The EMBO Journal Vol.16 No.12 pp.3494–3505, 1997 Alternative lipid remodelling pathways for glycosylphosphatidylinositol membrane anchors in Saccharomyces cerevisiae all reflect the lipid moieties present in phosphatidylinositol Gyo ¨ rgy Sipos, Fulvio Reggiori, (PI) which would appear as a natural starting point for Christine Vionnet and the GPI biosynthesis in the endoplasmic reticulum (ER) Andreas Conzelmann 1 (Roberts et al., 1988b; Luhrs and Slomiany, 1989; Bu ¨ti- Institute of Biochemistry, University of Fribourg, Rue du Muse ´e, kofer et al., 1990, 1992; Walter et al., 1990; McConville Pe ´rolles, CH-1700 Fribourg, Switzerland and Ferguson, 1993; Patnaik et al., 1993; Doering et al., 1 Corresponding author 1994; Redman et al., 1994; Brewis et al., 1995; Serrano e-mail: andreas.conzelmann@unifr.ch et al., 1995; Treumann et al., 1995; Kapteyn et al., 1996). In principle, this difference could come about by the Glycosylphosphatidylinositol (GPI)-anchored mem- specificity of the GlcNAc-transferase which initiates GPI brane proteins of Saccharomyces cerevisiae exist with biosynthesis by transferring GlcNAc onto PI or by the two types of lipid moiety—diacylglycerol or ceramide— prevalence of a rare kind of PI in the environment of both of which contain 26:0 fatty acids. To understand this enzyme. Alternatively, lipid moieties might become at which stage of biosynthesis these long-chain fatty exchanged either before or after transfer of the GPI onto acids become incorporated into diacylglycerol anchors, proteins (Englund, 1993). This latter model has been found we compared the phosphatidylinositol moieties isolated to apply to the blood stage form of Trypanosoma brucei from myo-[2- 3 H]inositol-labelled protein anchors and where longer fatty acids are replaced by myristic acid from GPI intermediates. There is no evidence for the through sequential deacylation/reacylation reactions on presence of long-chain fatty acids in any intermediate sn-2, then sn-1 shortly before the GPI is attached to the of GPI biosynthesis. However, GPI-anchored proteins protein (Masterson et al., 1990). A remodelling mechanism contain either the phosphatidylinositol moiety charac- has also been proposed to explain the prevalence of sn- teristic of the precursor lipids or a version with a long- 1-alkyl, 2-acylglycerol in mammalian cells (Singh et al., chain fatty acid in the sn-2 position of glycerol. The 1994). The situation in yeast is peculiar since two very introduction of long-chain fatty acids into sn-2 occurs different types of lipid moieties can be found: ceramides in the endoplasmic reticulum (ER) and is independent and another lipid which is sensitive to mild base treatment of the sn-2-specific acyltransferase SLC1. Analysis of and can be removed by PI-specific phospholipase C (PI- ceramide anchors revealed the presence of two types PLC), probably diacylglycerol. The ceramides are found of ceramide, one added in the ER and another more on the majority of yeast anchors; they mainly consist of polar molecule which is found only on proteins which C18:0 phytosphingosine and a C26:0 fatty acid and are have reached the mid Golgi. In summary, the lipid of different from the main ceramide found in the abundant GPI-anchored proteins can be exchanged by at least inositolphosphoceramides (IPCs) (Smith and Lester, 1974; three different remodelling pathways: (i) remodelling Lester and Dickson, 1993). Alternatively, Gas1p, a well- from diacylglycerol to ceramide in the ER as proposed characterized GPI protein of yeast, is made with a C26:0 previously; (ii) remodelling from diacylglycerol to a fatty acid (FA)-containing, base-sensitive lipid (Fank- more hydrophobic diacylglycerol with a long-chain hauser et al., 1993). In both types of lipid moieties the fatty acid in sn-2 in the ER; and (iii) remodelling to a C26:0 may be hydroxylated on C2. Complete GPIs (CPs) more polar ceramide in the Golgi. ready to be transferred to proteins are present only in very Keywords: ceramide/diacylglycerol/glycosylphos- low amounts in wild-type cells but can be detected in phatidylinositol/remodelling/Saccharomyces cerevisiae lipid extracts of [2- 3 H]mannose-labelled pmi40, a strain with a conditional defect in mannose biosynthesis incorp- orating elevated amounts of exogenously added [ 3 H]man- nose at 37°C (Sipos et al., 1994). CPs also become Introduction detectable in extracts from [ 3 H]inositol-labelled gaa1 and gpi8 cells which both have a conditional mutation affecting Numerous glycoproteins of Saccharomyces cerevisiae the addition of CPs to proteins (Benghezal et al., 1995; become attached to a glycosylphosphatidylinositol (GPI) Hamburger et al., 1995). In all these cases, the CPs are anchor and many ultimately appear in the cell wall or at completely susceptible to mild base treatment, supporting the plasma membrane (Hardwick et al., 1992; Stratford, the notion that the ceramide moieties may be introduced 1994; Cawley et al., 1995; Komano and Fuller, 1995). at a later stage by lipid remodelling of protein anchors. The biosynthesis of GPI-anchored proteins follows the The work presented here addresses the question of whether same basic rules in all eukaryotes, including yeast the C26:0 long-chain FAs of mild base-sensitive anchors (Conzelmann et al., 1990; Englund, 1993; Fankhauser are present already at the beginning of GPI biosynthesis, et al., 1993; Nuoffer et al., 1993). Nevertheless, different or whether they are introduced only at a later stage. organisms contain widely differing kinds of lipid moieties in their GPI anchors which, in most organisms, do not at It appeared impossible to obtain GPI intermediates in 3494 © Oxford University Press