Mechanisms of Sterol Uptake and Transport in Yeastଝ

G Model SBMB-3587; No. of Pages 9 ARTICLE IN PRESS

Journal of Steroid Biochemistry & Molecular Biology xxx (2010) xxx–xxx

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Journal of Steroid Biochemistry and Molecular Biology

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Review Mechanisms of sterol uptake and transport in yeastଝ

Nicolas Jacquier, Roger Schneiter ∗

Department of Medicine, Division of Biochemistry, University of Fribourg, Chemin du Musée 5, CH-1700 Fribourg, Switzerland article info abstract

Article history: Sterols are essential lipid components of eukaryotic membranes. Here we summarize recent advances in Received 12 July 2010 understanding how sterols are transported between different membranes. Baker’s yeast is a particularly Received in revised form attractive organism to dissect this lipid transport pathway, because cells can synthesize their own major 12 November 2010 sterol, ergosterol, in the membrane of the endoplasmic reticulum from where it is then transported to Accepted 30 November 2010 the plasma membrane. However, Saccharomyces cerevisiae is also a facultative anaerobic organism, which becomes sterol auxotroph in the absence of oxygen. Under these conditions, cells take up sterol from the Keywords: environment and transport the lipid back into the membrane of the endoplasmic reticulum, where the Cholesterol Sterol metabolism free sterol becomes esterified and is then stored in lipid droplets. Steryl ester formation is thus a reliable Lipid homeostasis readout to assess the back-transport of exogenously provided sterols from the plasma membrane to the Lipid transport endoplasmic reticulum. Structure/function analysis has revealed that the bulk membrane function of Endoplasmic reticulum the fungal ergosterol can be provided by structurally related sterols, including the mammalian choles- Lipid detoxification terol. Foreign sterols, however, are subject to a lipid quality control cycle in which the sterol is reversibly Lipid droplets acetylated. Because acetylated sterols are efficiently excreted from cells, the substrate specificity of the Steryl esters deacetylating enzymes determines which sterols are retained. Membrane-bound acetylated sterols are Saccharomyces cerevisiae excreted by the secretory pathway, more soluble acetylated sterol derivatives such as the steroid precursor pregnenolone, on the other hand, are excreted by a pathway that is independent of vesicle formation and fusion. Further analysis of this lipid quality control cycle is likely to reveal novel insight into the mechanisms that ensure sterol homeostasis in eukaryotic cells. © 2010 Published by Elsevier Ltd.

Contents

1. Introduction ...... 00 2. Uptake and export of sterols by mammalian cells ...... 00 3. Sterol transport in yeast ...... 00 4. Sterol uptake and transport back to the ER ...... 00 5. Sterol sensing and uptake by other fungal species ...... 00 6. Genetic screens for sterol uptake and transport mutants ...... 00 7. Sterol acetylation, a quality-control step that controls sterol efﬂux ...... 00 8. Perspectives ...... 00 References ...... 00

1. Introduction associated functions, including vesicle formation and protein sorting, endocytosis, homotypic membrane fusion, the activity of Sterols constitute an important class of lipids, which har- membrane embedded enzymes, the lateral aggregation between bor multiple essential functions ranging from signal transduction proteins and lipids, and the ion permeability of the membrane bar- to protein lipidation. The precise structure and concentration rier [1–5]. Due to their concentration-dependent modulation of of sterols within eukaryotic membranes affect many membrane these functions, the distribution of sterols between different membranes needs to be tightly controlled. The lowest concentration of the free sterol is typically found in the endoplasmic reticulum (ER), where cholesterol levels are maintained at around 5 mol.% of total ଝ Article from a special issue on Steroids and Microorganisms. lipids [6]. The sterol concentration then appears to increase along ∗ Corresponding author. Tel.: +41 26 300 8654; fax: +41 26 300 9735. E-mail address: [email protected] (R. Schneiter). the membranes of the secretory pathway to reach a maximum at

Please cite this article in press as: N. Jacquier, R. Schneiter, Mechanisms of sterol uptake and transport in yeast, J. Steroid Biochem. Mol. Biol. (2011), doi:10.1016/j.jsbmb.2010.11.014 G Model SBMB-3587; No. of Pages 9 ARTICLE IN PRESS 2 N. Jacquier, R. Schneiter / Journal of Steroid Biochemistry & Molecular Biology xxx (2010) xxx–xxx the plasma membrane, which harbors 90% of the free sterol pool of Absorption of nutritional sterols in the intestinal lumen by the the cell and where free sterols account for 30 mol.% of total lipids enterocytes requires the Niemann-Pick C1 like protein (NPC1L1), ∼ [7–12]. a glycosylated brush border membrane protein with 13 pre- Sterols are synthesized and mature in the ER by a cascade of cou- dicted transmembrane domains, including a sterol sensing domain pled enzymatic reactions. The final product is cholesterol in case [28,29]. Mice lacking NPC1L1 are resistant to diet-induced hyper- of animal cells, stigmasterol, sitosterol, and campesterol in case cholesterolemia, suggesting that inhibition of this cholesterol of plants and ergosterol in fungal cells [13]. While these mature uptake route by the NPC1L1-specific inhibitor, ezetimibe, may pro- sterols slightly differ in structure between the different kingdoms vide an effective treatment to reduce LDL-cholesterol levels [30]. of life, their basic functions appear to be conserved. It is interest- Ezetimibe also reduces plasma phytosterol levels in patients with ing to note that these kingdom-specific changes in sterol structure sitosterolemia, indicating that NPC1L1, appears to transport a wide are frequently accompanied by corresponding structural changes in range or sterols. Those foreign sterols then need to be exported the major sphingolipids synthesized, indicating that complex for- again through the action of the heterodimeric ATP-binding cassette mation between sterols and sphingolipids is one of the conserved transporters (ABC) composed of ABCG5 and ABCG8, mutations in properties of these lipids [14–16]. In addition to the free mem- either one of which cause sitosterolemia [31]. brane embedded sterol, sterols are also converted to a storage form Two related ABC transporters, ABCA1 and ABCG1 are required by esterification with long chain fatty acids. The resulting steryl for the major part of cholesterol efflux to serum or high-density esters are then stored in lipid droplets from where the free sterol lipoprotein (HDL) from macrophage foam cells [32]. Mutations in is liberated by the action of specific lipases [17]. Balance between ABCA1 are associated with Tangier disease, and result in the char- synthesis, transport, esterification of the free sterol and hydrolysis acteristic severe reduction in HDL levels. of steryl esters has to be tightly regulated to maintain sterol homeostasis [18]. To prevent the toxic accumulation of free sterols at its site of synthesis, the ER membrane, the free sterol has to be effi- 3. Sterol transport in yeast ciently transported to the plasma membrane [19]. This transport pathway is still incompletely understood but involves both vesic- Yeast is a powerful genetic model organism to study basic ular and non-vesicular components because it is ATP-dependent, cellular processes that are conserved in eukaryotic cells [33]. Sac- but only partially sensitive to brefeldin A, which disrupts vesicular charomyces cerevisiae was adopted early on to study the structure transport at the level of the Golgi apparatus [12,20,21]. The proposi- to function relation of different sterols by Bloch and colleagues tion that sterol transport between the ER and the plasma membrane and subsequently to identify the genes that participate in sterol involves both vesicular as well as non-vesicular components is also synthesis and in its regulation [13,34–36]. As in mammalian cells, consistent with the observation that lipid transport continues in ergosterol, the mature sterol made by yeast, is synthesized by yeast mutants that have a conditional block of vesicular transport ER-localized enzymes and is then transported to the plasma mem- between the two compartments [22]. brane, which harbors the highest level of the free ergosterol pool In this review, we will summarize the approaches that were of the cell [9,37]. This transport is fast because newly synthe- taken and the progress made over the past couple of years to under- sized ergosterol equilibrates with a half-life of 10–15 min with stand how sterols are transported between different intracellular the plasma membrane-localized sterol pool, which requires that membranes, how this lipid transport may be regulated, and how approximately 105 ergosterol molecules traffic in and out of the these membranes may establish their characteristic sterol concen- plasma membrane per second, a rate that is 10 times greater than tration – focusing on studies using yeast as a genetically tractable needed to generate a new plasma membrane during cell doubling model organism. [22,38]. Equilibration of the newly synthesized ER localized ergosterol pool with that of the plasma membrane is independent of the secretory pathway but requires ATP and a normal level and 2. Uptake and export of sterols by mammalian cells rate of sphingolipid synthesis [22]. This observation was taken to suggest that concentration of the available free sterol in the Mammalian cells take up cholesterol by receptor-mediated ER is comparable to that of the plasma membrane, and that the endocytosis of low-density lipoproteins (LDLs) containing observed transport reflects the equilibration of this “available” cholesteryl esters [23]. LDLs are then delivered to late endo- pool of sterols between the two compartments. In this model, somes or lysosomes where cholesteryl esters are hydrolyzed by the majority of the plasma membrane-localized pool of ergosterol an acidic lipase. The liberated free cholesterol is recycled to the is not freely available because it is trapped into complexes with plasma membrane or transported to the ER where it is esterified sphingolipids [39]. Formation of sterol–sphingolipid complexes and stored in lipid droplets. The transport of the free sterol from within the yeast plasma membrane is supported by the observation the plasma membrane and the endocytic compartment back to that the plasma membrane-localized pool of ergosterol in mitotic the ER closes the transport cycle between the ER and the plasma cells is not accessible to binding to the fluorescent sterol binding membrane and the level of free cholesterol that travels through polyene filipin [40]. Since various phospholipids also show differ- this route is buffered by the storage and release of sterols from ent affinities for sterols, the intracellular distribution of the free lipid droplets. The back transport of free cholesterol to the ER is sterol may be based on an equilibration of the concentration of inhibited by hydrophobic amines, progesterone, disruption of the the “active” sterol pool only, that is the fraction of sterols that is cytoskeleton or that of the acidic compartments, but not by ATP not engaged in formation of lipid complexes with either sphin- depletion, indicating that it occurs through a non-vesicular route golipids or phospholipids [41]. Such a hypothesis may also explain [24,25]. This cholesterol transport cycle is defective in certain lipid the wide variation of sterol content between different subcellular storage diseases, such as Wolman disease in which a defective membranes [42]. acidic lipase results in accumulation of cholesteryl esters in the Because sterols are insoluble in the aqueous environment, equi- lysosomal compartment or in Niemann-Pick type C disease where libration of the concentration of free/available sterols between transport of free cholesterol out of the lysosomal compartment is membranes of different organelles requires transfer of the lipid blocked. Functional homologues of these proteins are present in between the two membranes. This lipid transfer can occur through yeast and are implicated in sterol transport in fungal cells as well sites of membrane contact between the two organelles, or by tran- [3,4,12,17,26,27]. sient solubilization of the lipid through its binding to a soluble

Please cite this article in press as: N. Jacquier, R. Schneiter, Mechanisms of sterol uptake and transport in yeast, J. Steroid Biochem. Mol. Biol. (2011), doi:10.1016/j.jsbmb.2010.11.014 G Model SBMB-3587; No. of Pages 9 ARTICLE IN PRESS N. Jacquier, R. Schneiter / Journal of Steroid Biochemistry & Molecular Biology xxx (2010) xxx–xxx 3 lipid binding/transfer protein [43]. Membrane contact sites are properties of the cell wall that might prevent exogenous sterols zones where membranes from different organelles, usually the ER from reaching the plasma membrane [65]. Because the synthe- and a partner organelle come into close apposition [44]. Some of sis of sterols requires molecular oxygen, S. cerevisiae becomes the best-characterized inter-organelle contacts are those between sterol auxotroph when grown under anaerobic conditions and the nucleus and the vacuole and the junctions between the ER cells take up sterol from the environment, incorporate them into and mitochondria [45,46]. Disruption of the contacts between ER the plasma membrane and even transport them back to the ER and mitochondria impairs phospholipid transport between the two for esterification and storage in lipid droplets [64]. Anaerobic organelles, indicating that the close, protein-mediated apposition conditions can be mimicked by mutations that prevent the syn- of the two organelles is functionally important [46,47]. Since the thesis of heme, by gain-of-function alleles of the transcription protein complex that is required to establish the ER-mitochondria factor UPC2, or of its homologue ECM22, and by overexpres- encounter structure (ERMES) shows homology to characterized sion of the transcriptional regulator SUT1 allowing biochemical lipid transfer protein, these proteins appear not only to establish uptake studies to be performed under normal growth conditions the contact but may also catalyze lipid transfer between the two [66–68]. organelles [48]. The aerobic uptake of sterols in cells expressing the gain-of- On the other hand, exchange of lipids by soluble lipid transfer function allele of UPC2, UPC2-1 mutant is due to an upregulation proteins, requires pick-up of the lipid from a donor membrane, its of the plasma membrane-localized ABC transporters Pdr11 and transient solubilization and transfer through the aqueous space, Aus1 and that of the cell wall protein Dan1 [69,70]. Mutant cells followed by its deposition into a suitable acceptor membrane. lacking both of these two ABC transporters cannot grow under Prominent candidate proteins to exert lipid transfer between anaerobic conditions and are blocked in uptake and esterifica- membranes of different organelles are the oxysterol-binding pro- tion of exogenously provided sterols [69,71]. Thus, as exemplified tein (OSBP)-related proteins (ORPs). Yeast contains a family of by the requirement of ABCA1 and ABCG1 in promoting efflux of seven cytoplasmic proteins (Osh1–Osh7) which contain a 400- cholesterol to HDL and that of ABCG5 and ABCG8 in clearance of ∼ residue lipid binding pocket. Lack of all Osh proteins is lethal foreign sterols from the intestine and the promotion of choles- and lack of Osh functions causes alterations in intracellular sterol terol elimination from the body through hepatobiliary secretion, distribution [49–51]. Osh4 has a ␤-barrel structure with a cen- ABC transporters play a crucial role in promoting sterol uptake as tral hydrophobic tunnel that can accommodate a single sterol well as efflux. While a variety of models explaining the mechanism molecule with its 3# hydroxyl oriented towards the closed side of ABC-mediated cholesterol efflux to acceptor proteins or mem- of the tunnel. The other end of the tunnel is gated by a hinged branes have been proposed, an in-depth understanding of the lipid lid that closes upon sterol binding, shielding the hydrophobic transfer process mediated by these transporters is likely to require interior from the aqueous environment [52]. The structure of unequivocal identification of the substrate that is actually translo- Osh4 suggests that ORPs may function as lipid transfer pro- cated across the membrane [72]. Identification of the substrate is teins. Recent in vitro assays indicate that ORPs have at least two particularly pertinent in light of the fact that the function of some membrane-binding surfaces allowing them to bind to multiple of these transporters, for example that of ABCA1, is dependent on membranes simultaneously, extract sterols from one membrane the lipid composition of the surrounding membrane, cholesterol and transfer them to an acceptor thus facilitating sterol trans- may thus not only be a substrate of several ABC transporters, but fer [53,54]. Lipid transfer is stimulated by PI(4,5)P2, a known may also regulate ABC activity through its effects on the forma- effector of Osh4 [55]. But how exactly membrane association of tion of membrane rafts [73]. Thus, while Pdr11 and Aus1 are both ORPs is regulated is not yet well understood. However, Vps4, localized at the plasma membrane and thus may be more directly an ATPase of the AAA family (ATPases associated with a variety implicated in sterol influx they may also function to modify the of cellular activities), which plays a critical role in the forma- properties of the plasma membrane to allow sterol insertion into tion of multivesicular bodies, directly binds the yeast ORPs Osh6 the outer leaflet, flip-flop across the bilayer, or its extraction from and Osh7 and thereby may regulate their membrane association the cytoplasmic leaflet [71]. [56]. Transport of sterols from the plasma membrane to the ER occurs Osh proteins are also implicated in diverse cell signaling path- by a nonvesicular pathway and the rate of transport/esterification ways. For example, Rho-dependent polarized vesicular transport correlates with the affinity of the sterol to plasma membrane lipid requires Osh protein function [57] and vesicular transport through rafts [71,74]. Back-transport of the free sterol from the plasma the Golgi is affected by Osh4/Kes1 [58]. Effector kinases of the membrane to the ER, however, does not appear to be a prereq- Rho-type GTPase Cdc42, Ste20 and Cla4, which are members of uisite for its incorporation into the plasma membrane, because the p21-activated kinases (PAK) family, interact physically and an esterification deficient mutant is perfectly viable under sterol genetically with sterol biosynthetic components and downregu- auxotrophic conditions. late sterol levels under aerobic conditions as well as sterol uptake An excess of free sterols in the ER is buffered via ester- under anaerobic conditions, indicating a tight coordination of sterol ification with long chain fatty acids by two ER-localized levels with cell polarization [59–61]. Because bulk sterol transport acyl-CoA:cholesterol acyltransferases, Are1 and Are2, and the from the ER to the plasma membrane is reduced rather than inhib- resulting steryl esters are stored in lipid droplets [75–77]. These ited when Osh-function is depleted, it has been questioned whether steryl esters can be mobilized from lipid droplets to liberate free Osh proteins have a direct role in bulk transport of sterols in vivo or sterols by the action of three steryl-ester hydrolases: Yeh1, Yeh2 if they have a more regulatory role on sterol transport or modulate and Tgl1 [17,78,79]. The activities of these lipases are crucial to signal transduction [38,62,63]. regulate cellular levels of free ergosterol under various growth conditions [80]. Sterol esterification and storage in lipid droplets is a potential mechanism to avoid harmful levels of sterols in the ER 4. Sterol uptake and transport back to the ER membrane. The fact that sterol esterification is not essential under standard growth conditions may indicate that the regulation of syn- Yeast cells do not take up free sterols from the environment thesis and export from the ER are sufficient to prevent accumulation if cultivated in the presence of oxygen, i.e., under aerobic con- of free sterols in the ER membrane. An overview of the mecha- ditions [64]. The molecular mechanisms underlying this aerobic nisms that contribute to the regulation of sterol homeostasis and sterol-exclusion are not well understood, but could be caused by transport is given in Fig. 1.

Fig. 1. Basic pathways for the intracellular transport of sterols in yeast. Major cellular compartments are indicated, arrows indicate sterol transport pathways and proteins implicated in sterol transport, sterol conversion and the regulation of sterol-dependent transcription are speciﬁed.

When cells cannot esterify sterols, otherwise non-essential nuclear pore complexes with expansion of the ER membrane, lipid membrane proteins become essential. One of these is Arv1, which synthesis and possibly transport [86]. localizes to the ER and Golgi membranes and contains 6 putative transmembrane domains. Cells lacking Arv1 display defects 5. Sterol sensing and uptake by other fungal species in growth under anaerobic conditions and in sterol uptake, have defects in sphingolipid and phospholipid metabolism, and accumu- Unlike baker’s yeast, which is a facultative anaerobe, Schizosac- late intermediates of glycosylphosphatidylinositol (GPI) synthesis charomyces pombe is an obligate aerobe that adapts to changes in [81–83]. oxygen availability by regulating sterol levels through the activa- In addition to Arv1, a family of integral membrane proteins of tion of hypoxic transcription factors. These are homologues of the the nuclear envelope/ER membrane has recently been implicated human sterol regulatory element binding proteins (SREBP, Sre1) in membrane homeostasis of the ER. Cells lacking Apq12 are viable and Scap (SREBP cleavage-activating protein, Scp1) and sense the but cannot grow at low temperature, have aberrant nuclear pore sterol content in the ER to coordinate the expression of sterol complexes and a defect in mRNA export. Defects in NPC assem- biosynthetic enzymes with oxygen levels [87,88]. SREBPs are also bly can be overcome by supplementing cells with the membrane important for sensing oxygen levels in pathogenic fungi, such as fluidizing agent benzyl alcohol, suggesting that Apq12 impacts the Cryptococcus neoformans and Aspergillus fumigatus [89]. There are, flexibility of the nuclear membrane [84]. A second, essential mem- however, no obvious SREBP orthologs in S. cerevisiae or in any of brane protein, Brr6, shares at least partially overlapping functions the Candida clade species, which both mount a response to hypoxia with Apq12 and is also required for the assembly of functional by increasing the expression of ergosterol synthesis, remodeling of nuclear pores. Remarkably, cells having reduced function of Brr6 cell wall composition, induction of glycolytic genes and repression require sterol esterification for viability, display elevated levels of of respiratory chain components, ATP synthesis and the citric acid steryl esters, and accumulate free sterols in the ER membrane when cycle [90–93]. sterols are supplemented from the outside [85]. These proteins The response of pathogenic fungi to hypoxic conditions is impor- may thus coordinate nuclear functions, such as the assembly of the tant for several virulence attributes, ranging from infection to

Please cite this article in press as: N. Jacquier, R. Schneiter, Mechanisms of sterol uptake and transport in yeast, J. Steroid Biochem. Mol. Biol. (2011), doi:10.1016/j.jsbmb.2010.11.014 G Model SBMB-3587; No. of Pages 9 ARTICLE IN PRESS N. Jacquier, R. Schneiter / Journal of Steroid Biochemistry & Molecular Biology xxx (2010) xxx–xxx 5 biofilm formation [94,95]. The human fungal pathogen Candida and Lro1, a diacylglycerol acyltransferase (DGAT) that catalyzes the albicans, for example, can grow on superficial as well as inter- synthesis of triacylglycerol. Unexpectedly, Cax4/Cwh8 mutant cells nal sites in the infected host, areas that differ significantly in the also have greatly reduced levels of sphingolipids, particularly inos- availability of oxygen [94]. Under hypoxic conditions, C. albicans itolphosphorylceramides (IPCs) [108]. Because reduced levels of switches from yeast to hyphal growth, a phenotypic change that IPCs are generally observed upon inhibition of N-glycosylation, for has been associated with invasion and virulence. example upon treatment with tunicamycin, it seems plausible that Both systemic and superficial fungal infections are treated N-glycosylation, sphingolipid synthesis and sterol esterification, by drugs that specifically inhibit ergosterol biosynthesis. How- all of which are ER-localized processes are coordinately regulated ever, drug resistant isolates of Candida are being recovered with [106,108,109]. increased frequency from immunocompromised and immunosup- Mutations in other genes identified in this screen, such as those pressed patients [96]. Unlike S. cerevisiae, which does not take up in phospholipase C (Plc1), protein phosphatase type 2C (Ptc1), sterols in the presence of oxygen, some of the pathogenic fungi, and Arv1, on the other hand, resulted in elevated levels of free such as C. glabarata and A. fumigatus, import exogenous cholesterol sterols and the aberrant accumulation of filipin stained material from the host serum in the presence of oxygen to counteract the in an endosomal compartment, indicating that these functions are toxicity of sterol biosynthetic inhibitors [97–99]. In C. glabarata, this important for proper distribution of sterols [106]. Additional work drug resistance can even be induced by overexpression of the ABC is now required to define the causal relation between the func- transporter Aus1, indicating that it occurs by similar pathways as tion of these proteins and the observed mislocalization of sterols in does the anaerobic uptake of sterols by S. cerevisiae [96]. these mutants. A third approach took advantage of the aerobic sterol uptake of UPC2-1 cells to perform a suicide selection screen [110]. Therefore, 6. Genetic screens for sterol uptake and transport mutants cells were mutagenized using a transposon library, fed with tri- tiated cholesterol, stored to allow for radiations-induced damage, To identify components important for sterol uptake and trans- and the survivors were screened for reduced levels of cholesterol port, a number of genetic screens were performed over the uptake. This screen identified 23 nonessential genes with a puta- last couple of years. Taking advantage of the fact that sterol tive role in sterol transport. Many of the mutants had defects in the uptake and transport are essential for cell growth under anaero- expression or trafficking of the ABC transporters Aus1 and Pdr11. bic conditions, we screened the yeast deletion mutant collection For example, mutations in Mot3, a transcription factor required for mutants that fail to grow under anaerobic conditions. This for the repression of anaerobic/hypoxic genes under aerobic condi- allowed us to identify 17 mutants, which affect sterol uptake tions, displayed reduced levels of sterol uptake because it reduced and/or trafficking as assessed by genetic and biochemical analy- the expression of AUS1 and PDR11 [110,111]. The mutant that ses [100,101]. These mutants display reduced levels of uptake of affected cholesterol uptake independently of the expression and radio- and fluorescent-labeled cholesterol and they have greatly localization of Aus1 and Pdr11, contained an insertion in an unchar- reduced levels of esterified cholesterol. Surprisingly, the largest acterized gene, Ydr051c/Det1. Det1 mutant cells also affected the class of mutants isolated in this screen is affected in mitochondrial forward transport of ergosterol to the plasma membrane as ana- functions, indicating that at least one unidentified mitochondrial lyzed by methionine labeling of the newly synthesized ergosterol function is required for uptake of sterols by yeast under anaer- and its extraction from the plasma membrane with cyclodextrin, obic conditions. In vertebrate and insect cells, mitochondria are but they did not affect the subcellular distribution of ergosterol important for steroid biogenesis, which requires the import of [110]. Det1 is a soluble cytoplasmic protein and a recent bio- sterols into mitochondria and the export of steroid precursors from chemical screen identified it as an acid phosphatase with broad mitochondria to the ER [102]. In yeast, on the other hand, ergos- substrate specificity [112]. Interestingly, an ecdysteroid phosphate terol biosynthesis is required to maintain normal mitochondrial phosphatase and the related human proteins Sts-1 were recently morphology and the sterol content of mitochondria drops up to identified as members of the histidine phosphatase superfamily four-fold when cells are shifted to anaerobic conditions [103,104]. and shown to have phosphatase activity towards ecdysteroid- and Many of the uptake mutants identified in this screen as well as a steroid-phosphates [113]. Further studies will be required to exam- mutant in the two ABC transporters required for uptake, Aus1 and ine whether the phosphatase activity of Det1 is important for its Pdr11, have electron-dense mitochondrial inclusions when culti- function in sterol trafficking. vated under lipid auxotrophic conditions, suggesting that aberrant The fact that none of these screens allowed the identification of a mitochondria may result from impaired sterol uptake. How these bona-fide sterol transporter, indicates that there are likely multiple observations are mechanistically linked to sterol uptake/transport, pathways that ensure the proper distribution of sterols and that however, remains to be resolved [105]. these are at least partially redundant and can compensate for the A second screen took advantage of the susceptibility of sterol absence or defect in one of these pathways. transport and storage mutants to drugs that inhibit sterol synthesis, such as the HMG-CoA reductase inhibitor lovastatin, and hyper- sensitivity to the sterol-binding and pore-forming antifungal drug, 7. Sterol acetylation, a quality-control step that controls nystatin [106]. The screen uncovered 56 mutants from the homozy- sterol efflux gous diploid deletion collection that were sensitive to both drugs. Phenotypic analysis of these mutants revealed that seven of these Mammalian cells can reduce their intracellular cholesterol had severely reduced numbers of lipid droplets, indicating that load by delivering free cholesterol for lipidation of ApoA-1 and the drug sensitivity of these mutants is likely due to their reduced the maturation of high density lipoprotein (HDL) particles. This capacity to store steryl esters in lipid droplets. More detailed analy- reverse cholesterol transport pathway requires the ABC trans- sis of these strains revealed that defects in Cax4, encoding a dolichyl porters ABCA1 and ABCG1 and defects in ABCA1 cause Tangier pyrophosphate phosphatase that regenerates free dolichol, a lipid disease and the early onset of atherosclerosis [32]. No compa- required for N-linked glycosylation of proteins in the ER, resulted in rable pathway for the excretion of sterols has been described severely reduced incorporation of palmitate into steryl esters and in yeast. Since yeast is a unicellular organism, it is questionable triacylglycerols [106,107]. Reduced synthesis of these neutral lipids whether such an overflow pathway would be economically effi- was due to reduced levels of the respective acyltransferases, Are1 cient, because the synthesis of one sterol molecule requires more

Fig. 2. The sterol acetylation/deacetylation cycle. The components of the sterol acetylation/deacetylation cycle are depicted. Acetylation of sterols by Atf2 results in their excretion from the cells, whereas deacetylation of the acetylated lipid by Say1 will result in retainment of the lipid inside the cell. In this cycle, the substrate specificity of Say1 specifies the fate of the acetylated lipid. than 10 molecules of glucose. In yeast, an excess of sterol is buffered over by enzymes that are structurally unrelated to Atf2. In verte- by esterification and by the down-regulation of sterol biosynthesis. brates, sterols and steroids that are subject to detoxification and Cells lacking the capacity to esterify sterols display reduced levels of excretion are generally rendered more soluble by sulfonation- and the squalene epoxidase, Erg1, and incorporate less ergosterol into glucuronidation-reactions, suggesting that detoxification from a the plasma membrane [114]. single cell organism may follow fundamentally different rules than In addition to utilizing the buffering capacity of lipid droplets that from a multicellular organism, which does not only need to to store an excess of sterols as steryl esters, yeast has recently clear the substance from individual cells but also from the entire been shown to reversibly convert free sterols, steroid precur- circulation and hence needs to render the substance more soluble sors and sterol-like molecules to their acetylated derivatives. [119,120]. Remarkably, these acetylated sterols and their derivatives are efficiently excreted by the cells and rendered soluble in the culture 8. Perspectives media. Acetylation of sterols requires the alcohol acetyltransferase Atf2, whereas deacetylation depends on a membrane-anchored The identification of this novel acetylation cycle indicates that lipase Say1 (Fig. 2) [115–117]. Secretion of cholesteryl acetate sterols may be subject to others, as yet unidentified modifications requires ongoing vesicular transport but is independent of the that may confer novel functions to these lipids. One of these mod- ABC transporters Aus1 and Pdr11. Acetylation and deacetylation of ifications includes the glycosylation of sterols, which is important cholesterol is likely to take part in the lumen of the ER, where the for pexophagy in the methylotrophic yeast Pichia pastoris [121]. active site residues of both Say1 and Atf2 are located. The fact that Clearly sterols have evolved with the eukaryotic cell and have taken endogenously synthesized ergosterol is also acetylated by Atf2 but over a surprising number of diverse cellular functions and new ones rapidly deacetylated again by Say1 whereas non-mature intermedi- are likely to be uncovered in the future. While the picture of how ates of the ergosterol biosynthetic pathway or the steroid precursor exactly cells maintain sterol homeostasis is still far from complete, pregnenolone are only acetylated but not efficiently deacetylated additional stones are added to the mosaic at an ever increasing and thus are excreted by the cells, indicates that the acetylation pace. One particularly pertinent question is how the transbilayer cycle may act as a lipid proofreading cycle that controls the qual- distribution of sterols is established and maintained. Traditionally, ity/structure of the sterol within the ER membrane before it is sterols are believed to flip-flop rapidly between the two leaflets of incorporated into more stable lipid complexes and delivered to biophysical model membranes, the precise distribution of sterols the plasma membrane [116,117]. The formation of ER derived lipid in complex and energized cellular membranes, however, is much rafts, however, appears not to be affected in deacetylase mutants less clear [122–124]. This transmembrane distribution of sterols since the raft-associated and glycosylphosphatidylinositol (GPI)- is of course important as it affects the concentration of “active” anchored protein Gas1 matures with wild-type kinetics in both non-complexed sterols within the leaflet of a particular membrane, say1! and atf2! mutant cells (our unpublished observations) the potential role of ABC transporters to establish and maintain [118]. In addition, other ER functions such as the response to the the proper transbilayer distribution of either sterols or lipids that accumulation of unfolded proteins (UPR) or the generation of COPII complex the sterols, as well as the topology of the sterol transport vesicles also appear to be unaffected since acetylation-deficient processes, whether mediated by cytosolic lipid transfer proteins or cells are not hypersensitive against tunicamycin, which blocks membrane contact sites. The efficiency of lipid transfer, for example N-glycosylation and thus induces the UPR, and because the vac- by Osh proteins, could be limited by its direct access to an excess of uolar carboxypeptidase 1 matures with wild-type kinetics in these non-complexed sterols in the cytosolic leaflet of a donor membrane mutants cells (our unpublished observations). The lipid acetyla- or may also be enhanced by removing the transferred sterol from tion cycle, however, serves to protect cells from naturally occurring the cytosolic leaflet of the acceptor membrane. Resolving this trans- hydrophobic and steroid-like compounds, because mutants lacking bilayer distribution of sterols in membranes of different organelles either Say1 or Atf2 are hypersensitive against eugenol, a member in living cells is technically daunting because the measurement of the allylbenzene class of compounds that is present in clove oil, itself may already perturb the functional distribution. nutmeg, cinnamon and bay leaf and that is used as local antiseptic and anesthetic [116]. Interestingly, Say1 has homology to the human arylacetamide References deacetylase AADAC family of proteins. Expression of AADAC in yeast prevents the accumulation of cholesteryl acetate, indicat- [1] T.H. Haines, Do sterols reduce proton and sodium leaks through lipid bilayers? Prog. 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Please cite this article in press as: N. Jacquier, R. Schneiter, Mechanisms of sterol uptake and transport in yeast, J. Steroid Biochem. Mol. Biol. (2011), doi:10.1016/j.jsbmb.2010.11.014