Glycobiology

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Glycobiology, 2000, Vol. 10, No. 12 1271-1275
© 2000 Oxford University Press

Phosphatidylethanolamine is the donor of the phosphorylethanolamine linked to the 1,4-linked mannose of yeast GPI structures

Isabella Imhof, Elisabeth Canivenc-Gansel, Urs Meyer and Andreas Conzelmann¹

Institute of Biochemistry, University of Fribourg, CH-1700 Fribourg, Switzerland

Received on February 28, 2000; revised on July 13, 2000; accepted on July 18, 2000.

	Abstract

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
Glycosylphosphatidylinositol (GPI) anchors of all species contain the core structure protein-CO-NH-(CH₂)₂-PO₄-Man1–2Man1–6Man1–4GlcN1–6inositol-PO₄-lipid. In recent studies in yeast it was found that gpi10-1 mutants accumulate M2, an abnormal intermediate having the structure Man1–6[NH₂-(CH₂)₂-PO₄]Man1–4GlcN1–6(acyl)inositol-PO₄-lipid. It thus was realized that yeast GPI lipids, as their mammalian counterparts, contain an additional phosphorylethanolamine side chain on the 1,4-linked mannose. The biosynthetic origin of this phosphorylethanolamine group was investigated using gpi10-1 ept1 cpt1, a strain which is unable to synthesize phosphatidylethanolamine by transferring phosphorylethanolamine from CDP-ethanolamine onto diacylglycerol, but which still can make phosphatidylethanolamine by decarboxylation of phosphatidylserine. Gpi10-1 ept1 cpt1 triple mutants are unable to incorporate [³H]ethanolamine into M2 although metabolic labeling with [³H]inositol demonstrates that they make as much M2 as gpi10-1. In contrast, when labeled with [³H]serine, the triple mutant incorporates more label into M2 than gpi10-1. This result establishes that the phosphorylethanolamine group on the 1,4-linked mannose is derived from phosphatidylethanolamine and not from CDP-ethanolamine.

Key words: MCD4/glycosylphosphatidylinositol/phosphatidylethanolamine/Saccharomyces cerevisiae/ethanolaminephosphotransferase

	Introduction

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
Many glycoproteins of lower and higher eukaryotes are attached to the plasma membrane by means of a glycosylphosphatidylinositol (GPI) (Englund, 1993; McConville and Ferguson, 1993; Takeda and Kinoshita, 1995). The GPI biosynthesis seems to have been elaborated early in evolution since the carbohydrate structure linking the C-terminal end of GPI proteins to the lipid moiety is identical in GPI anchors from all organisms analyzed so far, namely protein-CO-NH-(CH₂)₂-PO₄-6Man1–2Man1–6Man1–4GlcNH₂-inositol-PO₄-lipid. GPI anchors from various species differ widely by the kind of side chains attached to this core structure and by their lipid moieties (McConville and Ferguson, 1993). This report concerns the phosphorylethanolamine (P-EtN) side chain which is often present on Man1 of the core structure (Figure 1). Indeed, a P-EtN is invariably found on Man1 of GPI proteins in mammals and in Torpedo californica, but not in Trypanosoma brucei, Leishmania major, or Plasmodium falciparum. (McConville and Ferguson, 1993). Moreover, P-EtN attached to Man1 has also been identified recently on the complete precursor lipid CP2 of Saccharomyces cerevisiae (Canivenc-Gansel et al., 1998). The presence of P-EtN on Man1 of CP2 came as a surprise in as much as a previous analysis of the pool of all GPI anchors of S.cerevisiae, prepared on the basis of their biochemical and biophysical properties and without the preliminary purification of any particular GPI protein had failed to reveal P-EtN on Man1 (Fankhauser et al., 1993). This result probably was partially due to inadvertent hydrolysis of P-EtN during anchor preparation since recent results obtained with refined techniques show that at least 10 to 20% of GPI proteins do contain a phosphodiester-linked group on the 1,4-linked mannose (I.Imhof, unpublished observations).

View larger version (11K): [in this window] [in a new window]   Fig. 1. Structure of the complete GPI precursor lipid CP2. The phosphodiester linked substituent X on Man2 has not yet been identified. P, Phosphate; R, alkyl.

 
It recently was shown, that the addition of this P-EtN in mammalian cells requires PIG-n, an integral membrane protein of the ER, which is the murine homologue of yeast MCD4 and bears a distinct homology with a large group of phosphodiesterases (Gaynor et al., 1999; Hong et al., 1999). Mcd4p and PIG-n can specifically be inhibited by fungal inhibitors such as YW3548 (Sütterlin et al., 1997). The acceptor for P-EtN transfer by PIG-n is Man1–4GlcN1–6(acyl)inositol-PO₄-lipid.

This report attempts to unambiguously identify the immediate donor substrate for the addition of P-EtN to Man1. In particular, we wanted to test, if CDP-ethanolamine or phosphatidylethanolamine (PE) was the donor in this reaction. Early studies had suggested that the bridging P-EtN on Man3 is not transferred directly from CDP-ethanolamine but from an other donor since washed trypanosomal microsomes were able to make complete GPI lipids in the presence of UDP-GlcNAc, GDP-Man, and ATP without any need for the addition of ethanolamine (EtN) or CDP-EtN (Masterson et al., 1989). The same was found to be true for mammalian and yeast microsomal systems (Hirose et al., 1992; Ueda et al., 1993; Canivenc-Gansel et al., 1998). PE was identified as the donor of P-EtN for transfer onto Man3 by an elegant study in yeast (Menon and Stevens, 1992) exploiting the fact that yeast can synthesize PE by two alternative pathways, either by decarboxylation of phosphatidylserine (PS) or by the transfer of EtN from CDP-EtN onto diacylglycerol, a reaction carried out by the two partially redundant enzymes Ept1p and Cpt1p (Hjelmstad and Bell, 1991) (Figure 2). It was shown that [³H]EtN is incorporated into the GPI protein Gas1p in wild type (wt) but not in an ept1 cpt1 strain (Menon and Stevens, 1992). This established that EtN cannot be transferred onto GPI intermediates directly from CDP-EtN but needs to first be added onto diacylglycerol to form PE (Menon and Stevens, 1992). The proposal that the bridging P-EtN is derived from PE has further been supported by metabolic labeling experiments in trypanosomes using a microsomal in vitro system or intact cells (Menon et al., 1993).

View larger version (21K): [in this window] [in a new window]   Fig. 2. Phosphorylethanolamine groups can be transferred to Man1 and Man3 from PE made either by decarboxylation of PS or from CDP-ethanolamine. DAG, Diacylglycerol; PI, phosphatidylinositol.

 
PE may be used for P-EtN transfer to Man3 since the later stages of GPI biosynthesis may occur in the ER lumen while early stages are certainly occurring on the cytosolic leaflet of the ER membrane (Vidugiriene and Menon, 1993, 1994; Menon and Vidugiriene, 1994; Vidugiriene and Menon, 1995; Watanabe et al., 1996; Takahashi et al., 1996; Nakamura et al., 1997). Thus, it appeared possible that the early addition of P-EtN to Man1–4GlcN1–6(acyl)inositol-PO₄-lipid would utilize the cytosolic pool of CDP-EtN. Here however we demonstrate, that the P-EtN on Man1 is equally derived from PE. To do so we use the same genetic approach that previously was used to identify the origin of the P-EtN on Man3 (Menon and Stevens, 1992).

	Results

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
When analyzing lipid extracts from [³H]inositol ([³H]Ins) labeled wt S. cerevisiae cells, it is impossible to detect polar GPI lipids having the TLC mobility of complete precursor lipids (CPs) as are found in other organisms (Sipos et al., 1997). Nevertheless, polar CPs become detectable in several gpi mutants which are deficient in the biosynthesis of GPI structures or their addition to proteins (Hamburger et al., 1995).

Preliminary experiments showed that [³H]Ins gets incorporated by gpi10-1 and ept1 cpt1 to the same extent whereas [³H]EtN is only incorporated by gpi10-1. Gpi10-1 and ept1 cpt1 were crossed and, as expected, all three mutations segregated independently. We chose to analyze the few tetratypes, i.e., tetrads containing both parental types as well as a triple mutant and a wt spore. The lipid profiles of such a tetrad is shown in Figure 3. As judged from the incorporation of [³H]Ins, gpi10-1 ept1 cpt1 synthesize as much M2 as gpi10-1 EPT1 CPT1 cells (Figure 3A, lanes 5 and 7). On the other hand, when the segregants of the same tetrad were labeled by [³H]EtN, M2 is only visible in gpi10-1 EPT1 CPT1 but not in gpi10-1 ept1 cpt1 (Figure 3B, lanes 2 and 4). The same result was obtained in two other tetrads in which the segregation pattern was the same (not shown). The labelings clearly show that the transfer of [³H]EtN onto diacylglycerol by Ept1p and/or Cpt1p is a prerequisite for its attachment to Man1 of M2 (Figure 3B). This result argues that the P-EtN group on the 1,4-linked mannose is not directly transferred from CDP-EtN but from PE. If this is true, we would expect that in gpi10-1 ept1 cpt1 triple mutants, this P-EtN group is derived exclusively from PE made by decarboxylation of PS. This was verified by metabolically labeling the same tetrad as analyzed in Figure 3 with [³H]serine. As shown in Figure 4, M2 could easily be detected after labeling of gpi10-1 mutants with [³H]serine. It also appeared that gpi10-1 ept1 cpt1 incorporated significantly more label into M2 than gpi10-1 and that even ept1 cpt1 cells contained some material comigrating with M2 (Figure 4, lane 3). Close inspection of scans indicated the presence of unrelated [³H]serine-labeled compounds in the region of M2. Therefore we also analyzed the lipid extracts by two-dimensional TLC. As can be seen in Figure 5, this procedure resolved the region of M2 into several labeled compounds, the lower of which can also be detected in wt cells. This two-dimensional TLC confirmed the impressions obtained from one-dimensional TLCs: M2 is more prominent in gpi10-1 ept1 cpt1 than gpi10-1 and ept1 cpt1 also contains a small amount of labeled M2. Close inspection even reveals a trace of M2 in wt cells. Our interpretation is that M2 is a physiological GPI intermediate which can be revealed in wt cells by [³H]serine labeling, which seems to be more sensitive than [³H]Ins labeling. It also appears that the block of the salvage pathway in the ept1 cpt1 mutants increases the incorporation of [³H]serine into M2 (compare Figure 5e with 5c, 5f with 5d). This could be explained by assuming that during labeling with [³H]serine the specific activity of PE gets significantly higher in ept1 cpt1 than in EPT1 CPT1 wt strains. Indeed the relative amount of c.p.m. in PE was 1.5-fold higher in ept1 cpt1 strains than EPT1 CPT1 strains.

View larger version (44K): [in this window] [in a new window]   Fig. 3. Analysis of lipids from yeast metabolically labeled with [3H]Ins or [3H]EtN. (A) 2.5OD600 of wt cells (W303–1B, lane 1), two haploid parental strains (HJ000 cpt1 ept1; FBY637 gpi10-1 lane 3) and the strains FBY37, FBY38, FBY39 and FBY310 representing the progeny of a single tetrad obtained by crossing HJ000 with FBY637 (lanes 4–7) were labeled with [3H]-Ins. (B) 10OD600 of the same strains were labeled with [3H]EtN: tetrad (lanes 1–4), HJ000 (lane5), FBY637 (lane6) and W303–1B (lane7). The genotypes are indicated at the bottom with X indicating a mutant allele. Extracted lipids from both labelings were separated by TLC with solvent system chloroform/methanol/0.25% KCl 55/45/10 (v/v/v) in (A) and chloroform/methanol/water 10/10/3 (v/v/v) in (B). It is unknown why ept1 cpt1 cells still make minor amounts of labeled lipids comigrating with PC and PE. It is conceivable that PS synthase (Cho1p) occasionally utilizes ethanolamine instead of serine as a substrate. PI, Phosphatidylinositol; IPC, inositolphosphorylceramide; MIPC, Mannosylinositolphosphorylceramide; O, origin.

 

View larger version (98K): [in this window] [in a new window]   Fig. 4. Analysis of lipids from yeast metabolically labeled with [3H]serine. The same tetrad (lanes 1–4), parental strains (lanes 5, 6) and wt strains (lane 7) as in Figure 3 were labeled. 6OD600 of cells were labeled and between 9 and 15% of the added radioactivity was recovered in the lipid extracts. The genotypes are indicated at the bottom with "x" indicating mutant alleles. Extracted lipids were separated by TLC with solvent system chloroform/methanol/0.25% KCl 55/45/10 (v/v/v).

 

View larger version (112K): [in this window] [in a new window]   Fig. 5. Two-dimensional analysis of yeast lipids metabolically labeled with [3H]Ser. The lipid extracts obtained in the experiment shown in Figure 4, lanes 1–6 were also analyzed by two-dimensional TLC with the first dimension running in chloroform/methanol/0.25% KCl 55/45/10 (v/v/v), the second in chloroform/methanol/water 10/10/3 (v/v/v).

 

	Discussion

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
The functional role of P-EtN side chains in yeast is not clear yet: data so far support the view that they are required for GPI biosynthesis since MCD4 is an essential gene and, in yeast, the mannosyltransferase adding Man3 apparently does not add onto Man1–6Man1–4GlcN1–6(acyl)inositol-PO₄-lipid unless the 1,4-linked mannose is substituted with a P-EtN (Sütterlin et al., 1998; Gaynor et al., 1999). In contrast to P-EtN on Man3, the P-EtN on Man1 seems not to be used to link GPI structures to proteins (Canivenc-Gansel et al., 1998). Deletion of the murine MCD4 homologue PIG-n does not abolish but only reduces the surface expression of GPI proteins although the synthesis of the complete GPI lipid H8 is completely abrogated (Hong et al., 1999).

The presence of an additional P-EtN on Man1 on CP2 and on some yeast GPI proteins led us to reevaluate the interpretation of data obtained using metabolic labeling with [³H]EtN in the past. (1) The previously reported absence of labeling of Gas1p in ept1 cpt1 (Menon and Stevens, 1992) could prove that the P-EtN on Man1 as well as the one on Man3 is derived from PE if it were known that Gas1p carries a P-EtN on Man1. Since this is presently unknown, these previous data cannot be interpreted in this sense. (2) Moreover, since Gas1p indeed may carry a P-EtN on Man1, the previously reported absence of labeling of Gas1p in ept1 cpt1 does not formally prove that PE derived from CDP-EtN can be used as a donor substrate for the addition of P-EtN onto Man3: This data can not rule out that normally only the PE derived from PS is a donor substrate for the addition of P-EtN onto Man3 whereas PE derived from CDP-EtN may be used solely for the addition of P-EtN onto Man1. However, in the trypanosomal system, where no other P-EtN than the one on Man3 is added, CDP-[³H]EtN efficiently labels the complete precursor in vitro, a finding that clearly establishes that PE made from CDP-[³H]EtN can be the donor for the transfer of P-EtN onto Man3. In view of the high degree of conservation of GPI biosynthetic enzymes among eukaryotic organisms, it thus is safe to conclude that PE made by either pathway can serve as a donor for P-EtN transfer onto Man3. Additionally, since psd1 psd2 double mutants, which almost completely lack the ability to make PE from PS (Trotter and Voelker, 1995) are still viable, one can conclude that PE made from CDP-EtN can probably be used for transfer of P-EtN onto Man3, at least in this mutant.

For the experiments reported here, we chose to work with gpi10-1 in order to analyze a well characterized GPI structure, lipid M2, which contains one single P-EtN and thus yields an unambiguous result. The data clearly establish that P-EtN added on Man1 is transferred from PE, and that PE made from CDP-EtN as well as PE made by decarboxylation of PS can be utilized for this biosynthetic step.

In summary, the present and previous data strongly argue that P-EtN residues on Man3 and Man1 stem from PE which can be made by either of the two biosynthetic pathways as depicted in Figure 2.

	Materials and methods

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
Strains, media, and materials
Saccharomyces strains are listed in Table I and were grown as described previously (Benghezal et al., 1996). Strain HJ000 was obtained from Dr. McMaster, Atlantic Research Centre, Department of Pediatrics, Dalhousie University, Halifax, Canada. The absorbance of dilute cell suspensions was measured in a 1 cm cuvette at 600 nm; one OD₆₀₀ unit of cells corresponds to 1–2.5•10⁷ cells. Reagents were purchased from the following sources: [2-³H]-myo-inositol, 20 Ci/mmol and [1-³H]-ethanolamine (30Ci/mmol) from Anawa Trading SA, (Zürich, Switzerland).

View this table: [in this window] [in a new window]   Table I. Yeast strains

 
The gpi10-1 ept1 cpt1 triple mutant was obtained by crossing FBY637 with HJ000 and using the leucine and uracil prototrophy to follow the cpt1::LEU2 and ept1::URA3 disruption alleles in the resulting haploid progeny. Leu⁺ ura⁺ prototrophs were labeled with [³H]Ins to identify the gpi10-1 ept1 cpt1 triple mutants.

Radiolabeling of GPI lipids and GPI proteins
Cells were precultured in SDCUA, resuspended in SDUA supplemented with amino acids, preincubated for 10 min and labeled with [2-³H]Ins (2 µCi/OD₆₀₀ of cells) for 60 min at 37°C as described (Canivenc-Gansel et al., 1998). Labeling of cells with [³H]EtN was done as described (Menon and Stevens, 1992) with slight modifications: cells were precultured in "complete synthetic medium" at 24°C. 10 OD₆₀₀ units of growing cells were resuspended in 1 ml of the same, preincubated for 10 min at 37°C, 50 µCi of [³H]EtN were added and cells were incubated in a shaking water bath at 37°C. After 90 min, cells were diluted with 1 volume of fresh medium and incubated for further 90 min. For [³H]Ser labeling cells were preincubated in SDYEUA (SDUA + 0.2% yeast extract) at 24°C and resuspended in SDUA supplemented with amino acids corresponding to auxotrophies, preincubated for 10 min at 37°C and labeled for 40 min (10µCi/ OD₆₀₀ of cells) followed by a dilution with 4 volumes of fresh medium and further incubation for 80 min. Labeling was stopped by addition of 10mM NaN₃/NaF (final concentration). Lipids were extracted with chloroform/methanol/water 10:10:3 (v/v/v) and desalted by butanol/water phase separation as described (Sipos et al., 1994). Lipid extracts were analyzed by ascending TLC using 0.2 mm-thick silica gel plates. Radioactivity was detected by one- and two-dimensional radioscanning and fluorography (Benghezal et al., 1995).

	Acknowledgements

 
We thank Dr. C.McMaster for yeast strains. This work was supported by Grant No. 3100–032515 from the Swiss National foundation.

	Abbreviations

Top Abstract Introduction Results Discussion Materials and methods Abbreviations References

 
CP, complete precursor; EtN, ethanolamine; GPI, glycosylphosphatidylinositol; Ins, myo-inositol; Man, mannose; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PS, phosphatidylserine; P-EtN, phosphorylethanolamine; wt, wild type.

	Footnotes

 
¹ To whom correspondence should be addressed

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