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Vol. 19, Issue 5, 2069-2082, May 2008

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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

Kentaro Kajiwara^*, Reika Watanabe, Harald Pichler^,, Kensuke Ihara^*, Suguru Murakami^*, Howard Riezman, and Kouichi Funato^*

*Department of Bioresource Science and Technology, Graduate School of Biosphere Science, Hiroshima University, Higashi-Hiroshima 739-8528, Japan; Department of Biochemistry, University of Geneva, Sciences II, CH-1211 Geneva, Switzerland; and Institute of Molecular Biotechnology, Graz University of Technology, A-8010 Graz, Austria

Submitted August 2, 2007; Revised February 1, 2008; Accepted February 8, 2008
Monitoring Editor: Sean Munro

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
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.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Glycosylphosphatidylinositol (GPI) is a complex glycolipid found in all eukaryotic cells. It is covalently attached to the C-terminal end of certain secretory proteins and acts as a membrane anchor (Kinoshita and Inoue, 2000; Ikezawa, 2002; Pittet and Conzelmann, 2007). Because many proteins anchored by GPI are poorly extracted with detergents at 4°C, they have been proposed to be localized in detergent-resistant membranes (DRMs), which may be related to microdomains called lipid rafts that have been proposed to play an important role in the trafficking of GPI-anchored proteins (Simons and Ikonen, 1997; Chatterjee and Mayor, 2001; Mayor and Riezman, 2004).

Biosynthesis of GPI takes place on the membranes of the endoplasmic reticulum (ER) (Kinoshita and Inoue, 2000; Pittet and Conzelmann, 2007), and this starts with the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to the inositol of phosphatidylinositol (PI) to generate N-acetylglucosaminyl-PI (GlcNAc-PI), followed by de-N-acetylation to form glucosaminyl-PI (GlcN-PI). The two reactions occur on the cytoplasmic side of the ER membrane (Vidugiriene and Menon, 1993; Orlean and Menon, 2007). The GlcN-PI is then acylated on the inositol ring to form glucosaminyl-acyl-PI (GlcN-acylPI) before or after transfer of the first mannose (Smith et al., 1997; Kinoshita and Inoue, 2000; Pittet and Conzelmann, 2007). Dolicholphosphomannose (Dol-P-Man) is the mannose donor for the core mannose residues (Menon et al., 1990). In Saccharomyces cerevisiae and Plasmodium falciparum, inositol acylation appears to be a prerequisite for mannosylation of GPIs (Doerrler et al., 1996; Gerold et al., 1999), whereas, in Trypansoma brucei and mammalian cells, it is not essential for addition of the first mannose (Guther and Ferguson, 1995; Smith et al., 1996; Murakami et al., 2003), although the mammalian mannosyltransferase (GPI-MT-I), PIG-M seems to prefer GlcN-acylPI to GlcN-PI. The PIG-M has a lumenally oriented, functionally important DXD motif that is found in many glycosyltransferases (Maeda et al., 2001). Based on the topological aspect of synthesis of GlcN-PI and subsequent mannosylation, it was suggested that the early GPI intermediates must flip from the cytoplasmic side to the ER lumen before the first mannosylation. In addition, genes coding for mammalian PIG-W and the yeast homologue GWT1, which are involved in inositol acylation of GPI were recently identified (Murakami et al., 2003; Umemura et al., 2003). They encode multi-spanning ER membrane proteins. Because the comparison of locations of predicted transmembrane domains and amino acid sequence of PIG-W homologues of various organisms showed that the location of conserved regions face the lumenal side of the ER, it was also proposed that inositol acylation occurs in the ER lumen; hence, inositol acylation is not required for flipping of GPI (Murakami et al., 2003). However, it remains unclear whether the catalytic site of PIG-W is located within the conserved regions. Mutations in the temperature-sensitive (ts) mutant alleles of GWT1, which were randomly generated by PCR mutagenesis, were not found in the conserved regions (Umemura et al., 2003). Thus, it is not yet known whether GlcN-PI or GlcN-acylPI, or both, are flipped across the ER membrane. After mannosylation and addition of phosphorylethanolamine (EtNP) residue(s), the entire GPI anchor precursor is attached to proteins by a GPI transamidase, which acts on the lumenal side of the ER (Kinoshita and Inoue, 2000; Ikezawa, 2002; Pittet and Conzelmann, 2007). Subsequently, the acyl group of the inositol is removed from the GPI-anchored proteins (Tanaka et al., 2004; Fujita et al., 2006a), and the GPI lipid moieties are remodeled to a more hydrophobic diacylglycerol or ceramide (Sipos et al., 1997; Reggiori et al., 1997). Most of the genes encoding enzymes involved in GPI biosynthetic pathway have been isolated and characterized (Kinoshita and Inoue, 2000; Pittet and Conzelmann, 2007). Recently, genes that are required for lipid remodeling of GPI-anchored proteins were also identified (Tashima et al., 2006; Bosson et al., 2006; Fujita et al., 2006b; Ghugtyal et al., 2007; Umemura et al., 2007). However, despite evidence that GPI precursor flipping is a protein-mediated process (Vishwakarma and Menon, 2005; Pomorski and Menon, 2006), the putative GPI flippase has not yet been identified.

Once the inositol-linked acyl chain is eliminated by a GPI inositol deacylase, the GPI-anchored proteins are transported from the ER to the Golgi apparatus via transport vesicles (Muniz and Riezman, 2000; Ikonen, 2001; Mayor and Riezman, 2004). In S. cerevisiae, GPI-anchored proteins are known to exit the ER in vesicles distinct from other secretory proteins (Muniz et al., 2001). Rab GTPase Ypt1p, the tethering factors Uso1p, COG complex Sec34p and Sec35p, and the ER v-SNAREs Bos1p, Bet1p, and Sec22p are necessary for the sorting of GPI-anchored proteins upon ER exit (Morsomme and Riezman, 2002; Morsomme et al., 2003). The lipid composition may have a function in sorting of GPI-anchored proteins into ER-derived vesicles because GPI-anchored proteins are associated with DRMs in the ER (Bagnat et al., 2000) and because ongoing ceramide synthesis is required for the efficient transport of GPI-anchored proteins (Horvath et al., 1994; Sutterlin et al., 1997a; David et al., 1998; Barz and Walter, 1999; Watanabe et al., 2002). Ceramide synthesis does not seem to be required for GPI-anchored protein transport in trypanosomes, but the anchors are not remodeled to ceramides in these cells (Sutterwala et al., 2007). Because the maturation of GPI-anchored proteins is delayed when yeast cells are incubated with myriocin, an inhibitor of serine palmitoyltransferase (SPT), or when a ts lcb1-100 mutant defective in SPT activity is incubated at nonpermissive temperature (Horvath et al., 1994; Sutterlin et al., 1997a), but not affected when yeast cells are treated with aureobasidin A (AbA), an inhibitor of yeast sphingolipid synthesis (Reggiori and Conzelmann, 1998), ceramide-rich domains rather than complex sphingolipid-rich domains could be important for the sorting step in the ER. This is also supported by the fact that ceramide is synthesized in the ER and then is delivered to the Golgi where it is converted to inositolphosphorylceramide (IPC), followed by mannosylation to form mannosyl IPC (MIPC) and mannosyl di(inositolphosphoryl)ceramide [M(IP)₂C], which are the three major yeast sphingolipids (Funato et al., 2002; Dickson et al., 2006). Previously, we have shown that ER-to-Golgi transport of ceramide for IPC synthesis is mediated by both vesicular and nonvesicular trafficking (Funato and Riezman, 2001).

Although the roles of the different pathways are poorly understood, the vesicular transport of ceramide could be directly coupled to transport of proteins. In addition, GPI anchor attachment to proteins and the lipid remodeling are necessary for efficient transport of GPI-anchored proteins to the Golgi (Hamburger et al., 1995; Doering and Schekman, 1996; Bosson et al., 2006; Fujita et al., 2006b). Recent studies have shown that mutant cells defective in GPI anchor synthesis or attachment block export of some detergent-insoluble transmembrane proteins from the ER (Okamoto et al., 2006), suggesting a role of GPI-anchored proteins in raft-dependent protein sorting. However, evidence indicating that GPI-anchored proteins are required for transport of raft lipid(s) per se is missing.

In this study we show that yeast ARV1, which has been known to be somehow involved in the regulation of sphingolipid and sterol metabolism (Tinkelenberg et al., 2000; Swain et al., 2002; Fores et al., 2006), genetically interacts with genes involved in GPI anchor synthesis and that Arv1p is required for the efficient synthesis of GlcN-acylPI bearing one mannose, whereas arv1 cells retain both Dol-P-Man synthesis and GPI-MT activity. We propose that the primary function of Arv1p is either to deliver GlcN-acylPI from the cytoplasmic side to the luminal side of the ER or to present it to the yeast PIG-M homologue GPI14 (Maeda et al., 2001). In addition, we present evidence that GPI assembly is required not only for GPI-anchored protein transport but also for ceramide transport from the ER as well as to maintain the proper intracellular distribution and amounts of sterols.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains, Media, Library Screen, Plasmids, and Reagents
The yeast strains and plasmids used in this study are listed in Supplementary Tables S1 and S2, respectively. Yeast cultures and genetic manipulations were carried out essentially as described by Sherman et al. (1983). To construct deletion strains, entire open reading frames were deleted and replaced with the designated genes. Deletions were confirmed by PCR. Yeast strains were grown in rich medium, YPUAD (Dulic et al., 1991), semisynthetic medium, SDYE (Horvath et al., 1994), or synthetic minimal medium, SD (Funato et al., 2003), supplemented with the appropriate nutrients to select for plasmids. pmi40 mutant cells were supplemented with 0.5% mannose (Pitkanen et al., 2004).

Temperature sensitivity was determined by spotting diluted yeast cultures on SD (–ura) plates at 25 and 37°C. To test drug sensitivity, cells were cultured on SD plates containing calcofluor white (CFW) or AbA. A genomic LEU2-marked library made in YEp13 (a gift from Dr. Y. Ohya, University of Tokyo, Japan) was used to isolate suppressors of the ts growth phenotype of arv1 mutant (FK137). A plasmid was isolated containing GPI15. GPI15, GPI1, GPI2, GPI3, and ERI1 genes were amplified from S. cerevisiae genomic DNA by PCR. GPI15 was cloned into BamHI/EcoRI sites of pRS426GPD, GPI1 into BamHI/XhoI, GPI2 into BamHI/SalI, GPI3 into SpeI/XhoI, and ERI1 into BamHI/XhoI of pRS426ADH vector. To construct a plasmid (pGAL-HA-GPI18) for expressing N-terminal two-hemagglutinin (HA)-tagged GPI18, the BamHI-SalI PCR fragment containing the open reading frame of GPI18 was ligated into pRS316-GAL1-2xHA-BS, a pRS316-based expression vector carrying the EcoRI site, the initiation codon, two HA-epitope–encoding regions, and multicloning sites (provided by Dr. K. Tanaka, Hokkaido University, Japan). The EcoRI-SalI fragment including the epitope-tagged GPI18 was subcloned into pRS416GAL1. A diploid heterozygous ARV1/arv1::LEU2 GPI18/gpi18::KAN^R double deletion strain was created by deleting ARV1 in the GPI18/gpi18::KAN^R diploid strain BY4743 MATa/ his31/his31 leu20/leu20 ura30/ura30 lys20 met150 gpi18::KAN^R (generated by the Saccharomyces Gene Deletion Project and provided by Dr. T. Kinoshita, Osaka University, Japan) and transformed with pGAL-HA-GPI18. The transformants were sporulated and tetrads dissected to obtain haploid strains arv1::LEU2 gpi18::KAN^R (FK1015), gpi18::KAN^R (FK1017), and wild-type (FK1019) harboring pGAL-HA-GPI18. A C-terminal triple HA-tagged ARV1 was amplified from genomic DNA (prepared from RH6076, which carried the epitope-tagged ARV1 gene) by PCR, and cloned into BamHI/XhoI sites of pRS426ADH. AbA and C₂-ceramide were purchased from Takara (Tokyo, Japan) and Matreya (State College, PA), respectively. YW3548 was prepared as described previously (Sutterlin et al., 1997b).

Labeling of Lipids In Vivo
In vivo labeling of lipids with [³H]myo-inositol (Perkin Elmer-Cetus Life Sciences, Boston, MA) or [³H]dihydrosphingosine (DHS; American Radiolabeled Chemicals, St. Louis, MO) was performed as described previously (Zanolari et al., 2000). Cells were grown overnight in SDYE, harvested and resuspended in SD without inositol for labeling with [³H]myo-inositol, or SD medium for labeling with [³H]DHS. The cells were preincubated for 15 min at 25°C and then labeled by 25 µCi of [³H]myo-inositol or 10 µCi of [³H]DHS. If present, C₂-ceramide (200 µM) was added at the start of the labeling. The incubation was stopped by the addition of 10 mM NaF and 10 mM NaN₃. The cells were then washed with cold water, and lipids were extracted with chloroform-methanol-water (10:10:3, vol/vol/vol). Half of the dried sample was treated by mild alkaline hydrolysis with 0.6 M NaOH, neutralized, and desalted by n-butanol partitioning. The lipids were analyzed by thin-layer chromatography (TLC) using solvent system I, chloroform-methanol-0.25% KCl (55:45:10, vol/vol/vol) for complex sphingolipid labeled with [³H]myo-inositol, and solvent system II, chloroform-methanol-4.2 N ammonium hydroxide (9:7:2, vol/vol/vol) for sphingolipid labeled with [³H]DHS. Radiolabeled lipids were visualized and quantified on a Cyclone Storage Phosphor System (Packard Instrument, Meriden, CT).

In vivo [³H]mannose labeling using the pmi40 and arv1 pmi40 double mutant strains was performed as described (Sipos et al., 1994; Sutterlin et al., 1997b). In brief, the cells were grown overnight in SDCU medium (2% glucose, 1% peptone, 0.67% yeast nitrogen base, 0.5% mannose, and the required nutrients), harvested, and resuspended in SPCU medium (0.1% glucose, 2% pyruvate, 0.67% yeast nitrogen base, and the required nutrients). Wild-type and arv1 mutant strains were grown in SDCU medium without mannose. For [³H]mannose labeling of strains transformed with pGAL-HA-GPI18, cells were first grown in SGYE (2% galactose, 0.67% yeast nitrogen base, 0.2% yeast extract, and the required nutrients) medium, then shifted to SDYE medium for 16 h at 25°C, and resuspended in SPCU medium. The cells were preincubated for 20 min at 25°C with 20 µg/ml tunicamycin in the presence or absence of YW3548 (10 µM) and then labeled with 25 µCi of [³H]mannose (Perkin-Elmer Cetus Life Sciences) for 30 min at 25°C. The labeling was stopped by the addition of NaF and NaN₃, and lipids were extracted with chloroform-methanol-water (10:10:3, vol/vol/vol), desalted by n-butanol partitioning, and analyzed by TLC using solvent system III, chloroform-methanol-water (10:10:3, vol/vol/vol).

Labeling of Lipids In Vitro
Membranes for in vitro labeling experiments with [¹⁴C]UDP-GlcNAc (Perkin-Elmer Cetus Life Sciences) or with [³H]GDP-mannose (GDP-Man; American Radiolabeled Chemicals) were prepared as described by Schonbachler et al. (1995), except that for cell breakage, TDP (50 mM Tris-HCl, pH 7.4, 5 mM DTT, and 1 mM PMSF) was used. The membranes were suspended in TDP containing 20% glycerol and stored at –80°C. Samples of membranes containing equivalent amounts of protein were assayed for the early steps of GPI anchor biosynthesis using [¹⁴C]UDP-GlcNAc as described (Schonbachler et al., 1995) with a few modifications. Two hundred micrograms of membranes were incubated with 50 nCi [¹⁴C]UDP-GlcNAc at 25°C in 50 µl of GPI buffer consisting of 100 mM Tris-HCl, pH 7.5, 1 mM EGTA, 3 mM Mg-acetate, 0.5 mM MnCl₂, 1 mM CoA, 1 mM ATP, 20 µg/ml tunicamycin, and 250 nM GDP-Man. Dol-P-Man synthesis was assayed in GPI buffer containing 0.5 mM UDP-GlcNAc and 0.25 µCi [³H]GDP-Man (250 nM). Lipids were extracted by the addition 750 µl of chloroform-methanol (1:2, vol/vol), desalted, and analyzed by TLC using solvent system IV, chloroform-methanol-1N ammonium hydroxide (10:10:3, vol/vol/vol) for [¹⁴C]UDP-GlcNAc labeled lipids (Watanabe et al., 1998), and solvent system III for the synthesis of Dol-P-Man (Sutterlin et al., 1997b).

For mannosylated GPI lipid synthesis assay, ER-enriched membranes were prepared as described (Funato and Riezman, 2001), and the membranes were incubated with GPI buffer, 0.5 mM UDP-GlcNAc and 0.5 µCi [³H]GDP-Man in a final volume of 100 µl (Sutterlin et al., 1997b). Some reactions contained 10 mM n-octyl β-D-glucopyranoside and 0.4 µg/ml dioctanoyl-PI [GlcN-PI(C8); a gift from Dr. M. A. Lehrman, University of Texas Southwestern Medical Center, Dallas, TX] with 6 x 10^–4 % Triton X-100, which was used to add GlcN-PI(C8). After 10-min incubation at 25°C, lipids were extracted by the addition 666 µl of chloroform-methanol (1:1, vol/vol), desalted, and analyzed by TLC using solvent system III.

Pulse-Chase Experiments for Protein Maturation and GPI Anchor Attachment
Radiolabeling and immunoprecipitation to measure maturation of GPI-anchored proteins (Gas1p, Yps1p) and carboxypeptidase Y (CPY) were performed as described previously (Sutterlin et al., 1997a). The samples were subjected to SDS-PAGE, analyzed, and quantified using the Cyclone Storage Phosphor System (Packard Instruments). The percentage of mature proteins was determined by taking the ratio of the mature form to the total signal (ER, Golgi, and mature forms). GPI anchor attachment was examined as described (Watanabe et al., 2002).

Fluorescence Microscopy
Labeling of cells with C₆-NBD-ceramide was performed as described (Levine et al., 2000). Cells were incubated with or without AbA (20 µg/ml) at 25°C for 15 min and labeled with C₆-NBD-ceramide (20 µM) at 25°C for 15 min in the presence of defatted BSA (5 mg/ml), followed by washing and back-extracting. The staining was visualized by fluorescence microscopy. For visualization of chitin (Sobering et al., 2004) and sterol distribution (Beh and Rine, 2004), cells were grown in SD medium at 25°C, washed in PBS, and stained with CFW (1 mg/ml) for 5 min and filipin complex (0.1 mg/ml) for 15 min, respectively. The cells were washed and observed by fluorescence microscopy with a UV filter.

Gas Liquid Chromatography–Mass Spectrometry Analysis of Sterols
Cells were grown in SD medium at 25°C. Total sterols including free sterols and sterol esters were extracted from whole cells and analyzed by gas liquid chromatography-mass spectrometry (GLC-MS) as described previously (Munn et al., 1999; Heese-Peck et al., 2002, Mullner et al., 2005). Cholesterol was used as an internal standard.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
An arv1 Mutant Is Deficient in Ceramide Transport from the ER to the Golgi Site of IPC Synthesis
ARV1 was identified in a screen for genes that are essential in the absence of yeast sterol esterification (Tinkelenberg et al., 2000). Arv1p is a multispanning integral membrane protein with six predicted transmembrane domains, and homologues have been found in a wide variety of eukaryotes. Because disruption of ARV1 leads to not only altered levels and distribution of sterols but also reduced sphingolipid levels, it has been proposed that this protein is involved in maintenance of cellular sterol and sphingolipid homeostasis (Swain et al., 2002). To obtain further evidence for the role of Arv1p in the regulation of sphingolipid homoeostasis, we re-examined sphingolipid biosynthesis in arv1 mutant cells, which were generated in our strain background. Consistent with the previous report, the arv1 mutant showed a strong reduction of sphingolipid synthesis, 20% of IPC-C/-D/MIPC and 50% of M(IP)₂C levels found in wild-type cells when cells were pulse-labeled with [³H]myo-inositol (Figure 1A). A similar reduction of IPC-C synthesis was also seen when the sphingolipid synthesis of arv1 mutant was assessed by [³H]DHS labeling (Figure 1B), confirming that this mutant exhibits a sphingolipid synthesis defect. As shown by a previous experiment (Swain et al., 2002), we also observed that the arv1 mutant cells made normal amounts of ceramides as determined by [³H]DHS pulse-labeling for 2 h (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 1. The arv1 mutant is defective in ceramide transport from the ER to the Golgi. (A and B) Wild-type (RH6082) and arv1 (RH6078) strains were labeled with [3H]myo-inositol (A) or with [3H]DHS (B) for 2 h at 25°C. The [3H]myo-inositol–labeled lipids were subjected (+) or not (–) to mild alkaline hydrolysis with NaOH. (C) The same strains as in A were labeled with C6-NBD-ceramide for 15 min at 25°C, with or without AbA as described in Materials and Methods, and the staining was visualized by fluorescence microscopy. (D) The same strains were also labeled with [3H]myo-inositol for the indicated times at 25°C in the presence of C2-ceramide. The labeled lipid products were applied to TLC plates using solvent system I (A and D) or solvent system II (B). Lipids were identified by their mobility and resistance to mild-base treatment. The band, which was identified as C2-IPC, appeared only when the precursor C2-ceramide was added, and the appearance was resistant to mild-base treatment and sensitive to the addition of AbA (not shown). Incorporation (%) into sphingolipids was determined as the percentage of total radioactivity into all lipids containing inositol (A and D). Incorporation of [3H]DHS into IPC-C was quantified, and the relative amounts were calculated by setting the amounts in wild-type cells to 100% (B). Results of a typical experiment are shown. Data in A and B are means ± SE for six and four independent experiments, respectively, and in E are quantification of D. PI, phosphatidylinositol; IPC-C and -D, inositolphosphorylceramide subclasses C and D; MIPC, mannosyldi(inositolphosphoryl) ceramide; Lyso-PI, lyso-phosphatidylinositol; DHS, dihydrosphingosine; PE, phosphatidylethanolamine; PHS, phytosphingosine; PC, phosphatidylcholine.

Considering that the arv1 mutant is deficient in ceramide transport, it is reasonable to assume that this block would entail the accumulation of intermediates in ceramide synthesis. The finding that arv1 mutant cells showed an increased incorporation of [³H]DHS into glycerophospholipids (Figure 1B) is consistent with this, because DHS incorporation is dependent upon its phosphorylation and subsequent cleavage by a long-chain base phosphate lyase (Funato et al., 2003; Saba, 2006). Higher amounts of DHS are likely to occur when ceramide synthesis is backed up, due to its lack of transport.

ARV1 Genetically Interacts with Genes Involved in GPI Anchor Synthesis
Although the arv1 mutant can grow at 25, 30, and 35°C, it fails to grow at 37°C (Swain et al., 2002, Figure 2, A and B). To gain further insight into the function of Arv1p in ceramide transport, we screened a 2 µ/LEU2-marked genomic yeast library for multicopy suppressors of the growth defect. A plasmid was isolated from this screen, which contained GPI15, and rescued the arv1 ts growth (Figure 2A). GPI15 encodes a protein involved in the synthesis of GlcNAc-PI, the first intermediate in GPI anchor biosynthesis (Yan et al., 2001). GlcNAc-PI is synthesized by the GPI-GlcNAc transferase, which is thought to act as a complex composed of six known components in yeast: Gpi1p, Gpi2p, Gpi3p, Gpi15p, Gpi19p, and Eri1p (Pittet and Conzelmann, 2007). To test whether an excess of GPI-GlcNAc transferase activity was required for the suppression, other genes were cloned into a 2 µ vector and transformed into the arv1 mutant cells. Overexpression of Gpi2p suppressed the arv1 growth defect at 37°C almost as well as Arv1p overexpression (Figure 2A). Partial rescue of the arv1 mutant growth defect was observed by overexpressing Gpi1p, Gpi3p, and Eri1p.

View larger version (58K): [in this window] [in a new window]   Figure 2. Overexpresssion of genes involved in GPI anchor synthesis suppresses the temperature-sensitive growth defect of arv1, and combining arv1 and gwt1-20 causes a synthetic growth defect. (A) Wild-type (RH6082) and arv1 (FK137) strains transformed with pRS426GPD, pRS426ADH, pGPI15, pARV1-HA, pGPI1, pGPI2, pGPI3, or pERI1 were spotted onto SD-ura plates and incubated for 4 d at 25 or 37°C. (B) Wild-type (W303-1B), gwt1-20, arv1 (FK245), and arv1 gwt1-20 (FK246) were streaked onto YPUAD plates and incubated for 3 d at 30, 35, or 37°C.

Arv1p Is Involved in GPI Anchoring of Proteins
These genetic interactions suggest that Arv1p may have a role in GPI anchor synthesis or anchoring. To test this hypothesis, we examined the kinetics of maturation of GPI-anchored proteins in an arv1 mutant and wild-type cells, because GPI anchor synthesis and anchoring are required for efficient transport of GPI-anchored proteins from the ER to the Golgi (Schonbachler et al., 1995; Hamburger et al., 1995; Doering and Schekman, 1996; Sobering et al., 2004). Gas1p, a GPI-anchored protein that undergoes O-linked and N-linked glycosylation, is synthesized as a 105-kDa glycoprotein in the ER, and after arrival at the Golgi compartment, fully glycosylated Gas1p has an apparent size of 125 kDa (Nuoffer et al., 1993). A pulse-chase experiment revealed that the maturation of Gas1p is retarded in arv1 mutant cells (Figure 3A). A similar result was observed for the maturation of another GPI-anchored protein, Yps1p, whose ER form is found at 85 kDa, and then is modified in the Golgi and migrates as a smear of 120–180 kDa (Sievi et al., 2001). By contrast, transport from the ER (P1) to the Golgi (P2) and onto the vacuole (mature form) of CPY (Stevens et al., 1982) in arv1 mutant cells occurs with wild-type kinetics. Because CPY is an N-glycosylated but not a GPI-anchored protein, these results indicate that protein N-glycosylation is normal and ER-to-Golgi transport of GPI-anchored proteins is specifically delayed in arv1 mutant.

View larger version (47K): [in this window] [in a new window]   Figure 3. arv1 mutant cells show GPI-anchored protein transport and attachment defects and display the same phenotypes as gpi mutants. (A) ER-to-Golgi transport of protein was examined by pulse-chase experiments as described in Materials and Methods. Wild-type (RH6082) and arv1 (RH6078) strains were labeled with [35S]methionine for 6 min at 25°C and chased for the indicated period of time. The percentage of mature Gas1p, Yps1p, or CPY is shown. (B) GPI anchor attachment was studied in wild-type (RH6082) and arv1 (RH6078) cells. The cells were labeled with [35S]methionine for 6 min and chased for 5 min at 25°C. Cell extracts were prepared and solubilized with Triton X-114. After partitioning into detergent (D1) and aqueous phases (A1), the detergent phase was divided and incubated in the presence or absence of PI-PLC to remove anchors. Phases were re-extracted to yield A2 and D2 fractions and processed for Gas1p immunoprecipitation followed by SDS-PAGE. The total amount of ER form of Gas1p quantified in each partition was set to 100%. (C) Wild-type (RH6082) and arv1 (FK137) strains were spotted onto YPUAD, YPUAD supplemented with 10 µg/ml CFW, and incubated for 4 d at 25°C. (D) Wild-type (RH6082), arv1 (FK137), gpi1 (DL2831), gpi2-7 (DL2828), gpi3-10 (DL2829), eri1/eri1 (DL2680), gwt1-20, gpi7 (FBY182) and gaa1-1 (RH401-7C) were stained for chitin with CFW (1 mg/ml). CFW staining was visualized by fluorescence microscopy. The images were taken with equal exposures. The morphology of the cells was observed by differential interference contrast (DIC).

The delay in maturation of GPI-anchored proteins could result from a defect in GPI anchor synthesis (Schonbachler et al., 1995; Sobering et al., 2004), a defect in GPI anchoring (Hamburger et al., 1995; Doering and Schekman, 1996) or a lack of stable membrane association of GPI-anchored proteins (Watanabe et al., 2002). Therefore, we first examined the membrane association of Gas1p. We found that in the arv1 mutant, a significantly larger fraction of Gas1p was released from the membranes under three different conditions, buffer alone, high pH, and detergent, compared with wild-type membranes, whereas the behavior of Gap1p, an integral membrane protein, and Ypt1p, a prenylated protein, were similar in arv1 and wild-type membranes (Figure S2A), indicating that association of GPI-anchored proteins to the membranes is weakened in arv1 mutant cells.

To test whether the weakened membrane association of Gas1p was due to inefficient GPI anchoring, we examined GPI anchoring of Gas1p by pulse-chase protein labeling and Triton X-114 phase separation (Watanabe et al., 2002). Anchored Gas1p partitioning into the detergent phase can only be shifted into the aqueous phase by treatment with PI-specific phospholipase C (PI-PLC), which removes the diacylglycerol moiety of GPI lipids. Unanchored Gas1p partitions into the aqueous phase and the portion remaining in the detergent phase is not affected by PI-PLC treatment (Nuoffer et al., 1991). After the first partition, in arv1 mutant cells, 40% of the ER form of Gas1p was partitioned into the aqueous phase (A1) in contrast to wild-type cells (13%; Figure 3B). Furthermore, among the fraction that was partitioned into the first detergent phase (D1), most of the ER form of Gas1p in wild-type cells was shifted from the detergent phase (D2, –PI-PLC) to the aqueous phase (A2, +PI-PLC) with PI-PLC treatment, confirming that they were indeed GPI-anchored. However in arv1 mutant cells, only small fraction of the ER form of Gas1p found in D1 fraction was sensitive to PI-PLC treatment, suggesting that they were not GPI-anchored Gas1p. These results demonstrate that GPI anchoring is significantly defective in the arv1 mutant. The anchoring deficiency was confirmed by examining the incorporation of [³H]myo-inositol or [³H]DHS into proteins in arv1 mutant (Figure S2B), both of which label the glycolipid portion of GPI-anchored proteins (Reggiori et al., 1997).

Because strains defective in GPI anchoring (Benghezal et al., 1995) as well as GPI anchor synthesis mutants (Taron et al., 2000; Umemura et al., 2003; Fujita et al., 2004) are hypersensitive to CFW, we reasoned that arv1 mutant cells might display the same phenotype. Indeed, the arv1 mutant cells showed hypersensitivity to CFW (Figure 3C). Hypersensitivity to CFW seen in gpi mutants is most likely due to elevated cell wall chitin levels that are thought to be brought about by either an up-regulated chitin synthase 3 activity in response to cell wall stress (Osmond et al., 1999; Valdivieso et al., 2000; Lesage et al., 2005) or an incidental increase of UDP-GlcNAc concentration (Sobering et al., 2004). Therefore, we looked for evidence of the elevated chitin levels by staining with CFW. For this, we tested six GPI anchor synthesis mutants (e.g., gpi1, gpi2-7, gpi3-10, eri1, gwt1-20, and gpi7) and one GPI-anchoring mutant (e.g., gaa1-1) that are hypersensitive to CFW (data not shown). All gpi mutants that we tested show hyperaccumulation of chitin in the cell wall (Figure 3D), suggesting that the hyperaccumulation of chitin is a common phenotype of gpi mutants. Similarly, arv1 mutant cells hyperaccumulated chitin. This is consistent with our working hypothesis that Arv1p is required for GPI anchoring.

The arv1 Mutant Accumulates GlcN-acylPI and Has a Defect in Synthesis of Man-GlcN-acylPI
It is possible that GPI-anchoring defects result from a block in GPI anchor synthesis. Therefore, we examined whether the arv1 mutant cells have a specific defect in GPI anchor synthesis. We assayed production of GlcNAc-PI, GlcN-PI, and GlcN-acylPI by using an in vitro assay with membranes and a radiolabeled donor, [¹⁴C]UDP-GlcNAc (Schonbachler et al., 1995; Sobering et al., 2004). All three GPI anchor intermediates were detected in membranes of arv1 mutant cells (Figure 4A), indicating that the arv1 mutant has enzyme activities for the first three steps of GPI anchor synthesis. As a control, membranes of the gwt1-20 mutant (Umemura et al., 2003) inefficiently generated GlcN-acylPI (data not shown). Remarkably, arv1 mutant membranes accumulated GlcN-acylPI when compared with wild-type membranes (Figure 4, A and B). The accumulation of GlcN-acylPI could not be detected in arv1 mutant cells by labeling with [³H]myo-inositol (Figure 1, A and D). The reason for this discrepancy is unclear but may be due to differences between in vitro and in vivo assays (e.g., different enzymatic reaction rates) or radioactive substrates used (e.g., different labeling efficiencies). Time courses for Dol-P-Man synthase activity in the same membranes revealed that the higher accumulation of GlcN-acylPI in the arv1 membranes is not due to the lack of Dol-P-Man (Figure 4C), suggesting that later steps of GPI anchor synthesis are affected in the arv1 mutant cells.

View larger version (74K): [in this window] [in a new window]   Figure 4. arv1 mutant cells accumulate GlcN-acylPI and have a strong reduction in mannosylated GlcN-acylPI synthesis. (A and B) In vitro assay for the early steps of GPI anchor biosynthesis was performed as described in Materials and Methods. Membranes from strains wild-type (RH6082) or arv1 (FK137) were incubated with [14C]UDP-GlcNAc for the indicated times at 25°C. The lipids were extracted and analyzed by TLC plates using solvent system IV. The positions of GlcNAc-PI, GlcN-PI, and GlcN-acylPI were marked in A. Radioactivity in each GPI lipid was quantified, and incorporation (%) was determined as percentage of the total radioactivity. Data in B are quantification of A. (C) The same membranes as in A were labeled with [3H]GDP-Man for the indicated times at 25°C. The lipids were extracted and analyzed by TLC plates using solvent system III. (D and E) Strains pmi40 (FK395), arv1 pmi40 (FK397), wild-type (RH6082), and arv1 (FK137) were grown as described in Materials and Methods (D). Wild-type [+ pGAL-HA-GPI18] (FK1019), gpi18 [+ pGAL-HA-GPI18] (FK1017), and arv1 gpi18 [+ pGAL-HA-GPI18] (FK1015) cells were grown in galactose-containing medium, and shifted to glucose-containing medium for 16 h at 25°C to repress GPI18 expression (E). The cells were labeled with [3H]mannose for 30 min at 25°C in the presence of YW3548 (10 µM) or methanol. The lipids were extracted and analyzed by TLC using solvent system III. To give better separation of Dol-P-Man and lipids a–d, some experiments were applied to a double development using the same solvent. Radioactivity in each lipid was quantified, and the relative amounts were calculated by setting the amounts in pmi40 with YW3548, wild-type with YW3548 cells (D) or in gpi18-pGAL-HA-GPI18 without or with YW3548 (E) to 100% and were expressed as percentages of the relative controls. Data in D and E are means ± range for two independent experiments. The relative amounts of total incorporation into lipids were also shown (E). ND, not detectable; a–d, mannolipids accumulated upon YW3548 treatment; e, lipid accumulated in Gpi18-depleted cells, preferentially in the absence of YW3548 [the mobility is consistent with that of (EtNP)Man-GlcN-acylPI (M1), which has been reported to accumulate in Gpi18p-depleted cells (Fabre et al., 2005)]); CP, complete GPI precursor.

To further investigate which step of GPI anchor biosynthesis is defective in the arv1 mutant cells, we carried out an analogous experiment with a specific inhibitor of GPI anchor synthesis, YW3548, that inhibits the addition of EtNP onto the first mannose of the GPI core structure and leads to the accumulation of a GPI lipid, Man-Man-GlcN-acylPI (Man2; Sutterlin et al., 1997b). Because a mutation in phosphomannose isomerase, pmi40, which causes mannose auxotrophy enhances [³H]mannose labeling of mannosylated GPI intermediates (Sipos et al., 1994), pmi40 and arv1 pmi40, mutant strains were created and analyzed for synthesis of mannosylated GPIs. When YW3548 was added to the pmi40 mutant (Figure 4D) or a pmi40 ts mutant (Figure S4A) cells, four major mannose-labeled lipids termed a, b, c, and d were accumulated, whereas synthesis of CP was significantly decreased. All a–d mannosylated lipids are acylated inositol ring-bearing GPI species because they were sensitive to GPI-phospholipase D (PLD; Figure S4, A and B), NaOH (Figure S4A), nitrous acid (Figure S4B) but resistant to PI-PLC (Figure S4, A and B). PI-PLC cleavage requires the two-position of inositol to be unacylated (Doerrler et al., 1996). Because c and d migrated just above MIPC at a position expected for Man2 (Sutterlin et al., 1997b) and because a and b were more hydrophobic than c and d (Figure 4D and Figure S4A), it appears that mannolipids c and d are Man2 species and that a and b species are GlcN-acylPI bearing one mannose (Man1). This was consistent with the fact that lipids (a and b) comigrated with the spot accumulated by shutting off GPI18 expression in the presence of YW3548 (Figure 4E), which would be predicted to be a Man1 species, as Gpi18p is required for addition of the second mannose during GPI assembly (Fabre et al., 2005; Pittet and Conzelmann, 2007).

GPI-PLD cleaves the linkage between the phosphate and inositol in GPI structures and generates phosphatidic acid and mannosylated GlcN-acyl-inositol. If lipid a and b species and lipid c and d species, respectively, have different acyl groups on the inositol ring, then treatment of these lipid mixtures with GPI-PLD should yield products of Man- and Man₂-GlcN-acyl-inositol consisting of at least two species. By treating the mixtures with GPI-PLD, we observed distinct subsets that migrated differently in our TLC system (Figure S4B). The GPI-PLD products derived from lipids (a and b) contained two bands that have slightly different mobilities (R_f = 0.46 and 0.44). The products from lipids c and d had multiple bands migrating at lower mobilities (R_f = 0.34–0.24). The subsets were also seen when [³H]mannose-radiolabeled total lipids were tested (Figure S4A). These results suggest the existence of multiple forms of mannosylated GlcN-acylPI carrying different acyl groups on the inositol.

Importantly, deletion of ARV1 in the pmi40 mutant strains prevented the accumulation of mannolipids (a and b) upon treatment with YW3548 (Figure 4D, left). In the absence of YW3548 a minor amount of these lipids was still observed in the pmi40 mutant, but was not detectable in the arv1 pmi40 mutant. Also, the arv1 mutation suppressed the accumulation of mannolipids (c and d) seen in the pmi40 mutant (Figure 4D, left) or wild-type (Figure 4D, right) cells in the presence of YW3548. The formation of lipids a and b, which accumulate in Gpi18p-depleted cells was reduced by disrupting ARV1 (Figure 4E). These results show indirectly that Arv1p is required for the efficient synthesis of Man1.

Arv1p Is Required for the Delivery of GlcN-acylPI to the GPI-MT
The Man1 synthesis defect could be explained by 1) a block in production and/or translocation of Dol-P-Man across the ER membrane; 2) a defect in the activity of GPI-MT transferring mannose from Dol-P-Man to GlcN-acylPI; and 3) a defect in the delivery of GlcN-acylPI to the GPI-MT. The first possibility is unlikely because underglycosylation of CPY, a phenotype seen in the dpm1 mutant defective in Dol-P-Man synthase (Helenius et al., 2002) was not observed in the arv1 mutant cells (Figure 3A). Indeed, no defect in Dol-P-Man production was observed when arv1 mutant cells were labeled with [³H]mannose (Figure 4D, right) or when arv1 mutant membranes were labeled with [³H]GDP-Man (Figure 4C).

To assess GPI-MT activity, we used ER-enriched membranes from wild-type and arv1 mutant cells labeled with [³H]GDP-Man. The membranes from wild-type cells generated two mannolipids comigrating with lipids b and d, respectively, whereas arv1 membranes inefficiently synthesized them (Figure 5A, lanes 1 and 3). The lipids synthesized in vitro were also sensitive to GPI-PLD but not to PI-PLC and were accumulated upon treatment with YW3548 (data not shown). These results indicate that arv1 mutant membranes have a reduced mannosylGlcN-acylPI synthesis activity, which is consistent with our in vivo results (Figure 4, D and E). Because the incorporation of [³H]GDP-Man into these lipids (b and d) was linear over 15 min and that of [³H]GDP-Man into Dol-P-Man was constant (Figure S5), all subsequent assays were performed at the incubation time of 10 min.

View larger version (69K): [in this window] [in a new window]   Figure 5. arv1 mutant cells have full Dol-P-Man synthesis and GPI-MT activity. (A) To measure GPI-MT activity, the ER-enriched membranes from wild-type (RH6082) and arv1 (FK137) were incubated with [3H]GDP-Man for 10 min at 25°C in the presence (lanes 2, 4, 8, and 10) or absence (lanes 1, 3, 5–7, and 9) of 10 mM n-octyl β-D-glucopyranoside. The experiments were also done in the presence (lanes 6–10) or absence (lanes 1–5) of 0.4 µg/ml GlcN-PI(C8). The lipids were extracted and analyzed by TLC using solvent system III. The detergent causes a slightly altered migration of Dol-P-Man and lipid b. (B) Radioactivity in each lipid was quantified, and the relative amounts were calculated by setting the amounts in wild-type membranes in the presence or absence of detergent to 100%. For the estimation of GPI-MT activity, the amounts of lipids (b and d) or lipid (f) were normalized by dividing by the amounts of Dol-P-Man. Because lipid g comigrated with MIPC, we could not obtain the proper amounts of lipid g for quantification. The results represent the means ± SD from four independent experiments. Lipids f and g represent Man-GlcN-acylPI(C8) and Man2-GlcN-acylPI(C8), respectively, and GPI designates more hydrophilic GPI intermediates than lipid d. Addition of 10 mM n-octyl β-D-glucopyranoside inhibited the incorporation into GPI to 24 ± 9% (wild-type) and 35 ± 19% (arv1) of the amounts without detergent (lanes 1–4; see also Figure S5).

gpi Mutants Affect Sphingolipid Synthesis and Sterol Amounts and Distribution
If the primary function of Arv1p protein was to transport ceramides out of the ER, the defect of Man1 synthesis seen in the arv1 mutant would result from the accumulation of ceramides in the ER. To investigate this possibility, we assayed mannosylGlcN-acylPI synthesis in cells treated with AbA and in sec12 mutant cells, which are defective in ER-to-Golgi protein transport at nonpermissive temperature of 37°C. When wild-type cells were labeled with [³H]mannose, lipids a and b were not detectable at 25°C (Figure 4D, right), but they were detected at 37°C (Figure S6). Neither AbA treatment nor the sec12 allele affected lipid a and b synthesis nor lipid c and d synthesis at 37°C. This result suggests that the accumulation of ceramides in the ER cannot be the reason for the defect of mannosylated GlcN-acylPI synthesis, thereby suggesting that Arv1p functions primarily in the delivery of GlcN-acylPI to the GPI-MT.

On the other hand, we consistently, found that other gpi mutants affect sphingolipid synthesis. When wild-type and gaa1 mutant cells were labeled with [³H]myo-inositol, the gaa1-1 mutant cells showed 30% of the level of IPC-C synthesis as found in wild-type cells (Figure 6A). The extent of reduction was similar to arv1 mutant cells. Moreover, gpi1, gpi2-7, and gpi3-10 mutant cells made 50–60% of the amount of IPC-C synthesized in the wild-type cells. These results underscore that a GPI anchor synthesis or anchoring defect causes a defect in sphingolipid biosynthesis.

View larger version (47K): [in this window] [in a new window]   Figure 6. gpi mutants have a reduced level of sphingolipids and are hypersensitive to aureobasidin A. (A) Wild-type (RH6082), arv1 (FK137), gpi1 (DL2831), gpi2-7 (DL2828), gpi3-10 (DL2829), and gaa1-1 (RH401-7C) were labeled with [3H]myo-inositol for 1 h at 25°C. The lipids were extracted and analyzed by TLC plates using solvent system I. Incorporation (%) into sphingolipids was determined as the percentage of total radioactivity into all lipids containing inositol, and the relative amounts of each species were calculated by setting the amount in wild-type cells to 100%. Data in A are means ± SE for three independent experiments. (B) AbA (final concentration 1 µg/ml, right) or ethanol (left) was spread onto YPUAD plates. Wild-type (RH6082), lac1 lag1 (RH5308), and arv1 (RH6078) were streaked on the plates and incubated for 3 d at 25°C. (C) The same strains as in A were spotted onto YPUAD, YPUAD supplemented with 0.1 µg/ml AbA or ethanol and incubated for 4 d at 25°C.

Because the ARV1 deletion has been shown to accumulate sterols in internal membrane fractions (Tinkelenberg et al., 2000; Beh and Rine, 2004), we asked whether gpi mutants affect intracellular sterol distribution. As detected by filipin staining, the majority of sterols in arv1 and gpi mutants were found in internal membranes of cells. Wild-type cells displayed much less internal filipin levels than the mutants (Figure 7A). In the mutant cells, internal filipin fluorescence was observed in ring-like perinuclear structures of the ER (Figure 7A, arrows). Fluorescence was also seen in the lipid particles, which can be easily visualized with differential interference contrast (DIC; Figure 7A, arrowheads). Although the number and size of lipid particles per cell varies among cells and experiments, gpi mutants seem to have an increased number of lipid particles.

View larger version (65K): [in this window] [in a new window]   Figure 7. gpi mutants affect intracellular distribution and level of total sterols. (A) Wild-type (RH6082), arv1 (FK137), gpi1 (DL2831), gpi2-7 (DL2828), gpi3-10 (DL2829), and gaa1-1 (RH401–7C) were grown to similar densities (7–10 x 106 cells/ml) and then stained for sterols with filipin complex (0.1 mg/ml). The staining was visualized by fluorescence microscopy, and the images were taken with equal exposures. The morphology of the cells and lipid particles were observed by DIC. Arrows and arrowheads indicate ring-like perinuclear ER and lipid particles, respectively. (B) The same strains as in A were grown likewise, and total sterol levels were analyzed by GLC/MS using cholesterol as an internal standard. The amounts of total sterols were determined in two independent experiments analyzed in duplicate, and data are shown as means; error bars, ± range.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
In this study, we have shown that yeast deleted for ARV1 accumulates GlcN-acylPI and is impaired for GlcN-acylPI mannosylation without having defects in Dol-P-Man synthesis or GPI-MT activity. Because N-glycosylation of Gas1p, Yps1p and CPY was not affected in arv1 mutant, the delivery of Dol-P-Man required for synthesis of N-linked oligosaccharides to the mannosyltransferases appears to be normal. Therefore, our results suggest a specific role of Arv1p in the delivery of GlcN-acylPI to the GPI-MT. Furthermore, we provide evidence that GPI anchor synthesis and attachment to proteins are not only required for GPI-anchored protein transport but also regulate ceramide transport from the ER and intracellular sterol distribution.

When we labeled pmi40 or pmi40 ts mutant cells with [³H]mannose, we detected four mannolipids, a–d, that were accumulated upon treatment with YW3548. On the basis of labeling experiments (Figure 4, D and E; Figure S4, A and B), we suggest that the lipids a and b are Man-GlcN-acylPI and c and d are Man₂-GlcN-acylPI species. The mannosylGlcN-acylPI species might contain different types of acyl groups on the inositol ring, which to our knowledge has not been documented in yeast GPI anchor synthesis (Sipos et al., 1997; Reggiori et al., 1997; Bosson et al., 2006; Fujita et al., 2006b). Several studies demonstrated that acyl chains linked to inositol of GPI are heterogeneous in trypanosomes (Guther et al., 1996) and mammalian cells (Houjou et al., 2007). At present, we cannot exclude that the appearance of multiple forms of mannosylGlcN-acylPI might be a secondary effect, because it was neither found in wild-type cells without YW3548 (data not shown) nor with membranes in our in vitro system (Figure 5A and Figure S5). However, it has been shown that yeast membranes can utilize acyl-CoAs containing different lengths of fatty acid (C14–C20) as exogenous substrates for inositol acylation (Costello and Orlean, 1992; Doerrler et al., 1996; Umemura et al., 2003), suggesting that the various species of mannosylated GlcN-acylPI bearing different acyl chain lengths can be generated in yeast.

Murakami et al. (2003) demonstrated that in mammalian cells, the first mannosylation occurs without inositol acylation, similar to the T. brucei GPI biosynthetic pathway (Guther and Ferguson, 1995; Smith et al., 1996). This indicates that mammalian GPI-MT-I can accept GlcN-PI as a substrate. On the other hand, yeast GPI-MT does not seem to act on GlcN-PI. Previous studies have shown that all mannosylated GPI lipids accumulating in yeast mutants, gaa1 (Hamburger et al., 1995), gpi11 (Taron et al., 2000), gpi7 (Benachour et al., 1999), smp3 (Grimme et al., 2001), gpi10 (Sutterlin et al., 1998), and gpi18 (Fabre et al., 2005) were acylated GPI species. Also, we never detected any mannosylated GPI species that was not acylated. Only two acylated species, Man-GlcN-acylPI and (EtNP)Man-GlcN-acylPI, were detected in Gpi18p-depleted cells (Figure 4E). These observations strongly support the idea that the substrate specificity of yeast GPI-MT is restricted to GlcN-acylPI.

Three possibilities could explain how the GPI precursor lipid flips across the ER membrane. First, only GlcN-acylPI synthesized on the cytoplasmic side of the ER may flip into the ER lumen. Second, both GlcN-PI and GlcN-acylPI can flip but only GlcN-acylPI can be mannosylated. Finally, GlcN-PI flipped into the ER lumen can be acylated to yield GlcN-acylPI. In the last model, inositol acylation would occur on the luminal side of the ER, and fatty acyl-CoAs must be present in the ER lumen. Consistent with this notion, it is known that secretory proteins such as Hedgehog and Wnt are acylated in the ER lumen by the related acyl-CoA–dependent acyltransferases (Hofmann, 2000; Orlean and Menon, 2007). However, fatty acyl-CoAs are synthesized in the cytosol or the cytoplasmic side of organelle membranes (Black and Dirusso, 2007), and it has been reported that they do not normally penetrate into microsomal membranes (Gooding et al., 2004). In addition, there is evidence that the yeast ER does not contain enzymes involved in carnitine-dependent acyl-CoA transport across membranes (Tehlivets et al., 2007). These findings lead to the proposal that fatty acyl-CoAs may not reside in the ER lumen. Thus, it remains to be determined if inositol acylation can occur on the luminal side of the ER at all.

What might be the biochemical function of Arv1p? If GlcN-acylPI is synthesized on the cytoplasmic side of the ER, it is possible that Arv1p functions as a GPI flippase or as its regulator. Based on the biochemical analysis using proteoliposomes from a detergent extract of ER with fluorescent GPI analogues (Vishwakarma and Menon, 2005