomes, in conjunction with site-specific photocrosslinking to identify ER membrane components that are proximal to the reporter during its conversion to a GPtdIns-anchored protein. We generated variants of the reporter protein such that upon in vitro translation in the presence of Nepsilon-(5-azido-2-nitrobenzoyl)-lysyl-tRNA, photoreactive lysine residues would be incorporated in the protein specifically near the GPtdIns-attachment site. We analyzed photoadducts resulting from UV irradiation of the samples. We show that proproteins can be crosslinked to the transamidase subunit Gpi8p, as well as to ER proteins of molecular mass approximately 60 kDa, approximately 70 kDa, and approximately 120 kDa. The identification of a photoadduct between a proprotein and Gpi8p provides the first direct evidence of an interaction between a proprotein substrate and one of the genetically identified transamidase subunits. The approximately 70-kDa protein that we identified may correspond to the other subunit Gaa1p, while the other proteins possibly represent additional, hitherto unidentified subunits of the mammalian GPtdIns transamidase complex.     

Gpi17p does not stably interact with other subunits of glycosylphosphatidylinositol transamidase in Saccharomyces cerevisiae

Homologues of Gpi8p, Gaa1p, Gpi16p, Gpi17p, and Cdc91p are essential components of the GPI transamidase complex that adds glycosylphosphatidylinositols (GPIs 1) to newly synthesized proteins in the ER. In mammalian cells, these five subunits remain stably associated with each other in detergent. In yeast, we find no stable stoichiometric association of Gpi17p with the Gpi8p-Gpi16p-Gaa1p core in detergent extracts. Random and site-directed mutagenesis generated mutations in several highly conserved amino acids but did not yield nonfunctional alleles of Gpi17p and a saturating screen did not yield any dominant negative alleles of Gpi17p. Moreover, Gpi8p becomes unstable when any one of the other subunits is depleted, whereas Gpi17p is slightly affected only by the depletion of Gaa1p. These data suggest that yeast Gpi17p may be able to exert its GPI anchoring function without interacting in a stable and continuous manner with the other GPI-transamidase subunits. Shutting down ER-associated and vacuolar protein degradation pathways has no effect on the levels of Gpi17p or other transamidase subunits.     

The glycosylphosphatidylinositol (GPI) signal sequence of human placental alkaline phosphatase is not recognized by human Gpi8p in the context of the yeast GPI anchoring machinery

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins involves the action of a GPI trans-amidase, which replaces the C-terminal GPI signal sequence (GPI-SS) of the primary translation product with a preformed GPI lipid. The transamidation depends on a complex of four proteins, Gaa1p, Gpi8p, Gpi16p and Gpi17p. Although the GPI anchoring pathway is conserved throughout the eukaryotic kingdom, it has been reported recently that the GPI-SS of human placental alkaline phosphatase (hPLAP) is not recognized by the yeast transamidase, but is recognized in yeast that contain the human Gpi8p homologue. This finding suggests that Gpi8p is intimately involved in the recognition of GPI precursor proteins and may also be responsible for the subtle taxon-specific differences in transamidase specificity that sometimes prevent the efficient GPI anchoring of heterologously expressed GPI proteins. Here, we confirm that the GPI signal sequence of hPLAP is indeed not recognized by the yeast GPI-anchoring machinery. However, in our hands, GPI attachment cannot be restored by the co-expression of human Gpi8p in yeast cells under any circumstances.     

Truncation of the caspase-related subunit (Gpi8p) of Saccharomyces cerevisiae GPI transamidase: dimerization revealed

Eukaryotic proteins can be post-translationally modified with a glycosylphosphatidylinositol (GPI) membrane anchor. This modification reaction is catalyzed by GPI transamidase (GPI-T), a multimeric, membrane-bound enzyme. Gpi8p, an essential component of GPI-T, shares low sequence similarity with caspases and contains all or part of the enzyme's active site [U. Meyer, M. Benghezal, I. Imhof, A. Conzelmann, Biochemistry 39 (2000) 3461-3471]. Structural predictions suggest that the soluble portion of Gpi8p is divided into two domains: a caspase-like domain that contains the active site machinery and a second, smaller domain of unknown function. Based on these predictions, we evaluated a soluble truncation of Gpi8p (Gpi8(23-306)). Dimerization was investigated due to the known proclivity of caspases to homodimerize; a Gpi8(23-306) homodimer was detected by native gel and confirmed by mass spectrometry and N-terminal sequencing. Mutations at the putative caspase-like dimerization interface disrupted dimer formation. When combined, these results demonstrate an organizational similarity between Gpi8p and caspases.     

Structural requirements for the recruitment of Gaa1 into a functional glycosylphosphatidylinositol transamidase complex

Glycosylphosphatidylinositol (GPI)-anchored proteins are synthesized on membrane-bound ribosomes, translocated across the endoplasmic reticulum membrane, and GPI-anchored by GPI transamidase (GPIT). GPIT is a minimally heterotetrameric membrane protein complex composed of Gaa1, Gpi8, PIG-S and PIG-T. We describe structure-function analyses of Gaa1, the most hydrophobic of the GPIT subunits, with the aim of assigning a functional role to the different sequence domains of the protein. We generated epitope-tagged Gaa1 mutants and analyzed their membrane topology, subcellular distribution, complex-forming capability, and ability to restore GPIT activity in Gaa1-deficient cells. We show that (i) detergent-extracted, Gaa1-containing GPIT complexes sediment unexpectedly rapidly at approximately 17 S, (ii) Gaa1 is an endoplasmic reticulum-localized membrane glycoprotein with a cytoplasmically oriented N terminus and a lumenally oriented C terminus, (iii) elimination of C-terminal transmembrane segments allows Gaa1 to interact with other GPIT subunits but renders the resulting GPIT complex nonfunctional, (iv) interaction between Gaa1 and other GPIT subunits occurs via the large lumenal domain of Gaa1 located between the first and second transmembrane segments, and (v) the cytoplasmic N terminus of Gaa1 is not required for formation of a functional GPIT complex but may act as a membrane-sorting determinant directing Gaa1 and associated GPIT subunits to an endoplasmic reticulum membrane domain.     

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.     

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.     

Active site determination of Gpi8p, a caspase-related enzyme required for glycosylphosphatidylinositol anchor addition to proteins

Glycosylphosphatidylinositol (GPI) anchors are attached to newly synthesized proteins in the ER by a transamidation reaction during which a C-terminal GPI attachment signal is replaced by a preformed GPI precursor lipid. This reaction depends on GAA1 and GPI8, the latter belonging to a novel cysteine protease family. Homologies between this family and other Cys proteinases, such as caspases, pointed to Cys199 and His157 as potential active site residues. Indeed, gpi8 alleles mutated at Cys199 or His157 are nonfunctional, i.e., they are unable to suppress the lethality of Deltagpi8 mutants. The overexpression of these nonfunctional alleles in wild-type cells leads to the accumulation of the free GPI precursor lipid CP2, delays the maturation of the GPI protein Gas1p, and arrests cell growth. The dominant negative effect of the Cys199 mutant cannot be overcome by the simultaneous overexpression of Gaa1p. Most GPI8 alleles mutated in other conserved regions of the protein can complement the growth defect of Deltagpi8, but nevertheless accumulate CP2. CP2 accumulation, a delay in Gas1p maturation and a slowing of cell growth can also be observed when Gpi8p is depleted to 50% of its normal level in wild-type cells. The dominant negative effect of nonfunctional and partially functional mutant alleles can best be explained by assuming that Gpi8p works as part of a homo- or heteropolymeric complex.     

Yeast Gaa1p is required for attachment of a completed GPI anchor onto proteins

Anchoring of proteins to membranes by glycosylphosphatidylinositols (GPIs) is ubiquitous among all eukaryotes and heavily used by parasitic protozoa. GPI is synthesized and transferred en bloc to form GPI- anchored proteins. The key enzyme in this process is a putative GPI:protein transamidase that would cleave a peptide bond near the COOH terminus of the protein and attach the GPI by an amide linkage. We have identified a gene, GAA1, encoding an essential ER protein required for GPI anchoring. gaal mutant cells synthesize the complete GPI anchor precursor at nonpermissive temperatures, but do not attach it to proteins. Overexpression of GAA1 improves the ability of cells to attach anchors to a GPI-anchored protein with a mutant anchor attachment site. Therefore, Gaa1p is required for a terminal step of GPI anchor attachment and could be part of the putative GPI:protein transamidase.     

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.     

Phosphatidylinositol glycan (PI-G) anchored membrane proteins. Amino acid requirements adjacent to the site of cleavage and PI-G attachment in the COOH-terminal signal peptide.

Secreted proteins are processed from a nascent form that contains an NH2-terminal signal peptide. During processing, the latter is cleaved by a specific NH2-terminal signal peptidase. The nascent form of phosphatidylinositol glycan (PI-G) tailed proteins contain both an NH2- and a COOH-terminal signal peptide. The two signal peptides have much in common, such as size and hydrophobicity. The COOH-terminal peptide is also cleaved during processing. We propose that the amino acid in a nascent protein that ultimately combines with the PI-G moiety be designated the omega site. Amino acids adjacent and COOH-terminal to the omega site would then be omega + 1, omega + 2, etc. In previous studies, we showed that allowable substitutions at the omega site of an engineered form of placental alkaline phosphatase (miniPLAP) are limited to 6 small amino acids. In the present study, mutations were made at the omega + 1 and omega + 2 sites. At the omega + 1 site, processing to varying degrees was observed with 8 of the 9 amino acids substituted for alanine, the normal constituent. Only the proline mutant showed no processing. By contrast, the only substituents permitted at the omega + 2 site were glycine and alanine, with only trace activity observed with serine and cysteine. Thus, just as there is a -1, -3 rule for predicting cleavage by NH2-terminal signal peptidase, there appears to be a comparable omega, omega + 2 rule for predicting cleavage/PI-G addition by COOH-terminal signal transamidase.     

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 omega-->omega + 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 omega-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 omega-->omega + 2.     

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.     

Low-resolution structure of the soluble domain GPAA1 (yGPAA170–247) of the glycosylphosphatidylinositol transamidase subunit GPAA1 from Saccharomyces cerevisiae

The GPI (glycosylphosphatidylinositol) transamidase complex catalyses the attachment of GPI anchors to eukaryotic proteins in the lumen of ER (endoplasmic reticulum). The Saccharomyces cerevisiae GPI transamidase complex consists of the subunits yPIG-K (Gpi8p), yPIG-S (Gpi17p), yPIG-T (Gpi16p), yPIG-U (CDC91/GAB1) and yGPAA1. We present the production of the two recombinant proteins yGPAA1^70–247 and yGPAA1^70–339 of the luminal domain of S. cerevisiae GPAA1, covering the amino acids 70–247 and 70–339 respectively. The secondary structural content of the stable and monodisperse yGPAA1^70–247 has been determined to be 28% α-helix and 27% β-sheet. SAXS (small-angle X-ray scattering) data showed that yGPAA1^70–247 has an R_g (radius of gyration) of 2.72±0.025 nm and D_max (maximum dimension) of 9.14 nm. These data enabled the determination of the two domain low-resolution solution structure of yGPAA1^70–247. The large elliptical shape of yGPAA1^70–247 is connected via a short stalk to the smaller hook-like domain of 0.8 nm in length and 3.5 nm in width. The topological arrangement of yGPAA1^70–247 will be discussed together with the recently determined low-resolution structures of yPIG-K^24–337 and yPIG-S^38–467 from S. cerevisiae in the GPI transamidase complex.     

Biosynthesis of glycosylphosphatidylinositol (GPI)-anchored membrane proteins in intact cells: specific amino acid requirements adjacent to the site of cleavage and GPI attachment

Mutational studies were previously carried out at the omega site intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the omega + 1 and omega + 2 sites in a cell- free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidyl-inositol (GPI)-anchored form. We have now mutated the omega + 1 and omega + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the omega + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the omega + 1 site was promiscuous, with only proline being inactive. These and the previous studies indicate that the omega and omega + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.     

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.