Inositol Deacylation of Glycosylphosphatidylinositol-anchored Proteins Is Mediated by Mammalian PGAP1 and Yeast Bst1p

Abstract

The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis. The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins. However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment. Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear. Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositol-specific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1. PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif. Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase. The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER. Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi. The inositol moiety of mammalian glycosylphosphatidylinositol (GPI) is acylated at an early step in GPI biosynthesis. The inositol acylation is essential for the generation of mature GPI capable of attachment to proteins. However, the acyl group is usually absent from GPI-anchored proteins (GPI-APs) on the cell surface due to inositol deacylation that occurs in the endoplasmic reticulum (ER) soon after GPI-anchor attachment. Mammalian GPI inositol-deacylase has not been cloned, and the biological significance of the deacylation has been unclear. Here we report a GPI inositol-deacylase-deficient Chinese hamster ovary cell line established by taking advantage of resistance to phosphatidylinositol-specific phospholipase C and the gene responsible, which was termed PGAP1 for Post GPI Attachment to Proteins 1. PGAP1 encoded an ER-associated, 922-amino acid membrane protein bearing a lipase consensus motif. Substitution of a conserved putative catalytic serine with alanine resulted in a complete loss of function, indicating that PGAP1 is the GPI inositol-deacylase. The mutant cells showed a clear delay in the maturation of GPI-APs in the Golgi and accumulation of GPI-APs in the ER. Thus, the GPI inositol deacylation is important for efficient transport of GPI-APs from the ER to the Golgi. Many eukaryotic cell surface proteins with various functions are anchored to the membrane via glycosylphosphatidylinositol (GPI) 1The abbreviations used are: GPI, glycosylphosphatidylinositol; GPI-APs, GPI-anchored proteins; ER, endoplasmic reticulum; PI-PLC, phosphatidylinositol-specific phospholipase C; GST, glutathione S-transferase; CHO, Chinese hamster ovary; ALDH, aldehyde dehydrogenase; PBS, phosphate-buffered saline; DAF, decay-accelerating factor; FACS, fluorescence-activated cell sorter. (1Tiede A. Bastisch I. Schubert J. Orlean P. Schmidt R.E. Biol. Chem. 1999; 380: 503-523Google Scholar, 2McConville M.J. Menon A.K. Mol. Membr. Biol. 2000; 17: 1-16Google Scholar, 3Ikezawa H. Biol. & Pharm. Bull. 2002; 25: 409-417Google Scholar). GPI-anchored proteins (GPI-APs) on mammalian cells are usually sensitive to bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), leading to the release of the protein portions. Therefore, PI-PLC is often used as a tool to determine whether proteins are GPI-anchored. In contrast, precursors of the GPI-anchor present in the endoplasmic reticulum (ER) are resistant to PI-PLC due to an acyl chain linked to the 2-position of inositol (4Hirose S. Prince G.M. Sevlever D. Ravi L. Rosenberry T.L. Ueda E. Medof M.E. J. Biol. Chem. 1992; 267: 16968-16974Google Scholar). The inositol ring of GPI is acylated at an early step in GPI biosynthesis by the action of PIG-W protein, an acyltransferase that adds a palmitoyl chain to the inositol of glucosaminyl-phosphatidylinositol, the second intermediate in the pathway (5Murakami Y. Siripanyapinyo U. Hong Y. Kang J.Y. Ishihara S. Nakakuma H. Maeda Y. Kinoshita T. Mol. Biol. Cell. 2003; 14: 4285-4295Google Scholar). The inositol acylation is critical for the attachment of GPI to proteins, because mutant cells defective in PIG-W express only very low levels of GPI-APs (5Murakami Y. Siripanyapinyo U. Hong Y. Kang J.Y. Ishihara S. Nakakuma H. Maeda Y. Kinoshita T. Mol. Biol. Cell. 2003; 14: 4285-4295Google Scholar). It is very likely that the acyl group is required for a later step in the pathway when “bridging” ethanolamine phosphate, which links GPI to the protein, is added to the third mannose to generate mature GPI. Soon after the attachment of GPI to proteins, the inositol is usually deacylated in the ER and becomes sensitive to PI-PLC (6Chen R. Walter E.I. Parker G. Lapurga J.P. Millan J.L. Ikehara Y. Udenfriend S. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9512-9517Google Scholar). Human erythrocytes represent an exception, in which the inositol remains acylated, and all the GPI-APs are resistant to PI-PLC (7Walter E.I. Roberts W.L. Rosenberry T.L. Ratnoff W.D. Medof M.E. J. Immunol. 1990; 144: 1030-1036Google Scholar, 8Deeg M.A. Humphrey D.R. Yang S.H. Ferguson T.R. Reinhold V.N. Rosenberry T.L. J. Biol. Chem. 1992; 267: 18573-18580Google Scholar, 9Rudd P.M. Morgan B.P. Wormald M.R. Harvey D.J. van den Berg C.W. Davis S.J. Ferguson M.A. Dwek R.A. J. Biol. Chem. 1997; 272: 7229-7244Google Scholar). A possible reason for the lack of deacylation in human erythrocytes is that GPI-APs bearing three acyl chains are more stably associated with the membrane than those bearing two acyl chains, and thus the maintenance of GPI-APs during the long life of erythrocytes is ensured. The enzyme involved in inositol deacylation of GPI-APs has not been characterized. Furthermore, the reason why the deacylation usually occurs is unclear. In order to identify a gene for the GPI inositol-deacylase, we isolated a mutant Chinese hamster ovary (CHO) cell line defective in inositol deacylation and cloned the gene responsible, PGAP1 (for Post GPI Attachment to Proteins 1). Rat PGAP1 is an ER membrane protein with a catalytic serine-containing motif that is conserved in a number of lipases. Cells and Culture—Wild-type CHO-K1, 3B2A (10Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Google Scholar), and C10 cells (see below for characterization) were cultured in Ham's F-12 medium supplemented with 10% fetal bovine serum and 0.5 mg/ml G418 (3B2A and C10 cells only). To establish an RD3 rescued cell line, C10 cells were electroporated with 20 μg of linearized pME-Puro-PGAP1 for the expression of PGAP1, selected in Ham's F-12 medium with 5 μg/ml of puromycin for 1 week, and cloned by limiting dilution. Plasmids—The plasmids pME-Py (an expression vector with a polyoma origin of replication), pME-Py-FLAG-ALDH (ALDH, microsomal aldehyde dehydrogenase) (11Masaki R. Yamamoto A. Tashiro Y. J. Cell Biol. 1994; 126: 1407-1420Google Scholar), and pME-Py-GST-FLAG-hPIG-M were described previously (12Maeda Y. Tanaka S. Hino J. Kangawa K. Kinoshita T. EMBO J. 2000; 19: 2475-2482Google Scholar, 13Maeda Y. Watanabe R. Harris C.L. Hong Y. Ohishi K. Kinoshita K. Kinoshita T. EMBO J. 2001; 20: 250-261Google Scholar). A plasmid pME-Puro-PGAP1 was constructed by subcloning the XhoI/XbaI fragment of pME-Py-PGAP1 into the XhoI/XbaI site of pME-Puro. To fuse a FLAG tag at the N terminus of PGAP1, the rat DPM2 cDNA of pME-Py-FLAG-rDPM2 was replaced with a cDNA of PGAP1 whose start codon was replaced by a SalI recognition sequence. A glutathione S-transferase (GST)-tagged PGAP1 was constructed in a similar way using pME-Py-GST-rDPM2. We termed these plasmids pME-Py-FLAG-PGAP1 and pME-Py-GST-PGAP1, respectively. To obtain the Bst1p expression vector pME-Py-BST1, we amplified BST1 with the primers 5′-GCGGCCGCCACCATGGGTATCAGGAGATTAGTTAGTGTT-3′ (forward) and 5′-TCTAGACTAATGTATTGTTTCGAAAAATAGCAAC-3′ (reverse) from a Saccharomyces cerevisiae genomic DNA library, and we cloned it into the pGEM-T Easy vector (Promega). The BST1 was sequenced and subcloned into the NotI/XbaI site of pME-Py. We substituted serine 174 in PGAP1 with alanine by site-directed mutagenesis using a Quick Change Site-directed Mutagenesis Kit (Stratagene). After sequencing, the ClaI/BglI fragment of the mutated plasmid was used to replace the corresponding part of the wild-type pME-Py-PGAP1 to obtain pME-Py-PGAP1 (S174A). To construct pME-Puro-FLAG-folate receptor 1 (FR1), we amplified the human FR1 sequence with primers 5′-CCAGAAGCTTGCTCAGACAAGGATTGCATGGG-3′ (forward) and 5′-GGAGTGCGGCCGCTAGCTGAGCAGCCACAGCAGCA-3′ (reverse) from human cDNA library. The HindIII/NotI-cut fragment was subcloned into HindIII/NotI-cut pME-Puro-FLAG-CD59 to replace CD59 with FR1. Establishment of a GPI Inositol-deacylase-defective Cell Line, C10—3B2A cells (107) were treated with 0.4 mg/ml ethyl methanesulfonate for 1 day, followed by a 6-day culture without ethyl methanesulfonate. Cells were treated with 1 unit/ml PI-PLC (Molecular Probes) for 1 h at 37 °C, and the PI-PLC-resistant cells were enriched by auto-MACS (Miltenyi Biotec) using biotinylated anti-CD59 (5H8) and anti-DAF (IA10) antibodies and cloned by limiting dilution. Cloning of PGAP1 cDNA—C10 cells (1.2 × 108) were mixed with 360 μg each of a rat C6 glioma cDNA library (10Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Google Scholar) and pcDNA-PyT(ori-) plasmids (10Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Google Scholar) in 4.8 ml of Opti-MEM I and electroporated in 12 cuvettes at 960 microfarads and 300 V using Gene Pulser (Bio-Rad). After 1 day, cells (107/ml) were treated with 25 mg/ml Pronase (Sigma) in Opti-MEM I for 2 h at 37 °C. Transfected cells were resuspended in Opti-MEM I at 2 × 107/ml 1.5 days after the Pronase treatment, treated with 1 unit/ml of PI-PLC for 1 h at 37 °C, and then stained with a biotinylated anti-CD59 antibody (5H8) and phycoerythrin-conjugated streptavidin (Biomeda). Approximately 6,000 cells with low negative CD59 staining were collected using a cell sorter (FACS-Vantage, BD Biosciences). Plasmids (2 × 105 clones) were recovered from these cells by Hirt's method (14Hirt B. J. Mol. Biol. 1967; 26: 365-369Google Scholar). Pooled plasmids (15 μg) were transfected with 45 μg of pcDNA-PyT(ori-) plasmids into 1.8 × 108 C10 cells, as described above, in 9 cuvettes. After another cycle of cell sorting and plasmid recovery, the plasmids were electroporated into Escherichia coli and fractionated to 576 pools (20-30 clones/pool). One of these pools restored PI-PLC sensitivity in C10 cells, and 3 positive colonies were obtained from this pool. Human PGAP1 cDNA—Human PGAP1 cDNA was amplified by PCR using forward (5′-CTCGAGCCACCATGTTTCTTCACTCAGTTAATCTCTG-3′) and reverse (5′-GCGGCCGCTTACATAAAGTTGCATAATGCATGG-3′) primers. The primers were designed based on the sequences of FLJ12377 (DDBJ/GenBank™/EMBL accession number AK022439) and IMAGE:1676930 (3′) (DDBJ/GenBank™/EMBL accession number AI076615), respectively. PI-PLC Sensitivity Assay—Cells (106) were treated with or without 50 μl of 1 unit/ml PI-PLC in Opti-MEM I (Invitrogen) for 1-1.5 h at 37 °C. The cells were stained for CD59 and DAF and analyzed with a FACSCalibur (BD Biosciences). Triton X-114 Partitioning of FLAG-tagged CD59—C10 cells (2 × 106) were transiently transfected with 3.2 μg of pME-Py-FLAG-CD59 (15Hong Y. Ohishi K. Inoue N. Kang J.Y. Shime H. Horiguchi Y. van der Goot F.G. Sugimoto N. Kinoshita T. EMBO J. 2002; 21: 5047-5056Google Scholar) using LipofectAMINE 2000 Transfected cells (106) were treated with or without μl of 1 unit/ml PI-PLC for 1 h at 37 °C, in 1 ml of Triton X-114 in Opti-MEM I for on and then for at 37 with The cell were into and by at × for at 25 °C. FLAG-tagged CD59 was from each using (Sigma) and analyzed by of and C10 cells were transfected with expression After 2 cells (106) were treated with or without 1 (Sigma) in at °C. After the were collected by at × for 1 h at °C, and resuspended in μl of by treated with or without 3 of PI-PLC for 1.5 h at 37 °C. proteins were to Triton X-114 and analyzed by as described of cells × transfected with pME-Py-FLAG-PGAP1 were and the of and ER were as described previously Y. S. Watanabe R. Ohishi K. Kinoshita T. EMBO J. 1998; 17: Scholar). was and from each using and then analyzed by The of the were using a of and RD3 cells were for at 37 in and medium (Invitrogen) supplemented with 10% fetal bovine with and for at 37 °C, and then with 1.5 and for at 37 °C. Cells were with and in ml of Triton DAF was using a anti-DAF antibody (IA10) and Biosciences). DAF was by and analyzed using a of PGAP1 cells were transiently transfected with or pME-Py-GST-FLAG-hPIG-M and on after 1 of After 2 cells were with in for and then in in for 20 were with bovine serum in for complete Triton was added to the and for at of the cells were in 5 μg/ml for The cells were then with a antibody or antibody followed by or (Molecular Probes) The cells were a with a Establishment of a PI-PLC-resistant Cell Line, identify a GPI inositol-deacylase, we established a cell line whose surface GPI-APs were not by PI-PLC, because GPI-APs on the cell surface resistant to PI-PLC the cells a GPI inositol-deacylase. We 3B2A cells, cells CD59 and DAF (10Nakamura N. Inoue N. Watanabe R. Takahashi M. Takeda J. Stevens V.L. Kinoshita T. J. Biol. Chem. 1997; 272: 15834-15840Google Scholar), treated with PI-PLC, and enriched the cells whose CD59 and DAF were resistant to PI-PLC by Cell We obtained after limiting and used a mutant cell line, termed in the We the PI-PLC resistance of C10 cells by The of CD59 and DAF on C10 mutant cells were similar to those of wild-type 3B2A cells and were not by PI-PLC a and the on 3B2A cells were by and We the PI-PLC resistance of the FLAG-tagged CD59 from C10 cells by Triton X-114 of the mature from 3B2A cells was into the without PI-PLC 3 and the of the mature was into the after with PI-PLC 1 and In contrast, a part of the proteins from C10 cells was into the after PI-PLC and The of mature in C10 cells that the maturation of was in these mutant cells (see below for We two of a and a was in cell and was 3 and It that to GPI-anchored due to the was PI-PLC and only in C10 mutant cells and an GPI-anchored bearing acylated To obtain that the PI-PLC resistance of GPI-APs on C10 mutant cells is due to the inositol we the acyl chain by C10 cells with J.P. Roberts W.L. Rosenberry T.L. J. Scholar) and then whether GPI-APs We transfected C10 and 3B2A cells with FLAG-tagged of the was after treatment, likely because FR1 with GPI-anchor was and because FR1 with GPI-anchor We analyzed PI-PLC sensitivity of the by Triton X-114 on C10 cells was resistant to PI-PLC the of after on 3B2A was sensitive to PI-PLC without as that the inositol acylation of GPI-APs on C10 mutant cells was the for PI-PLC Cloning and of PGAP1 obtain that rescued the in C10 mutant cells, we transfected C10 mutant cells with a rat cDNA library, collected cells with a cell and recovered the plasmid was used for the second of We obtained three with the and that these plasmids were from the We termed the gene We established a cell line, termed which stably The PI-PLC sensitivity of CD59 on RD3 cells was than that of 3B2A wild-type cells and indicating that PGAP1 rescued the in C10 mutant The of RD3 to PI-PLC due to the of The PGAP1 cDNA restored PI-PLC sensitivity on mutant indicating that all mutant cells were defective in PGAP1 gene not The PGAP1 cDNA of and the encoded acid (DDBJ/GenBank™/EMBL accession number The sequence the start codon with We a lipase consensus motif with a putative catalytic serine that PGAP1 is the A of the acid sequence that PGAP1 has by using a The lipase motif in a after the the N the human we that the sequence of the of the protein FLJ12377 was with that of However, the sequence of the was not similar to PGAP1, and the sequence was than that of The sequence of FLJ12377 was in genomic sequence of human PGAP1 gene that it was a of We whose sequences are to the of To obtain the sequence of the human we amplified it from a human cDNA library by PCR and the sequence (DDBJ/GenBank™/EMBL accession number The of the acid sequences of human and rat PGAP1 was and lipase consensus sequences were S. cerevisiae Bst1p an of the for of PGAP1 and an S. cerevisiae of PGAP1 that was as BST1 accession number M.J. Mol. Biol. Cell. Scholar, S. J. Cell Biol. 2001; Scholar). The acid of rat PGAP1 and Bst1p was and the lipase consensus sequence was To whether BST1 was a we transiently transfected C10 mutant cells with BST1 cDNA and the PI-PLC We a of the PI-PLC sensitivity of the indicating that Bst1p is an of PGAP1 ER and of PGAP1 FLAG or at the N terminus of PGAP1, and we whether the PI-PLC sensitivity of C10 mutant and PGAP1 restored the not To determine the of PGAP1, we transfected cells with fractionated the cell by and the of membrane The membrane and Golgi were fractionated into 1 and 2 and the ER was fractionated into was by and showed a similar to the ER Thus, PGAP1 is an ER We the membrane of the acyl group on the inositol moiety of GPI is the ER PGAP1 is the the lipase motif to the this is the N terminus of PGAP1 on the of the ER. To we transiently transfected into cells treated the cells with for of the membrane or with Triton for of the and ER and stained the cells for the tag and To the of the we used which was with and FLAG at the N The of the on was established previously Y. Watanabe R. Harris C.L. Hong Y. Ohishi K. Kinoshita K. Kinoshita T. EMBO J. 2001; 20: 250-261Google Scholar). The tag of PGAP1 was in the cells and the staining and were not when cells were with Triton In contrast, was only stained after with Triton as and The staining of the was similar to that of the indicating that the N terminus of PGAP1 on the and that the lipase motif is to the of the ER. Site-directed Mutagenesis of a whether the lipase motif in PGAP1 is we substituted the putative catalytic serine 174 with alanine We transiently transfected C10 mutant cells with FLAG-tagged wild-type or PGAP1 in with FLAG-tagged to the We the FLAG-tagged proteins and analyzed by The of to were indicating a expression of The C10 mutant cells transfected with wild-type were sensitive to 3B2A cells a and C10 cells transfected with were resistant to PI-PLC those transfected with an vector and that the putative catalytic serine 174 is required for the of Thus, we that PGAP1 is the GPI inositol-deacylase The for the of the of the inositol deacylation of The that an of in C10 cells in that the inositol deacylation was important for the maturation of To this we analyzed the maturation of DAF using a The ER of DAF is to the Golgi and and of the to mature DAF In C10 mutant cells, the of mature DAF with a in the of the ER of DAF during the In contrast, in RD3 rescued cells, the in the mature DAF and in the ER of DAF were than those in C10 mutant cells 3B2A cells showed the as RD3 cells not of the showed that the C10 mutant cells a clear delay in the maturation of the DAF 20 in RD3 rescued cells, was required in C10 cells indicating that the in the inositol deacylation the maturation of The of the mature DAF in C10 and RD3 cells were Therefore, the of the was not We then analyzed DAF in the cell by and a accumulation of the ER of DAF in C10 cells that the inositol deacylation is important for the maturation of of DAF and CD59 the transport followed by in the Golgi. the of the was not we that the inositol deacylation is important for the transport of The biosynthesis of GPI-APs into two In the the of the GPI-anchor is and to the proteins, in the generation of In the second the GPI and protein of the GPI-APs are during transport from site of generation in the ER to the cell than 20 involved in the been cloned and is the involved in the second We a number of in the as (for as T. Inoue N. Chem. Biol. 2000; Scholar). Here we report the mammalian gene involved in the second the second of after the attachment of the GPI-anchor to proteins, we termed this gene The in the present are as that PGAP1 an GPI inositol-deacylase, a for the report that inositol deacylation occurs in the ER soon after the generation of GPI-APs (6Chen R. Walter E.I. Parker G. Lapurga J.P. Millan J.L. Ikehara Y. Udenfriend S. Medof M.E. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9512-9517Google and that inositol deacylation is important for efficient transport of A for in the of mutant cells, the transport of DAF as by the generation of Golgi of DAF was The for of DAF to mature was 20 in wild-type cells, it was in the mutant In S. the BST1 gene is to PGAP1 M.J. Mol. Biol. Cell. Scholar). PI-PLC sensitivity in cells was restored by of that Bst1p is a of with this maturation of a GPI-anchored protein whose maturation is the transport to the Golgi A. U. H. EMBO J. 1994; Scholar), was in S. J. Cell Biol. 2001; Scholar), DAF in C10 mutant BST1 of gene was by that by of a gene for a of M.J. Mol. Biol. Cell. Scholar). In mutant cells, deacylated GPI-APs not into and in the ER, the are The that the in BST1 rescued the by of that GPI-APs been from the ER via M. H. J. Cell Biol. 2000; Scholar) and M. R. A. Mol. Biol. Cell. 1999; Scholar), of are to of for GPI-anchored protein in S. and are M.J. Mol. Biol. Cell. Scholar, M. R. A. Mol. Biol. Cell. 1999; Scholar). with a that surface transport of GPI-APs is essential for of S. it is that of GPI-APs with that accumulation of GPI-APs in the ER is involved in the of It is possible that the of by is that the acyl chain on inositol of GPI-APs in mutant with the efficient with the in a lack of accumulation of Thus, inositol deacylation required for efficient of GPI-APs into transport the with the GPI in mammalian cells and the S. an acyl chain is added to the 2-position of inositol early in the GPI pathway Orlean P. J. Biol. Chem. 1992; 267: Scholar). The acylation is by PIG-W in mammalian cells (5Murakami Y. Siripanyapinyo U. Hong Y. Kang J.Y. Ishihara S. Nakakuma H. Maeda Y. Kinoshita T. Mol. Biol. Cell. 2003; 14: 4285-4295Google Scholar) and by the PIG-W in S. cerevisiae M. M. K. K. K. K. Y. J. Biol. Chem. 2003; Scholar). The for these is V.L. H. J. Biol. Chem. 1994; Scholar, J. M.A. J. Biol. Chem. Scholar). the acyl chain remains of GPI to proteins. In T. inositol acylation and deacylation are more from those in mammalian and and the and In T. the acyl chain is added step because it is added to Ferguson M.A. EMBO J. 14: Scholar, S. Ferguson M.A. J. Biol. Chem. Scholar). However, the palmitoyl chain and added at of the later Ferguson M.A. EMBO J. 14: Scholar). The of the GPI-anchor in is not acylated, in the GPI-anchor is gene that for inositol in T. has been cloned S. Ferguson M.A. EMBO J. 2001; 20: Scholar). of has an not a It is to mammalian that from bacterial is than PGAP1 and has sequence with is PGAP1 has Therefore, and PGAP1 are not We a sequence that has with PGAP1 in the T. Cloning and or of this gene are required to determine whether it is an inositol that for the inositol deacylation in the T. S. Ferguson M.A. EMBO J. 2001; 20: Scholar). We and Kinoshita for