The N-glycosylation defect of cwh8Δ yeast cells causes a distinct defect in sphingolipid biosynthesis

Pittet, Martine; Uldry, Danièle; Aebi, Markus; Conzelmann, Andreas

doi:10.1093/glycob/cwj043

Cited by 21 publications

(22 citation statements)

References 37 publications

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“…S5) (13), and lyso-IPCs therefore seem to be made by Aur1p. Aur1p has previously been shown to use not only the physiological C26-and C24-containing ceramides but also ceramides with shorter fatty acids of 2, 6, or 16 carbon atoms (13,56), and this report clearly shows that in the absence of other substrates, Aur1p can even use free LCBs, albeit with a preference for LCB-C20 over LCB-C18. However, we could not find ions corresponding to mannosylated lyso-IPCs, suggesting that lyso-IPCs are not a substrate for Csg1p or Csh1p.…”

Section: Discussionmentioning

confidence: 54%

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Yeast Cells Lacking All Known Ceramide Synthases Continue to Make Complex Sphingolipids and to Incorporate Ceramides into Glycosylphosphatidylinositol (GPI) Anchors

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Roubaty

Ejsing

et al. 2011

Journal of Biological Chemistry

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In yeast, the inositolphosphorylceramides mostly contain C26:0 fatty acids. Inositolphosphorylceramides were considered to be important for viability because the inositolphosphorylceramide synthase AUR1 is essential. However, lcb1⌬ cells, unable to make sphingoid bases and inositolphosphorylceramides, are viable if they harbor SLC1-1, a gain of function mutation in the 1-acyl-glycerol-3-phosphate acyltransferase SLC1. SLC1-1 allows the incorporation of C26:0 fatty acids into phosphatidylinositol (PI), thus generating PI؆, an abnormal, C26-containing PI, presumably acting as surrogate for inositolphosphorylceramide. Here we show that the lethality of the simultaneous deletion of the known ceramide synthases LAG1/LAC1/LIP1 and YPC1/YDC1 can be rescued by the expression of SLC1-1 or the overexpression of AUR1. Moreover, lag1⌬ lac1⌬ ypc1⌬ ydc1⌬ (4⌬) quadruple mutants have been reported to be viable in certain genetic backgrounds but to still make some abnormal uncharacterized inositol-containing sphingolipids. Indeed, we find that 4⌬ quadruple mutants make substantial amounts of unphysiological inositolphosphorylphytosphingosines but that they also still make small amounts of normal inositolphosphorylceramides. Moreover, 4⌬ strains incorporate exogenously added sphingoid bases into inositolphosphorylceramides, indicating that these cells still possess an unknown pathway allowing the synthesis of ceramides. 4⌬ cells also still add quite normal amounts of ceramides to glycosylphosphatidylinositol anchors. Synthesis of inositolphosphorylceramides and inositolphosphorylphytosphingosines is operated by Aur1p and is essential for growth of all 4⌬ cells unless they contain SLC1-1. PI؆, however, is made without the help of Aur1p. Furthermore, mannosylation of PI؆ is required for the survival of sphingolipid-deficient strains, which depend on SLC1-1. In contrast to lcb1⌬ SLC1-1, 4⌬ SLC1-1 cells grow at 37°C but remain thermosensitive at 44°C.

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Section: Discussionmentioning

confidence: 54%

“…On the other hand, as stated before, Aur1p accepts ceramides with C20:0 down to C2:0 fatty acids as substrates (13,29,56). Thus, if the unknown ceramide synthase of 4⌬ cells would make ceramides with shorter fatty acids, they should be transformed into IPCs.…”

Section: Discussionmentioning

confidence: 82%

Yeast Cells Lacking All Known Ceramide Synthases Continue to Make Complex Sphingolipids and to Incorporate Ceramides into Glycosylphosphatidylinositol (GPI) Anchors

Vionnet

Roubaty

Ejsing

et al. 2011

Journal of Biological Chemistry

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“…In cax4⌬ cells and in cells where N-linked glycosylation has been otherwise compromised, sphingolipid composition is significantly altered (52). Specifically, in cax4⌬ cells there is a considerable reduction in inositolphosphorylceramides (IPCs), which represent a major class of sphingolipids (52).…”

Section: Resultsmentioning

confidence: 99%

“…Specifically, in cax4⌬ cells there is a considerable reduction in inositolphosphorylceramides (IPCs), which represent a major class of sphingolipids (52). To test whether the effect of CAX4 on sphingolipid composition has a bearing on neutral lipid storage, lipid droplets in lcb1-100 ts (YJN63), lcb2 ts (YJN64), and wild-type (W303-1A) cells were visualized using Nile red fluorescence microscopy.…”

Section: Resultsmentioning

confidence: 99%

Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae

et al. 2008

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The pandemic of lipid-related disease necessitates a determination of how cholesterol and other lipids are transported and stored within cells. The first step in this determination is the identification of the genes involved in these transport and storage processes. Using genome-wide screens, we identified 56 yeast (Saccharomyces cerevisiae) genes involved in sterol-lipid biosynthesis, intracellular trafficking, and/or neutral-lipid storage. Direct biochemical and cytological examination of mutant cells revealed an unanticipated link between secretory protein glycosylation and triacylglycerol (TAG)/steryl ester (SE) synthesis for the storage of lipids. Together with the analysis of other deletion mutants, these results suggested at least two distinct events for the biogenesis of lipid storage particles: a step affecting neutral-lipid synthesis, generating the lipid core of storage particles, and another step for particle assembly. In addition to the lipid storage mutants, we identified mutations that affect the localization of unesterified sterols, which are normally concentrated in the plasma membrane. These findings implicated phospholipase C and the protein phosphatase Ptc1p in the regulation of sterol distribution within cells. This study identified novel sterol-related genes that define several distinct processes maintaining sterol homeostasis.Both cholesterol biosynthesis and storage are controlled in response to levels and localization of regulatory pools of sterols (33,37,54,65). In response to high cholesterol levels in the endoplasmic reticulum (ER) membrane, the enzyme acyl coenzyme A (CoA):sterol O-acyltransferase (ASAT) initiates sterol esterification and storage by covalently coupling fatty acids to cholesterol. Through an active process, the esterified cholesterol is amalgamated with other neutral lipids into lipid storage droplets that are released from the ER membrane (42, 88). The trafficking of unesterified sterols also affects the sterol distribution in regulatory pools. Although cholesterol is synthesized in the ER, the highest level of unesterified cholesterol is found in the plasma membrane (33) and maintenance of normal sterol levels requires the efficient transport of cholesterol from the ER membrane to the plasma membrane. The maintenance of cholesterol levels in the plasma membrane is affected by sorting from endosomal compartments and recycling back to the cell surface (33, 54), and feedback regulation of cholesterol on its own biosynthesis and storage also controls levels of cellular sterols (16, 68). These findings suggest that the maintenance of cellular cholesterol homeostasis requires the regulatory integration of cholesterol synthesis, storage, and transport pathways.As in mammalian cells, the budding yeast Saccharomyces cerevisiae synthesizes its own cholesterol-like lipids but, under normal aerobic conditions, yeast does not internalize exogenous sterol lipids. Apart from this difference, other elements of sterol homeostasis, including lipid storage and transport pathways, app...

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“…Ceramides are found in the GPI anchors of certain plants (e.g., pears), Trypanosoma cruzi, Paramecium, Aspergillus fumigatus, and Dictyostelium, sometimes as the sole anchor lipid (3,27). Recent studies show that, similar to the case in yeast, the first steps of GPI biosynthesis in A. fumigatus and T. cruzi do not use ceramide as the lipid support, suggesting that ceramide is added by remodeling at a later step not only in yeast but also in other species (2,11).…”

Section: Discussionmentioning

confidence: 99%

Incorporation of Ceramides intoSaccharomyces cerevisiaeGlycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

et al. 2009

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After glycosylphosphatidylinositols (GPIs) are added to GPI proteins of Saccharomyces cerevisiae, a fatty acid of the diacylglycerol moiety is exchanged for a C 26:0 fatty acid through the subsequent actions of Per1 and Gup1. In most GPI anchors this modified diacylglycerol-based anchor is subsequently transformed into a ceramide-containing anchor, a reaction which requires Cwh43. Here we show that the last step of this GPI anchor lipid remodeling can be monitored in microsomes. The assay uses microsomes from cells that have been grown in the presence of myriocin, a compound that blocks the biosynthesis of dihydrosphingosine (DHS) and thus inhibits the biosynthesis of ceramide-based anchors. The lipid moieties of the glycosylphosphatidylinositol (GPI) lipid at the stage when it is transferred by the transamidase to GPI proteins are different from those found on mature GPI anchors of Saccharomyces cerevisiae. The free GPI lipids contain a phosphatidylinositol (PI) moiety, which comigrates in thin-layer chromatography (TLC) with the free PI of yeast membranes and therefore probably contains the typical C 16:0 and C 18:1 fatty acids found in yeast PI (7,12,26,29,31). In contrast, the majority of mature protein-linked GPI anchors contain a ceramide moiety, and a minor fraction contains a diacylglycerol modified to have C 26:0 fatty acids in sn2 (9, 31). Ceramides of GPI anchors contain phytosphingosine (PHS) and C 26 fatty acids, as do the bulk of the free sphingolipids in yeast membranes, which are inositolphosphorylceramides (IPCs) and derivatives thereof (9, 32). Thus, all mature GPI proteins of yeast contain large lipid moieties with C 26 fatty acids, in the form of either a ceramide or a special diacylglycerol, and these lipids are introduced by remodeling enzymes (remodelases) that replace the primary lipid moiety of the anchor.

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The N-glycosylation defect of cwh8Δ yeast cells causes a distinct defect in sphingolipid biosynthesis

Cited by 21 publications

References 37 publications

Yeast Cells Lacking All Known Ceramide Synthases Continue to Make Complex Sphingolipids and to Incorporate Ceramides into Glycosylphosphatidylinositol (GPI) Anchors

Yeast Cells Lacking All Known Ceramide Synthases Continue to Make Complex Sphingolipids and to Incorporate Ceramides into Glycosylphosphatidylinositol (GPI) Anchors

Genome-Wide Analysis of Sterol-Lipid Storage and Trafficking in Saccharomyces cerevisiae

Incorporation of Ceramides intoSaccharomyces cerevisiaeGlycosylphosphatidylinositol-Anchored Proteins Can Be Monitored In Vitro

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