Where sterols are required for endocytosis

Sterols are essential membrane components of eukaryotic cells. Interacting closely with sphingolipids, they provide the membrane surrounding required for membrane sorting and trafficking processes. Altering the amount and/or structure of free sterols leads to defects in endocytic pathways in mammalian cells and yeast. Plasma membrane structures functioning in the internalization step in mammalian cells, caveolae and clathrin-coated pits, are affected by cholesterol depletion. Accumulation of improper plasma membrane sterols prevents hyperphosphorylation of a plasma membrane receptor in yeast. Once internalized, sterols still interact with sphingolipids and are recycled to the plasma membrane to keep an intracellular sterol gradient with the highest amount of free sterols at the cell periphery. Interestingly, cells from patients suffering from sphingolipid storage diseases show high intracellular amounts of free cholesterol. We propose that the balanced interaction of sterols and sphingolipids is responsible for protein recruitment to specialized membrane domains and their functionality in the endocytic pathway.     

Distribution and Functions of Sterols and Sphingolipids

Sterols and sphingolipids are considered mainly eukaryotic lipids 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, plants, 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 membrane, 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 sterol and sphingolipid functions in eukaryotes providing evidence that these two lipid classes function together. Intermediates in sphingolipid biosynthesis and degradation are involved in signaling pathways, whereas sterol structures are converted to hormones. Both lipids have been implicated in regulating membrane trafficking.Typical examples of eukaryotic lipids, sterols, and sphingolipids can both be found in membranes from simple unicellular fungi and protists to multicellular animals and plants. Their versatile use as structural elements but also as signaling molecules has probably played an important role during the evolution of this large and diverse group of organisms. There are also many eukaryotes that have lost the ability to synthesize sterols de novo including nematodes, insects, and marine invertebrates, which have to take up sterols with their diet. Sterol biosynthesis has also been reported in a number of bacteria. Sphingolipids are more widely spread among prokaryotes than sterols and also show a greater variety of structures among the different eukaryotes.In this short review we will first give an overview about the diversity of sterol and sphingolipid structures and their distribution in nature. Then we will discuss their subcellular distribution. A brief technical section will add some information on the separation and detection of these lipid molecules. Subsequently, we will summarize different genetic approaches to study the functions of sterols and sphingolipids, and finally, we will discuss the functional and possible physical interactions of the two lipid classes within the cell. Far from being comprehensive, we will focus only on a few interesting aspects and try to give new view points, which are less frequently discussed.     

Sphingolipids as modulators of membrane proteins

The diversity of the transmembranome of higher eukaryotes is matched by an enormous diversity of sphingolipid classes and molecular species. The intrinsic properties of sphingolipids are not only suited for orchestrating lateral architectures of biological membranes, but their molecular distinctions also allow for the evolution of protein motifs specifically recognising and interacting with individual lipids. Although various reports suggest a role of sphingolipids in membrane protein function, only a few cases have determined the specificity of these interactions. In this review we discuss examples of specific protein–sphingolipid interactions for which a modulator-like dependency on sphingolipids was assigned to specific proteins. These novel functions of sphingolipids in specific protein–lipid assemblies contribute to the complexity of the sphingolipid classes and other molecular species observed in animal cells. This article is part of a Special Issue entitled New Frontiers in Sphingolipid Biology.     

Cholesterol regulation of rab-mediated sphingolipid endocytosis

Despite a tight regulation of its intracellular content, cholesterol is found accumulated in pathological conditions such as sphingolipidosis as well as after cell treatment with drugs like hydrophobic amines. Furthermore, cellular cholesterol increases when cultured cells approach confluence. Under these conditions, the endocytic pathways of plasma membrane sphingolipids are differently affected. In this short review, we will summarize recent results from our laboratory as well as those of other groups, indicating that the intracellular accumulation of cholesterol inhibits the dissociation of rab GTPases from the target membranes, causing the alteration of rab-mediated membrane traffic.     

The cell biology of glycosphingolipids

Glycosphingolipids, a family of heterogeneous lipids with biophysical properties conserved from fungi to mammals, are key components of cellular membranes. Because of their tightly packed backbone, they have the ability to associate with other sphingolipids and cholesterol to form microdomains called lipid rafts, with which a variety of proteins associate. These microdomains are thought to originate in the Golgi apparatus, where most sphingolipids are synthesized, and are enriched at the plasma membrane. They are involved in an increasing number of processes, including sorting of proteins by allowing selectivity in intracellular membrane transport. Apart from being involved in recognition and signaling on the cell surface, glycosphingolipids may fulfill unexpected roles on the cytosolic surface of cellular membranes.     

Serinc, an activity-regulated protein family, incorporates serine into membrane lipid synthesis

Cell membranes contain various transporter proteins, some of which are responsible for transferring amino acids across membrane. In this study, we report another class of carrier proteins, termed Serinc1-5, that incorporates a polar amino acid serine into membranes and facilitates the synthesis of two serine-derived lipids, phosphatidylserine and sphingolipids. Serinc is a unique protein family that shows no amino acid homology to other proteins but is highly conserved among eukaryotes. The members contain 11 transmembrane domains, and rat Serinc1 protein co-localizes with lipid biosynthetic enzymes in endoplasmic reticulum membranes. A Serinc protein forms an intracellular complex with key enzymes involved in serine and sphingolipid biosyntheses, and both functions, serine synthesis and membrane incorporation, are linked to each other. In the rat brain, expression of Serinc1 and Serinc2 mRNA was rapidly up-regulated by kainate-induced seizures in neuronal cell layers of the hippocampus. In contrast, myelin throughout the brain is enriched with Serinc5, which was down-regulated in the hippocampus by seizures. These results indicate a novel mechanism linking neural activity and lipid biosynthesis.     

The phosphoinositol sphingolipids of Saccharomyces cerevisiae are highly localized in the plasma membrane.

To investigate the vital function(s) of the phosphoinositol-containing sphingolipids of Saccharomyces cerevisiae, we measured their intracellular distribution and found these lipids to be highly localized in the plasma membrane. Sphingolipids were assayed in organelles which had been uniformly labeled with [3H]inositol or 32P and by chemical measurements of alkali-stable lipid P, of long chain bases, and of very long chain fatty acids. We have developed an improved method for the preparation of plasma membranes which is based on the procedure of Duran et al. (Proc. Natl. Acad. Sci. USA 72:3952-3955, 1975). On the basis of marker enzyme and DNA assays carried out with a number of preparations, the plasma membranes contained less than 10% vacuolar membranes (alpha-mannosidase) and nuclei (DNA); the contamination by the endoplasmic reticulum (NADPH-cytochrome c reductase) varied from 0 to 20%. The plasma membrane preparations showed a 13-fold increase in the specific activity of vanadate-sensitive ATPase, compared with that in the homogenate, with a yield ranging from 50 to 80%. A comparison of the distribution of the ATPase with that of sphingolipids assayed by a variety of methods showed that 80 to 100% of the sphingolipids are localized in the plasma membrane; the sphingolipids constitute about 30% of the total phospholipid content of the plasma membrane. Minor amounts of sphingolipids that were found in isolated mitochondria and nuclei can be attributed to the presence of small amounts of plasma membrane in these fractions. These results suggest that one or more essential functions of these lipids is in the plasma membrane. Furthermore, sphingolipids may be useful chemical markers of the plasma membrane of S. cerevisiae.     

Sphingolipid trafficking--sorted out?

Studies of intracellular membrane traffic have traditionally focused on the protein components of membranes, but what about lipids? Recent findings have drawn attention to the transport of one type of lipid, the sphingolipids. Their unique physical properties may allow them to aggregate into microdomains in membranes that concentrate sphingolipids into specific transport pathways. Gerrit van Meer and Koert Burger consider here the routes of sphingolipid biosynthesis and transport, and the role of proteins in their targeting. The following article by Deborah Brown turns the tables to review the evidence suggesting that sphingolipid domains are important in specific targeting of GPI-anchored proteins to the plasma membrane.     

Association of sterol- and glycosylphosphatidylinositol-linked proteins with Drosophila raft lipid microdomains

In vertebrates, the formation of raft lipid microdomains plays an important part in both polarized protein sorting and signal transduction. To establish a system in which raft-dependent processes could be studied genetically, we have analyzed the protein and lipid composition of these microdomains in Drosophila melanogaster. Using mass spectrometry, we identified the phospholipids, sphingolipids, and sterols present in Drosophila membranes. Despite chemical differences between Drosophila and mammalian lipids, their structure suggests that the biophysical properties that allow raft formation have been preserved. Consistent with this, we have identified a detergent-insoluble fraction of Drosophila membranes that, like mammalian rafts, is rich in sterol, sphingolipids, and glycosylphosphatidylinositol-linked proteins. We show that the sterol-linked Hedgehog N-terminal fragment associates specifically with this detergent-insoluble membrane fraction. Our findings demonstrate that raft formation is preserved across widely separated phyla in organisms with different lipid structures. They further suggest sterol modification as a novel mechanism for targeting proteins to raft membranes and raise the possibility that signaling and polarized intracellular transport of Hedgehog are based on raft association.     

Down-regulating sphingolipid synthesis increases yeast lifespan

Knowledge of the mechanisms for regulating lifespan is advancing rapidly, but lifespan is a complex phenotype and new features are likely to be identified. Here we reveal a novel approach for regulating lifespan. Using a genetic or a pharmacological strategy to lower the rate of sphingolipid synthesis, we show that Saccharomyces cerevisiae cells live longer. The longer lifespan is due in part to a reduction in Sch9 protein kinase activity and a consequent reduction in chromosomal mutations and rearrangements and increased stress resistance. Longer lifespan also arises in ways that are independent of Sch9 or caloric restriction, and we speculate on ways that sphingolipids might mediate these aspects of increased lifespan. Sch9 and its mammalian homolog S6 kinase work downstream of the target of rapamycin, TOR1, protein kinase, and play evolutionarily conserved roles in regulating lifespan. Our data establish Sch9 as a focal point for regulating lifespan by integrating nutrient signals from TOR1 with growth and stress signals from sphingolipids. Sphingolipids are found in all eukaryotes and our results suggest that pharmacological down-regulation of one or more sphingolipids may provide a means to reduce age-related diseases and increase lifespan in other eukaryotes.     

Sphingolipid/cholesterol regulation of neurotransmitter receptor conformation and function

Like all other monomeric or multimeric transmembrane proteins, receptors for neurotransmitters are surrounded by a shell of lipids which form an interfacial boundary between the protein and the bulk membrane. Among these lipids, cholesterol and sphingolipids have attracted much attention because of their well-known propensity to segregate into ordered platform domains commonly referred to as lipid rafts. In this review we present a critical analysis of the molecular mechanisms involved in the interaction of cholesterol/sphingolipids with neurotransmitter receptors, in particular acetylcholine and serotonin receptors, chosen as representative members of ligand-gated ion channels and G protein-coupled receptors. Cholesterol and sphingolipids interact with these receptors through typical binding sites located in both the transmembrane helices and the extracellular loops. By altering the conformation of the receptors (“chaperone-like” effect), these lipids can regulate neurotransmitter binding, signal transducing functions, and, in the case of multimeric receptors, subunit assembly and subsequent receptor trafficking to the cell surface. Several sphingolipids (especially gangliosides) also exhibit low/moderate affinity for neurotransmitters. We suggest that such lipids could facilitate (i) the attachment of neurotransmitters to the post-synaptic membrane and in some cases (ii) their subsequent delivery to specific protein receptors. Overall, various experimental approaches provide converging evidence that the biological functions of neurotransmitters and their receptors are highly dependent upon sphingolipids and cholesterol, which are active partners of synaptic transmission. Several decades of research have been necessary to untangle the skein of a complex network of molecular interactions between neurotransmitters, their receptors, cholesterol and sphingolipids. This sophisticated crosstalk between all four distinctive partners may allow a fine biochemical tuning of synaptic transmission.     

Role of very-long-chain fatty acids in plant development,when chain length does matter

Very long chain fatty acids (VLCFAs) are essential components for eukaryotes. They are elongated by the elongase complex in the endoplasmic reticulum and are incorporated into four major lipid pools (triacylglycerols, waxes, phospholipids, complex sphingolipids). Functional analysis of several components of the elongase complex demonstrated the essential role of VLCFAs in plants, invertebrates and vertebrates. Although VLCFAs changes in the triacylglycerol pool has no consequence for plant development, modifications of the nature and levels of VLCFAs in waxes, phospholipids and complex sphingolipids have, collectively, profound effects on embryo, leaf, root and flower development. VLCFAs levels in epicuticular waxes are critical for the regulation of epidermal fusions during organogenesis. VLCFAs phospholipids and sphingolipids are involved in membrane structure and dynamics regulating cell size but also division and differentiation. This review summarizes the recent findings in plants but also in other organisms, highlighting the importance of very long acyl chain length during development.     

Cargo trafficking from the trans‐Golgi network towards the endosome

The trans‐Golgi network (TGN) is a major sorting, packing and delivering station of newly synthesised proteins and lipids to their final destination. These cargo molecules follow the secretory pathway, which is a vital part of cellular trafficking machinery in all eukaryotic cells. This secretory pathway is well conserved in all eukaryotes from low‐level eukaryotes, such as yeast, to higher level eukaryotes like mammals. The molecular mechanisms of protein sorting by adaptor proteins, membrane elongation and transport to the final destinations by motor proteins and the cytoskeleton, and membrane pinching‐off by scission proteins must be choreographically managed for efficient cargo delivery, and the understanding of these detailed processes is not yet completed. Functionally, defects in these mechanisms are associated with the pathology of prominent diseases such as acute myeloid leukaemia, Charcot–Marie–Tooth disease, I‐cell disease and Wiskott–Aldrich syndrome. The present review points out the recent advances in our knowledge of the molecular mechanisms involved in the transportation of the cargo from the TGN towards the endosome.     

Fungal cell membrane—promising drug target for antifungal therapy

Increase in invasive fungal infections over the past few years especially in immunocompromised patients prompted the search for new antifungal agents with improved efficacy. Current antifungal armoury includes very few effective drugs like Amphotericin B; new generation azoles, including voriconazole and posaconazole; echinocandins like caspofungin and micafungin to name a few. Azole class of antifungals which target the fungal cell membrane are the first choice of treatment for many years because of their effectiveness. As the fungal cell membrane is predominantly made up of sterols, glycerophospholipids and sphingolipids, the role of lipids in pathogenesis and target identification for improved therapeutics were largely pursued by researchers during the last few years. Present review focuses on cell membrane as an antifungal target with emphasis on membrane biogenesis, structure and function of cell membrane, cell membrane inhibitors, screening assays, recent advances and future prospects.     

Membrane rafts as a novel target in cancer therapy

Membrane rafts are distinct plasma membrane microdomains that are enriched in sphingolipids and cholesterol. They organize receptors and their downstream molecules and regulate a number of intracellular signaling pathways. This review presents information on the dependence of several growth factor receptor signaling pathways on membrane rafts. It also discusses the involvement of rafts in the regulation of differentiation, apoptosis and cell migration connected with invasiveness and metastasis. Examples of known synthetic and naturally occurring substances that are known to affect lateral membrane organization in tumor cell growth are discussed as potential or actual therapeutics.     

Lipid signalling in plant responses to abiotic stress

Lipids are one of the major components of biological membranes including the plasma membrane, which is the interface between the cell and the environment. It has become clear that membrane lipids also serve as substrates for the generation of numerous signalling lipids such as phosphatidic acid, phosphoinositides, sphingolipids, lysophospholipids, oxylipins, N‐acylethanolamines, free fatty acids and others. The enzymatic production and metabolism of these signalling molecules are tightly regulated and can rapidly be activated upon abiotic stress signals. Abiotic stress like water deficit and temperature stress triggers lipid‐dependent signalling cascades, which control the expression of gene clusters and activate plant adaptation processes. Signalling lipids are able to recruit protein targets transiently to the membrane and thus affect conformation and activity of intracellular proteins and metabolites. In plants, knowledge is still scarce of lipid signalling targets and their physiological consequences. This review focuses on the generation of signalling lipids and their involvement in response to abiotic stress. We describe lipid‐binding proteins in the context of changing environmental conditions and compare different approaches to determine lipid–protein interactions, crucial for deciphering the signalling cascades.     

Mutagenesis of the putative sterol-sensing domain of yeast Niemann Pick C-related protein reveals a primordial role in subcellular sphingolipid distribution

Lipid movement between organelles is a critical component of eukaryotic membrane homeostasis. Niemann Pick type C (NP-C) disease is a fatal neurodegenerative disorder typified by lysosomal accumulation of cholesterol and sphingolipids. Expression of yeast NP-C-related gene 1 (NCR1), the orthologue of the human NP-C gene 1 (NPC1) defective in the disease, in Chinese hamster ovary NPC1 mutant cells suppressed lipid accumulation. Deletion of NCR1, encoding a transmembrane glycoprotein predominantly residing in the vacuole of normal yeast, gave no phenotype. However, a dominant mutation in the putative sterol-sensing domain of Ncr1p conferred temperature and polyene antibiotic sensitivity without changes in sterol metabolism. Instead, the mutant cells were resistant to inhibitors of sphingolipid biosynthesis and super sensitive to sphingosine and C2-ceramide. Moreover, plasma membrane sphingolipids accumulated and redistributed to the vacuole and other subcellular membranes of the mutant cells. We propose that the primordial function of these proteins is to recycle sphingolipids and that defects in this process in higher eukaryotes secondarily result in cholesterol accumulation.     

Sphingomonads involved in the biodegradation of xenobiotic polymers

Sphingomonads involved in the microbial degradation of xenobiotic polymers are introduced. The metabolism of polyethylene glycol was the primary focus of the study. Several others, including polyvinyl alcohol, polyethylene and polyaspartate were also studied. It is suggested that these xenobiotic polymers are metabolized by intracellular enzymes located in the periplasmic space or bound to membranes, indicating that transport of these polymers through outer membranes is requisite for their metabolism. Involvement of specific membrane structures of sphingomonads such as unusual sphingolipids is suggested for membrane transport of xenobiotic compounds, especially hydrophobic materials. Received 01 May 1999/ Accepted in revised form 17 July 1999     

Membrane domains and polarized trafficking of sphingolipids

The plasma membrane of polarized cells consists of distinct domains, the apical and basolateral membrane, that are characterized by a distinct lipid and protein content. Apical protein transport is largely mediated by (glyco)sphingolipid--cholesterol enriched membrane microdomains, so called rafts. In addition changes in the direction of polarized sphingolipid transport appear instrumental in cell polarity development. Knowledge is therefore required of the mechanisms that mediate sphingolipid sorting and the complexity of the trafficking pathways that are involved in polarized transport of both sphingolipids and proteins. Here we summarize specific biophysical properties that underly mechanisms relevant to sphingolipid sorting, cargo recruitment and polarized trafficking, and discuss the central role of a subapical compartment, SAC or common endosome (CE), as a major intracellular site involved in polarized sorting of sphingolipids, and in development and maintenance of membrane polarity.     

Longins and their longin domains: regulated SNAREs and multifunctional SNARE regulators

Longins are the only R-SNAREs that are common to all eukaryotes and are characterized by a conserved N-terminal domain with a profilin-like fold called a longin domain (LD). These domains seem to be essential for regulating membrane trafficking and they mediate unexpected biochemical functions via a range of protein-protein and intramolecular binding specificities. In addition to the longins, proteins involved in the regulation of intracellular trafficking, such as subunits of the adaptor and transport protein particle complexes, also have LD-like folds. The functions and cellular localization of longins are regulated at several levels and the longin prototypes TI-VAMP, Sec22 and Ykt6 show different distributions among eukaryotes, reflecting their modular and functional diversity. In mammals, TI-VAMP and Ykt6 are crucial for neuronal function, and defects in longin structure or function might underlie some human neurological pathologies.