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Distribution and Functions of and

J. Thomas Hannich1, Kyohei Umebayashi1, and Howard Riezman

Department of , University of Geneva, CH-1211 Geneva 4, Switzerland Correspondence: [email protected]

Sterols and sphingolipids are considered mainly eukaryotic even though both are present in some prokaryotes, with sphingolipids being more widespread than sterols. Both sterols and sphingolipids differ in their structural features in vertebrates, , and fungi. Interestingly, some invertebrates cannot synthesize sterols de novo and seem to have a reduced dependence on sterols. Sphingolipids and sterols are found in the plasma mem- brane, but we do not have a clear picture of their precise intracellular localization. Advances in lipidomics and subcellular fractionation should help to improve this situation. Genetic approaches have provided insights into the diversity of and func- tions in providing evidence that these two classes function together. Intermediates in sphingolipid and degradation are involved in signaling path- ways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.

ypical examples of eukaryotic lipids, sterols, . Sphingolipids are more widely spread Tand sphingolipids can both be found in among prokaryotes than sterols and also show membranes from simple unicellular fungi and a greater variety of structures among the differ- protists to multicellular and plants. ent eukaryotes. Their versatile use as structural elements but In this short review we will first give an also as signaling molecules has probably played overview about the diversity of sterol and sphin- an important role during the evolution of this golipid structures and their distribution in large and diverse group of organisms. There nature. Then we will discuss their subcellular are also many eukaryotes that have lost the abil- distribution. A brief technical section will add ity to synthesize sterols de novo including nem- some information on the separation and detec- atodes, insects, and marine invertebrates, which tion of these lipid molecules. Subsequently, have to take up sterols with their diet. Sterol bio- we will summarize different genetic approaches synthesis has also been reported in a number of to study the functions of sterols and sphingo- lipids, and finally, we will discuss the functional and possible physical interactions of the two 1These authors contributed equally to this article. lipid classes within the cell. Far from being

Editor: Kai Simons Additional Perspectives on The Biology of Lipids available at www.cshperspectives.org Copyright # 2011 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a004762 Cite this article as Cold Spring Harb Perspect Biol 2011;3:a004762

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J.T. Hannich et al.

comprehensive, we will focus only on a few to biosynthesis (Ohyama et al. interesting aspects and try to give new view 2009). Both synthase and lano- points, which are less frequently discussed. sterol synthase are closely related to each other and to the bacterial -hopene cyclases, which produce the bacterial polycyclic hope- STRUCTURES AND DISTRIBUTION AMONG noids without a requirement for molecular oxy- SPECIES gen and which are most likely the evolutionary Sterol biosynthesis probably evolved after the ancestors of the sterol biosynthetic enzymes. emergence of oxygen about 2.5 billion years Cycloartenol and are further metabo- ago (Galea and Brown 2009). The first dedi- lized via (de-) methylations and double-bond cated step, which is common to all sterol bio- modifications to give rise to the final sterol synthetic pathways, is the mono-oxygenation products: several C-24 alkylated , of squalene catalyzed by Squalene Epoxidase/ C28 , C29 sitosterol, and stigma- Erg1p and uses molecular oxygen. Even at this sterol in plants (Benveniste 1986), a single early step in the pathway, the cyclization of the C-24 methylated C28 in most fungi linear precursor, which gives rise to the polycy- (Weete et al. 2010), and the single nonmethy- clic sterol ring structure, shows a diversification lated C27 in animals (Gaylor 2002) between kingdoms, with cycloartenol as the (Table 1). Most invertebrates including the product in most algae and plants and lanosterol important genetic model organisms in fungi and animals (Volkman 2003). Recently, melanogaster and Caenorhabditis elegans have it was found that some plants also have the abil- lost the ability to synthesize sterols (Clayton ity to produce lanosterol, which can contribute 1964; Kurzchalia and Ward 2003). They are

Table 1. Sterol and sphingolipid structures and distribution among species.

Auxotrophic Organism Plants Fungi Vertebrates Bacteria invertebrates Main sterol Campesterol (R = methyl) Ergosterol, Cholesterol Modifications of Generally absent, Sitosterol (R = ethyl) some exceptions dietary sterols very few exceptions (R = ethyl, Δ22)

R

H H H

H H

H HHHHH HO HO HO

Main sphingoid Several bases including: Variety of structures Sphingosine D. melanogaster : Mostly absent, Δ8∗ S. cerevisiae: Δ4* C sphingosine some exceptions base d18:1 ∗ (d18:1 ), 14 Δ4Δ8∗ sphinganine (d18:0 ) C. elegans: d18:2 ∗ several others present Δ ∗ phytosphingosine (t18:0 ) C iso-sphingosine t18:1 8 17 P. pastoris: 9-methyl- sphinga-4,8,-dienine

Higher -Inositolphosphoryl -Inositolphosphoryl -Sphingomyelin -Sphingomyelin (C. elegans) Mostly absent, sphingolipids glycolipids glycolipids -Glycosphingolipids -Phosphoethanolamine some exceptions with -Glycosphingolipids -Glycosphingolipids (mainly glucosyl-, some ceramide (D. melanogaster) glucosyl ceramide (mainly glucosyl-, some S. cerevisiae: only galactosylceramide series) -Glucosyl ceramide series series + + + mannosylceramide series) IPC , MIPC , M(IP)2C

SREBP Not found Oxygen sensor, SREBP1a/c regulate fatty Regulates fatty acid and Not found but not found in acid & fat metabolism S. cerevisiae SREBP2 regulates sterol metabolism

Shorthand nomenclature for sphingoid bases “aXX:YDZ”: a-number of hydroxylgroups (d ¼ 2, t ¼ 3); XX-number of carbon atoms; Y-double bonds, DZ-position of double bonds. þIPC-inositolphosphorylceramide, MIPC-mannosylinositolphosphorylceramide,

M(IP)2C-mannosyldiinositolphosphorylceramide.

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Sterols and Sphingolipids

dependent on external sterol sources and use C22 (Han and Jiang 2009) as well as 1-deoxy some of the remaining conserved sterol biosyn- and 1-deoxymethyl long chain bases can be thetic enzymes for sterol modifications, includ- found (Penno et al. 2010). Differences in double ing hormone production (Vinci et al. bonds and base hydroxylation additionally give 2008) and regulation (Hannich et al. 2009). rise to sphinganine (d18:0), phytosphinganine Another very important feature of sterol (t18:0), and their analogues. Also in plants C18 biosynthesis in vertebrates is its regulation by sphingoid bases, especially with D8 and D4D8 the membrane bound basic helix-loop-helix double bonds, predominate (Pata et al. 2010). (bHLH) transcription factor SREBP (Osborne Many fungi additionally methylate the alkyl and Espenshade 2009). The vertebrate genome chain at C-9, giving rise to C19 bases (Ternes contains two copies encoding three isoforms et al. 2006), but the baker’s yeast S. cerevisiae with SREBP-2 regulating mainly cholesterol produces only sphinganine and phytosphinga- biosynthesis and SREBP-1a and c regulating nine (mainly d18:0 and t18:0, some d20:0, and mainly fatty acid and metabolism t20:0). In invertebrates other chain lengths are as well as fat storage. Invertebrates have one abundant. Flies produce mainly C14 and some copy of SREBP, which has been shown to regu- C16 straight chain bases (Acharya and Acharya late fatty acid and phospholipid metabolism 2005), whereas nematodes build their sphingo- in flies and fatty acid metabolism and fat accu- lipids from C17 iso-branched sphingoid bases mulation in worms (see Ye and Debose-Boyd (Chitwood et al. 1995). Free LCBs exist only at 2011). Although no SREBP homolog has yet very low levels in the cell. Most of these can be been found in Saccharomyces cerevisiae, other phosphorylated to form sphingosine-1-phos- fungi like Schizosaccharomyces pombe also ex- phate and other phosphorylated sphingoid press a copy that serves as a sterol-dependent bases. The majority of sphinganine is trans- oxygen sensor that regulates oxygen-dependent formed into dihydroceramide (likewise for steps in ergosterol, heme, and sphingolipid other sphingoid bases) by N-acylation with fatty metabolism (Todd et al. 2006). So far no acids ranging from C14 to C26 or perhaps even SREBP homolog has been described in plants up to C36 in rare cases. The majority of incorpo- even though a number of membrane-bound rated fatty acids are saturated or monounsatu- bHLH transcription factors still await further rated and can be modified with an a and one characterization. additional hydroxylation. Great diversity bet- A detailed description of sphingolipid ween species is created by different polar head biosynthesis can be found in recent reviews groups that are attached to the C-1 hydroxyl (Hannun and Obeid 2008; Futerman and group of the ceramide backbone to create high- Riezman 2005). Sphingolipids are a very diverse er sphingolipids, including phosphosphingo- class of lipids, which is predicted to include tens lipids, glycosylinositolphosphorylceramides, and of thousands or maybe more distinct structures glycosphingolipids. Phosphorylation leads to (Merrill et al. 2009). This diversity is due to ceramide-phosphate but also the phosphobases combinations of structural varieties of sphin- phosphocholine and phosphoethanolamine can goid bases, the N-acylated fatty acid, and the be attached. These give rise to sphingomyelin, polar head group. The basic structure of the which is abundant in vertebrates and some central building block, the sphingoid/long- invertebrates like C. elegans, and phosphoetha- chain base (LCB), depends on the substrate spe- nolamine ceramide, which is only produced in cificity of its biosynthetic enzymes. very low amounts in mammalian cells (Vacaru The main vertebrate LCB is D-erythro- et al. 2009) but replaces sphingomyelin in sphingosine (short d18:1D4E for (2S,3R,4E)- D. melanogaster (Acharya and Acharya 2005). 2-aminooctadec-4-ene-1,3-diol; see Table 1 Plants and fungi have a series of glycolipids legend for an explanation of the shorthand that are derived from inositolphosphoryl nomenclature) but in addition also substantial ceramide (IPC), which can have a diverse range amounts of C20 and minor amounts of C12 to of more or less complex (modified) sugar chains

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J.T. Hannich et al.

attached to it (Pata et al. 2010). Plants, fungi, metabolic feature is therefore used as a signature invertebrates, and vertebrates all also transfer for different eukaryotic traces in paleogeology sugars directly onto the ceramide moiety to (Summons et al. 1988). form glycosphingolipids. In plants, glucosyl- Production of sphingolipids in bacteria is ceramide predominates and serves as a basic more widespread. The first crystal structure structure for series of up to tetraglycosylcer- for a sphingolipid biosynthetic enzyme was amides but also some mannosylceramide series obtained from a soluble bacterial serine pal- are produced (Sperling and Heinz 2003). mitoyl transferase (SPT) (Yard et al. 2007). Glucosyl- and to a lesser degree galactosylcer- Recently, the sphingosine 1 phosphate lyase amides are also widely spread among fungi, structure was determined from Symbiobacte- even though S. cerevisiae does not show any rium thermophilum (Bourquin et al. 2010). glycosylceramides. In invertebrates and verte- In some species like Sphingomonas capsulata, brates, hundreds of different head group struc- glycosphingolipids functionally replace lipo- tures are formed by adding different chains of polysaccharide in the outer membrane (Geiger sugar molecules and their derivatives to the et al. 2010). It can be assumed that sphingolipid first sugar moiety, which is mainly glucose. biosynthesis first evolved in prokaryotes from In addition, vertebrates also show a series other members of the a-oxoamine synthase of galactosylceramide-based glycolipids. In S. family, which fuse organic acids to amino acids, cerevisiae the higher sphingolipids are inositol- and probably was already present in the LECA. phosphoryl ceramide (IPC), and its derivatives So far, no prokaryotes with both sphingolipid mannosyl inositolphosphoryl ceramide (MIPC), and sterol synthesis have been discovered to and mannosyl di-inositolphosphoryl ceramide our knowledge. (M(IP)2C) (Dickson and Lester 1999). Very few studies have shown unequivocally DISTRIBUTION WITHIN CELLS the presence of sterols in prokaryotes, but both biochemical and genomic analysis have It is a long-standing dogma that concentrations identified a fairly elaborated sterol biosynthetic of sterols increase along the secretory pathway, pathway in Methylococcus capsulatus producing being very low in the ER, higher in the Golgi, also demethylated sterols (Bird et al. 1971; and highest in the plasma membrane. Sterols Lamb et al. 2007) and a very basic pathway in have also been shown to be enriched in early Gemmata obscuriglobus that only produces and recycling endosomes, but not late endo- cycloartenol and its isomer (Pearson somes. In lipid storage diseases such as et al. 2003). Most likely the last eukaryotic com- Niemann-Pick type C (NPC), sterol and sphin- mon ancestor (LECA) produced an elaborate golipids are abnormally accumulated in late set of sterols via the cycloartenol pathway and endocytic compartments (Mesmin and Max- differences with modern day eukaryotes are field 2009). In our opinion this dogma still characterized by small changes, including side needs to be confirmed by unequivocal experi- chain methylations (Desmond and Gribaldo mental data. Many studies on sterol localization 2009). Although these rare observations of bac- depend on binding to filipin, but we do not have terial sterol biosynthesis were initially explained sufficient molecular insights into how filipin by horizontal gene transfer from an early eukar- binds cholesterol and other sterols as the struc- yote, the dynamic endo-membrane system of ture of the complex has not been determined. G. obscuriglobus and the recent discovery of its There is also substantial experimental data membrane coat-like proteins, both additional suggesting that the evaluation of sterol concen- key eukaryotic features, have tempted others to tration by filipin binding is dependent on mem- suggest a close relationship of these bacteria to brane composition (see Jin et al. 2008, as an the origin of eukaryogenesis (Santarella-Mellwig example). The sterol content of a specific organ- et al. 2010). To date no side-chain C-24 alkyla- elle can also be measured by subcellular fractio- tion has been observed in prokaryotes and this nation and lipid quantification, however many

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Sterols and Sphingolipids

of the previous studies, while using state of the compounds, including fluorescent filipin, art purification techniques, did not analyze which has long been used for this purpose. In pure fractions. As will be discussed later, current both yeast and mammalian cells, filipin stains advances in mass spectrometry and organelle the plasma membrane and intracellular com- immunopurification should provide compre- partments, probably the endosomes and per- hensive information about the lipid composi- haps the Golgi. Using filipin, genome-wide tion of isolated organelles, thereby allowing us screening for genes regulating sterol localiza- to address the reality of the dogma. Recently, tion was conducted in both yeast and mamma- highly purified ER membrane was prepared, lian cells. These studies identified many genes, and subsequent quantification by mass spec- suggesting the complexity of intracellular sterol trometry showed a very delicate threshold localization. For example, in yeast mutants of sterol regulatory element-binding protein deleted for phospholipase C (PLC1) or protein (SREBP)-mediated cholesterol sensing (Rad- phosphatase type 2C (PTC1) gene, intense hakrishnan et al. 2008): the ER cholesterol con- filipin staining was observed in intracellular centration above or below 5 mol% is sharply compartments, probably late endosomes (Fei distinguished. In addition, a quantitative shot- et al. 2008). Thus, signal transduction pathways gun lipidomics approach (Ejsing et al. 2009) involving these proteins may sense and/or con- showed that post-Golgi secretory vesicles are trol sterol homeostasis. The screening of mam- enriched in sterols and sphingolipids, consis- malian cells identified a novel endolysosomal tent with the idea that lipid sorting occurs in protein TMEM97 whose knockdown reduced the trans-Golgi network (Klemm et al. 2009). the intensity of filipin staining as well as uptake In this study, the vesicles were purified from a of fluorescent LDL, and this protein was co- conditional yeast mutant. It is highly difficult immunoprecipitated with NPC1 (Bartz et al. to isolate organelles without contamination, 2009). Independent of such screening, abnor- for example separation of Golgi and endosomes mal filipin staining was observed in yeast is generally difficult and even immunoisolation mutants defective in GPI biosynthesis (Schon- techniques will have to rely on proteins other bachler et al. 1995; Kajiwara et al. 2008). Some than the many that cycle between the two com- gpi mutants are also defective in sphingolipid partments. Due to the high sensitivity of mass biosynthesis, possibly affecting the step of spectrometry it is no longer necessary to isolate ceramide transport, suggesting that GPI-anch- large amounts of purified organelles for analysis ored proteins may facilitate sterol and sphingo- and therefore, more sophisticated and selective lipid transport to the cell surface. The existence techniques can now be used. It is important to of the GPI moiety, whose structure is remodeled develop such techniques in order to gain insight from a diacylglycerol to ceramide in yeast may into normal and abnormal states, such as nor- stabilize or participate in sterol-sphingolipid mal and NPC endosomes. interactions affecting transport. In model membranes, under optimal condi- Ergosterol biosynthesis is a common target tions of lipid composition and temperature, ster- of drugs, such as polyene anti- ols and sphingolipids are laterally segregated biotics amphotericin B, natamycin, and nysta- from other lipids to form different physical tin. Recently, a natural antifungal compound phases, including a liquid-ordered phase. The theonellamide (TNM) was shown to target 3b- concept of “” states that these phase sep- hydroxysterol (Ho et al. 2009; Nishimura et al. arations also occur in biological membranes, 2010). TNM is an unusual bicyclic peptide and incorporating specific proteins that preferentially represents a novel class of sterol-binding com- interact with these two lipids. Currently, lipid pounds. As described below, yeast cells can be rafts are considered to be highly dynamic nano- stained using a fluorescent version of TNM. scale structures (Lingwood and Simons 2010). The initial steps of sphingolipid biosynthe- As mentioned above, sterols have been sis, including sphingoid base and ceramide for- localized by making use of sterol-binding mation, take place in the endoplasmic reticulum

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J.T. Hannich et al.

(Futerman and Riezman 2005). Higher sphin- columns in which lipids elute as lipid classes. golipids are produced in the Golgi apparatus Absolute quantification would require a stand- where they are transported by different mecha- ard curve for every analyzed molecule at the nisms. In mammalian cells, ceramides for glyco- respective ionization condition or a corre- sphingolipid production seem to be transported sponding stable isotope labeled standard for via a different pathway than SM, which is pro- each molecule. Although both possibilities are duced from a specific ceramide pool trans- not realistic in a vertebrate system the second ported via a nonvesicular pathway mediated one is actually feasible for the rather simple by the CERT protein (Hanada et al. 2003). In sphingolipidome of S. cerevisiae (Ejsing et al. yeast cells both vesicular and nonvesicular ER 2009). In most systems a sufficient number of to Golgi ceramide transport exists (Funato and internal standards containing unnatural and Riezman 2001). Most higher sphingolipids are stable isotope labeled lipids can ensure semi- transported to the plasma membrane. quantitative lipid analysis. In addition, the precise identification of most sterols cannot be determined based solely METHODS OF SEPARATION AND on mass measurements. Different chromato- DETECTION graphic methods have been developed to in- Mass spectrometric shotgun lipidomics as crease sensitivity by removing ion-suppressing described by Schwudke et al. (2011) is a power- background and to differentiate molecular ful analytical technique with many advantages structures based not only on mass and fragmen- but also limitations with respect to sterol and tation but also on retention time, which is deter- sphingolipid analysis even though careful sam- mined by structural differences even between ple preparation and high dilutions can reduce isomeric species. In the case of sterols, gas chro- ion suppression by glycerophospholipids in matography coupled to an EI-source has proven the nonseparated extract. Utilization of a chip- to give reliable results (Guan et al. 2009). based nano-spray setup provides high reprodu- cibility, whereas removal of phosphoglyceroli- GENETIC APPROACHES TO STEROL AND pids and triacylglycerides by base treatment SPHINGOLIPID FUNCTION makes it possible to detect a variety of sphingo- lipids (Jiang et al. 2007), although there are Both sterols and sphingolipids play important isomeric species that cannot be distinguished structural roles in many cellular membranes. (like glucosyl and galactosyl ceramides) and Sterols have been shown to influence fluidity many overlapping isotopic species that could and permeability of membranes, both in vitro be resolved by high mass accuracy fourrier and in vivo (Haines 2001; Emter et al. 2002). transformation mass spectrometry often show Still, at least some eukaryotic cells, like cultured signals that are too weak to gain structural in- insect cells, can function without almost any formation based on fragmentation. Different sterol in their membranes (Silberkang et al. protocols based on high performance liquid 1983) and it has been proposed that sphingo- chromatography coupled to ESI-MS have been lipids with dien-sphingoid bases can replace tailored for specific lipid classes (Sullards et al. sterols in the membrane (Carvalho et al. 2010). 2007). Although column separation increases Certain glycosphingolipids have also been sensitivity and structural information it also proposed to serve a structural role, for example introduces problems to the analysis, including in the chilling response of plants (Pata et al. changing ionization conditions during the 2010). Another very important role of sterols course of the analysis and column memory. that has to be kept in mind when studying their For quantitative analysis it is important to cellular functions is the production of different ensure that internal standards elute under the signaling molecules. Multicellular organisms same ionization conditions as the respective rely on sterol-derived hormones like the analytes, which is easier on normal-phase of vertebrates, brassinosteroids of plants and

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Sterols and Sphingolipids

ecdysteroids, or other molting hormones of the defects in erg mutants. However, results invertebrates. Other sterol-derived signaling measuring plasma membrane fluidity by fluo- molecules include D, bile acids, and rescence anisotropy of TMA-DPH did not oxysterols (Kurzchalia and Ward 2003). correlate with the severity of the mutant pheno- Yeast genetics has been a powerful tool to types (Guan et al. 2009). It was postulated that elucidate the biosynthesis and functions of ster- defective protein-lipid interactions in the erg ols and sphingolipids. Mutants defective in var- mutants caused the defects. One hypothesis is ious steps of these metabolic pathways were that some membrane proteins cannot fit into isolated. Many efforts have focused on mutants the altered membranes and their folding or (erg2, erg3, erg4, erg5, and erg6) defective in the activities are impaired. One of the noticeable last five steps of the biosynthesis, because each is phenotypes of the erg6 mutant is reduced a nonessential step. In these mutants, ergosterol uptake of tryptophan from the medium (Gaber is absent and specific sterol intermediates are et al. 1989). This is because the high-affinity accumulated. The erg mutants are defective in tryptophan permease Tat2 is inappropriately many cellular processes, indicating that ergo- ubiquitinated and missorted to the vacuole sterol cannot be replaced by the sterol inter- (Fig. 1) (Umebayashi and Nakano 2003). The mediates. The differences between ergosterol missorting of Tat2 is common to other erg and the intermediates are the desaturation sta- mutants (Daicho et al. 2009; Guan et al. tus of the A and B rings and the side chain mod- 2009), indicating that the sorting of Tat2 is ifications. Despite the slight differences from very sensitive to sterol structures, strictly requir- one another, each erg mutant has distinct ing ergosterol. One possibility is that Tat2 inter- defects. For example, endocytic internalization acts with sterol-sphingolipid complexes and, in from the plasma membrane is strongly affected the erg mutants, Tat2 is misfolded and placed in the erg2 mutant, defective in the C-8 sterol under ubiquitin surveillance. Identification of isomerase activity, but not drastically altered other membrane proteins that undergo ubiqui- in the erg6 mutant, defective in the C-24 sterol tination in the erg mutants would help us to methyltransferase activity (Munn et al. 1999; explain their diverse phenotypes. Alternatively, Heese-Peck et al. 2002). This highlights the the erg mutants may have a defect in signaling, structural importance of sterol. Because yeast such as amino acid sensing, so that they always cells do not take up sterol from the medium sort Tat2 to the vacuole. There have been two under aerobic conditions, the sterol composi- examples of signaling involved in Tat2 sorting. tion of each mutant is clearly defined and can First, plasma membrane localization of Tat2 is be quantitatively determined by GC-MS. Nota- regulated by the nutrient availability, which is bly, changes in ergosterol structure in the differ- sensed by the target of rapamycin (TOR) kinase ent erg mutants confer different sphingolipid (Fig. 1). Following starvation, TOR is inacti- compositions, without affecting glycerophos- vated and Tat2 is sorted to the vacuole in a pholipids, as determined by mass spectrometry ubiquitin-dependent manner (Beck et al. 1999). (Guan et al. 2009). Thus, cells adjust their Downstream from TOR, the Npr1 kinase and sphingolipid profile in response to changes in the type 2A phosphatase-associated protein sterol structures, strongly suggesting that sterols Tap42 are involved in this regulation (Schmidt and sphingolipids interact, perhaps physically. et al. 1998). Second, addition of the exoge- Using combinations of mutations in sterol and neous LCB phytosphingosine (PHS) inhibits sphingolipid biosynthesis a number of synthetic tryptophan uptake, most likely by transducing and suppression phenotypes were found. These a signal that down-regulates Tat2 (Fig. 1) findings led to the conclusion that sterols and (Skrzypek et al. 1998). In addition to being sub- sphingolipids interact functionally in biological strates of ceramide synthases, LCBs are known membranes. as signaling molecules that activate yeast It is theoretically possible that changes in PDK1 kinase homologs Pkh1 and Pkh2 as well membrane fluidity could account for many of as AGC kinase homologs Ypk1 and Ypk2 (Sun

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Starvation Tat2 PHS Tat2 Plasma Plasma Ub membrane Ub membrane

TOR Ub (TOR?) Ub inactivated signaling? Tat2 Tat2 Golgi/endosome Golgi/endosome

Vacuole Vacuole

erg mutants Plasma membrane

Tat2 misfolding? Ub Nutrient sensing problems? Tat2 Golgi/endosome

Vacuole

Figure 1. Regulation of Tat2 sorting to the vacuole. (Upper left) Following starvation, the TOR signaling pathway is inactivated. This leads to ubiquitin-dependent sorting of both newly synthesized and plasma membrane- localized Tat2 to the vacuole. (Upper right) Exogeneous PHS down-regulates Tat2, possibly by ubiquitination and vacuolar sorting. Signals activated by PHS remain to be elucidated. (Lower)Inerg mutants, Tat2 is inap- propriately ubiquitinated and sorted to the vacuole, not the plasma membrane. One possible reason is that Tat2 is misfolded by the altered sterol and sphingolipid composition. Alternatively, the sterol mutants may be defective in nutrient sensing.

et al. 2000; Friant et al. 2001; Liu et al. 2005). sphingolipids may influence amino acid sensing Although the putative signaling pathway through TORC1. Notably, TORC1 is associated between the exogeneous PHS and Tat2 down- with the vacuole/lysosome membrane (Sturgill regulation has not been characterized yet, this et al. 2008; Sancak et al. 2010). Its activity and/ may be linked to the TOR pathway. Aronova or localization could be sensitive to the lipid et al. (2008) showed that TOR complex 2 content of this organelle. (TORC2) regulates ceramide synthesis by acti- Lipid rafts have also been proposed to act as vating the Ypk2 kinase. Considering that Tat2 signaling platforms, clustering signaling mole- localization is influenced by TOR signaling, it cules for efficient transmission. During mating, is interesting that sterols and sphingolipids haploid yeast cells are polarized into pear- have recently been implicated in regulation of shaped “shmoos,” and MAPK signaling is TORC1 and TORC2. In yeast, it was shown activated to direct signaling required for mat- that TORC2 activity is synthetically impaired ing. At the shmoo tip, both filipin staining in a sterol and sphingolipid double mutant and the MAPK scaffold protein Ste5 are concen- (Guan et al. 2009). In mammalian cells, trated (Pryciak and Huntress 1998; Bagnat and cholesterol trafficking defects such as NPC defi- Simons 2002). One of the common pheno- ciency reduce the activities of the two TORCs types among the erg2–6 mutants is reduced (Xu et al. 2010). Since TORC1 couples amino mating efficiency, including the MAPK signal- acid availability to cell growth, sterols and ing and subsequent plasma membrane fusion.

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Strikingly, the erg3 mutant is defective in the 1996). Although the C7-C8 double bond polarized plasma membrane localization of introduced by Erg2/D8-D7 sterol isomerase Ste5 (Jin et al. 2008). Thus, correct sterol com- persists in ergosterol, it is removed by 7-dehy- position is required for the polarized recruit- drocholesterol reductase (DHCR7) to form ment of a signaling molecule. Ste5p binds to cholesterol. Mutations in the DHCR gene cause PI(4,5)P2 through its PH domain, and this is Smith-Lemli-Opitz syndrome with develop- important for its correct localization (Garren- mental abnormalities (Fitzky et al. 1999). ton et al. 2006). It seems that PI(4,5)P2 is con- Thus, unlike yeast, the C7-C8 double bond centrated at the shmoo tip in wild-type cells, has to be removed in mammals. These diseases but not in the erg6 mutant (Jin et al. 2008). Fur- tell us that correct sterol biosynthesis is vital ther work is needed to understand how sterol also in mammals. It is not clear whether the influences the PI(4,5)P2 distribution. phenotypes seen in yeast and human mutants Although ergosterol is enriched in the are due to the lack of cholesterol or the presence plasma membrane, the localization of sterol of sterol intermediates. If the former is the intermediates seems to be somewhat different. case the defects might be alleviated by feeding When fission yeast cells are stained with the cholesterol. If the latter, this strategy would not fluorescent TNM, the cell periphery is clearly work. As mentioned above, the yeast erg mutants labeled in wild-type but the labeling is very are defective in mating. When the erg4 mutant weak in the erg2 or erg3 mutant (Nishimura was forced to take up ergosterol under an anae- et al. 2010). The binding of TNM to the sterol robic condition, it restored shmoo formation fractions prepared from these mutants was nor- but not subsequent cell fusion (Aguilar et al. mal, strongly suggesting that the sterol inter- 2010). However, under these conditions, de mediates are not preferentially localized to the novo ergosterol synthesis is shut down because plasma membrane. Consistent with this, the of lack of oxygen and the only source of ergo- erg3 mutant of the budding yeast shows abnor- sterol is that taken up from the medium. mal filipin staining with significant labeling of Sphingolipids, especially LCBs, ceramides cytoplasmic spots (Fei et al. 2008). The reason and their phosphorylated forms are important for the altered localization is unclear, but the signaling molecules (Fyrst and Saba 2010) structural changes in sterol-sphingolipid com- whose numerous functions in vertebrates will plexes may facilitate clearance from the plasma not be described in detail here. Sphingosine 1 membrane or provide limited access to sterol phosphate is an extracellular signaling mole- transport proteins. In any case, reduced sterol cule that binds to G-protein coupled receptors levels in the plasma membrane could affect and controls cell migration and many other the sorting and function of plasma membrane physiological parameters. Recently, intracellular proteins such as Tat2. functions of sphingosine 1 phosphate in verte- The above results clearly indicate that subtle brates have been revealed in control of protein differences in sterol structure drastically affect ubiquitination (Alvarez et al. 2010) and tran- various membrane-related cellular events in scription (Hait et al. 2009). In yeast, recent stud- yeast. Also in mammals, it has been reported ies integrating genomic, transcriptomic, and that defects in late cholesterol biosynthetic steps lipidomic data have revealed a role for phyto- cause severe diseases. Mutations in a D8-D7 sphinganine 1 phosphate in regulating genes sterol isomerase were identified in human involved in mitochondrial respiration (Cowart X-linked dominant chondrodysplasia punctata et al. 2010). Another sphingolipid connection as well as its relevant disease in mice (Derry to mitochondrial function comes from the et al. 1999). This enzyme isomerizes a double dual localization of the yeast sphingomyelinase bond in the B ring, the same reaction as cata- homolog, Isc1p, which is found in both ER lyzed by yeast Erg2p. Indeed, both the murine and mitochondria (Vaena de Avalos et al. 2004). and human enzymes complement the yeast Genetic approaches have allowed the identi- erg2 mutant to produce ergosterol (Silve et al. fication of most of the enzymes in sphingolipid

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metabolism in yeast (Dunn et al. 2000; Dickson identification of CERT (Hanada et al. 2003) et al. 2006) and many homologs have been and insights derived from the analysis of found in other systems (Futerman and Riezman knockout mice, including mutants in glyco- 2005). Sphingolipids play a large number of sylceramide synthase (Yamashita et al. 1999; roles in yeast, many of which have been recently Sprong et al. 2001) and ceramide synthase 2 reviewed (Dickson 2008). A number of roles for (Imgrund et al. 2009; Pewzner-Jung et al. LCBs in yeast have been identified by use of a 2010a,b). Mutants in ceramide synthases in temperature-sensitive mutant in serine palmi- D. melanogaster (Bauer et al. 2009) and C. ele- toyltransferase (lcb1-100) activity, which was gans (Deng et al. 2008) have also provided new originally isolated as an endocytosis mutant functions for sphingolipids in these organisms, (end8) (Munn and Riezman 1994). LCBs were including regulation of the response to anoxia shown to activate the Pkh1 and 2 kinases to reg- (Menuz et al. 2009), while mutants in other ulate the actin cytoskeleton and the internaliza- steps of sphingolipid synthesis have been linked tion step of endocytosis (Friant et al. 2000, to gut development (Marza et al. 2009). We are 2001; Zanolari et al. 2000). Interestingly, sphin- still early in the period of the use of genetics to golipid metabolism also affects the endocytic study sphingolipid and sterol function in ani- pathway in D. melanogaster, genetically inter- mals and together with the new tools of lipido- acting with components of the internalization mics we can expect an exciting period in the machinery, such as dynamin (Rao and Acharya years to come. 2008). The lcb1-100 also shows defects in maturation and trafficking, including the plasma membrane ATPase (Lee ACKNOWLEDGMENTS et al. 2002; Gaigg et al. 2005, 2006) and The work of the authors was supported by GPI-anchored proteins (Horvath et al. 1994; grants from the Swiss National Science Founda- Bagnat et al. 2000; Watanabe et al. 2002). In tion (HR), SystemsX.ch (evaluated by the both cases, the evidence suggests that the SNSF), the European Science Foundation synthesis of ceramides is important. For the (EuroMembrane), and an EMBO long-term fel- GPI-anchored proteins this could be because lowship (J.T.H). of the fact that most GPI-anchored proteins are remodeled to contain ceramide (Reggiori et al. 1997) and that this step is required for their REFERENCES concentration into ER exit sites (Castillon et al. Acharya U, Acharya JK. 2005. Enzymes of sphingolipid 2009). Sphingolipids also play roles in regula- metabolism in Drosophila melanogaster. Cell Mol Life tion of translation affecting formation of RNA Sci 62: 128–142. processing bodies (Cowart et al. 2010) and reg- Aguilar PS, Heiman MG, WaltherTC, Engel A, Schwudke D, Gushwa N, Kurzchalia T, Walter P. 2010. Structure of ulating translation initiation during the heat sterol aliphatic chains affects yeast cell shape and cell shock response (Meier et al. 2006). fusion during mating. Proc Natl Acad Sci 107: 4170– S. cerevisiae mutants with a defect in synthe- 4175. sis of MIPC show a hypersensitivity to calcium Alvarez SE, Harikumar KB, Hait NC, Allegood J, Strub GM, Kim EY, Maceyka M, Jiang H, Luo C, Kordula T, et al. ions (Beeler et al. 1997), but MIPC synthesis 2010. Sphingosine-1-phosphate is a missing cofactor does not seem to be required for targeting of for the E3 ubiquitin ligase TRAF2. Nature 465: 1084– plasma membrane proteins (Lisman et al. 1088. 2004). However, in S. pombe MIPC does seem Aronova S, Wedaman K, Aronov PA, Fontes K, Ramos K, Hammock BD, Powers T. 2008. Regulation of ceramide to play a role in plasma membrane protein biosynthesis by TOR complex 2. Cell Metab 7: 148–158. localization (Nakase et al. 2010). Bagnat M, Simons K. 2002. Cell surface polarization during In mammalian systems a large variety of yeast mating. Proc Natl Acad Sci 99: 14183–14188. functions have been ascribed to sphingolipids Bagnat M, Keranen S, Shevchenko A, Simons K. 2000. Lipid rafts function in biosynthetic delivery of proteins and functional insights have also been de- to the cell surface in yeast. Proc Natl Acad Sci 97: rived from genetic approaches, including the 3254–3259.

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Distribution and Functions of Sterols and Sphingolipids

J. Thomas Hannich, Kyohei Umebayashi and Howard Riezman

Cold Spring Harb Perspect Biol 2011; doi: 10.1101/cshperspect.a004762 originally published online March 30, 2011

Subject Collection The Biology of Lipids

Role of Lipids in Virus Replication Membrane Organization and Lipid Rafts Maier Lorizate and Hans-Georg Kräusslich Kai Simons and Julio L. Sampaio Model Answers to Lipid Membrane Questions Shotgun Lipidomics on High Resolution Mass Ole G. Mouritsen Spectrometers Dominik Schwudke, Kai Schuhmann, Ronny Herzog, et al. Glycosphingolipid Functions Glycosphingolipid Functions Clifford A. Lingwood Clifford A. Lingwood Regulation of Cholesterol and Fatty Acid Phosphoinositides in Cell Architecture Synthesis Annette Shewan, Dennis J. Eastburn and Keith Jin Ye and Russell A. DeBose-Boyd Mostov Lipid-Mediated Endocytosis Synthesis and Biosynthetic Trafficking of Helge Ewers and Ari Helenius Membrane Lipids Tomas Blom, Pentti Somerharju and Elina Ikonen Fluorescence Techniques to Study Lipid Lipid Polymorphisms and Membrane Shape Dynamics Vadim A. Frolov, Anna V. Shnyrova and Joshua Erdinc Sezgin and Petra Schwille Zimmerberg Lysosomal Lipid Storage Diseases Specificity of Intramembrane Protein−Lipid Heike Schulze and Konrad Sandhoff Interactions Francesc-Xabier Contreras, Andreas Max Ernst, Felix Wieland, et al. Distribution and Functions of Sterols and Dynamic Transbilayer Lipid Asymmetry Sphingolipids Gerrit van Meer J. Thomas Hannich, Kyohei Umebayashi and Howard Riezman

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