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.
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Abstract |
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Key words: MCD4/glycosylphosphatidylinositol/phosphatidylethanolamine/Saccharomyces cerevisiae/ethanolaminephosphotransferase
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Introduction |
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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
).
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Results |
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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.
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Discussion |
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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.
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Materials and methods |
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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
).
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Acknowledgements |
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Abbreviations |
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Footnotes |
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References |
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