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Despite the essential roles of sphingolipids both as structural components of membranes and critical signalling molecules, we have a limited understanding of how cells sense and regulate their levels. Here we reveal the function in sphingolipid metabolism of the ORM genes (known as ORMDL genes in humans)-a conserved gene family that includes ORMDL3, which has recently been identified as a potential risk factor for childhood asthma. Starting from an unbiased functional genomic approach in Saccharomyces cerevisiae, we identify Orm proteins as negative regulators of sphingolipid synthesis that form a conserved complex with serine palmitoyltransferase, the first and rate-limiting enzyme in sphingolipid production. We also define a regulatory pathway in which phosphorylation of Orm proteins relieves their inhibitory activity when sphingolipid production is disrupted. Changes in ORM gene expression or mutations to their phosphorylation sites cause dysregulation of sphingolipid metabolism. Our work identifies the Orm proteins as critical mediators of sphingolipid homeostasis and raises the possibility that sphingolipid misregulation contributes to the development of childhood asthma.

The pathogenesis and progression of many tumors, including hematologic malignancies is highly dependent on enhanced lipogenesis. De novo fatty-acid synthesis permits accelerated proliferation of tumor cells by providing membrane components but these may also alter physicochemical properties of lipid bilayers, which can impact signaling or even increase drug resistance in cancer cells. Cancer type-specific lipid profiles would permit us to monitor and interpret actual effects of lipid changes, potential fingerprints of individual tumors to be explored as diagnostic markers. We have used the shotgun MS approach to identify lipid patterns in different types of acute myeloid leukemia (AML) patients that either show no karyotype change or belong to t(8;21) or inv16 types. Differences in lipidomes of t(8;21) and inv(16) patients, as compared to AML patients without karyotype change, presented mostly as substantial modulation of ceramide/sphingolipid synthesis. Furthermore, between the t(8;21) and all other patients we observed significant changes in physicochemical membrane properties. These were related to a marked alteration in lipid saturation levels. The discovered differences in lipid profiles of various AML types improve our understanding of the pathobiochemical pathways involved and may serve in the development of diagnostic tools. [ABSTRACT FROM AUTHOR]

The lipid raft hypothesis proposes lateral domains driven by preferential interactions between sterols, sphingolipids, and specific proteins as a central mechanism for the regulation of membrane structure and function; however, experimental limitations in defining raft composition and properties have prevented unequivocal demonstration of their functional relevance. Here, we establish a quantitative, functional relationship between raft association and subcellular protein sorting. By systematic mutation of the transmembrane and juxtamembrane domains of a model transmembrane protein, linker for activation of T-cells (LAT), we generated a panel of variants possessing a range of raft affinities. These mutations revealed palmitoylation, transmembrane domain length, and transmembrane sequence to be critical determinants of membrane raft association. Moreover, plasma membrane (PM) localization was strictly dependent on raft partitioning across the entire panel of unrelated mutants, suggesting that raft association is necessary and sufficient for PM sorting of LAT. Abrogation of raft partitioning led to mistargeting to late endosomes/lysosomes because of a failure to recycle from early endosomes. These findings identify structural determinants of raft association and validate lipid-driven domain formation as a mechanism for endosomal protein sorting. [ABSTRACT FROM AUTHOR]

Aims: We tested whether characteristic changes of the plasma lipidome in individuals with comparable total lipids level associate with future cardiovascular disease (CVD) outcome and whether 23 validated gene variants associated with coronary artery disease (CAD) affect CVD associated lipid species. Methods and Results: Screening of the fasted plasma lipidome was performed by top-down shotgun analysis and lipidome compositions compared between incident CVD cases (n = 211) and controls (n = 216) from the prospective population-based MDC study using logistic regression adjusting for Framingham risk factors. Associations with incident CVD were seen for eight lipid species (0.21≤q≤0.23). Each standard deviation unit higher baseline levels of two lysophosphatidylcholine species (LPC), LPC16∶0 and LPC20∶4, was associated with a decreased risk for CVD (P = 0.024–0.028). Sphingomyelin (SM) 38∶2 was associated with increased odds of CVD (P = 0.057). Five triglyceride (TAG) species were associated with protection (P = 0.031–0.049). LPC16∶0 was negatively correlated with the carotid intima-media thickness (P = 0.010) and with HbA1c (P = 0.012) whereas SM38∶2 was positively correlated with LDL-cholesterol (P = 0.0*10−6) and the q-values were good (q≤0.03). The risk allele of 8 CAD-associated gene variants showed significant association with the plasma level of several lipid species. However, the q-values were high for many of the associations (0.015≤q≤0.75). Risk allele carriers of 3 CAD-loci had reduced level of LPC16∶0 and/or LPC 20∶4 (P≤0.056). Conclusion: Our study suggests that CVD development is preceded by reduced levels of LPC16∶0, LPC20∶4 and some specific TAG species and by increased levels of SM38∶2. It also indicates that certain lipid species are intermediate phenotypes between genetic susceptibility and overt CVD. But it is a preliminary study that awaits replication in a larger population because statistical significance was lost for the associations between lipid species and future cardiovascular events when correcting for multiple testing. [ABSTRACT FROM AUTHOR]

Mass spectrometry-based shotgun lipidomics has enabled the quantitative and comprehensive assessment of cellular lipid compositions. The yeast Saccharomyces cerevisiae has proven to be a particularly valuable experimental system for studying lipid-related cellular processes. Here, by applying our shotgun lipidomics platform, we investigated the influence of a variety of commonly used growth conditions on the yeast lipidome, including glycerophospholipids, triglycerides, ergosterol as well as complex sphingolipids. This extensive dataset allowed for a quantitative description of the intrinsic flexibility of a eukaryotic lipidome, thereby providing new insights into the adjustments of lipid biosynthetic pathways. In addition, we established a baseline for future lipidomic experiments in yeast. Finally, flexibility of lipidomic features is proposed as a new parameter for the description of the physiological state of an organism. [ABSTRACT FROM AUTHOR]

Previous work has showed that ergosterol and sphingolipids become sorted to secretory vesicles immunoisolated using a chimeric, artificial raft membrane protein as bait. In this study, we have extended this analysis to three populations of secretory vesicles isolated using natural yeast plasma membrane (PM) proteins: Pma1p, Mid2p and Gap1*p as baits. We compared the lipidomes of the immunoisolated vesicles with each other and with the lipidomes of the donor compartment, the trans-Golgi network, and the acceptor compartment, the PM, using a quantitative mass spectrometry approach that provided a complete lipid overview of the yeast late secretory pathway. We could show that vesicles captured with different baits carry the same cargo and have almost identical lipid compositions; being highly enriched in ergosterol and sphingolipids. This finding indicates that lipid raft sorting is a generic feature of vesicles carrying PM cargo and suggests a common lipid-based mechanism for their formation. [ABSTRACT FROM AUTHOR]

Biological membranes are compartmentalized for functional diversity by a variety of specific protein-protein, protein-lipid, and lipid-lipid interactions. A subset of these are the preferential interactions between sterols, sphingolipids, and saturated aliphatic lipid tails responsible for liquid-liquid domain coexistence in eukaryotic membranes, which give rise to dynamic, nanoscopic assemblies whose coalescence is regulated by specific biochemical cues. Microscopic phase separation recently observed in isolated plasma membranes (giant plasma membrane vesicles and plasma membrane spheres) (i) confirms the capacity of compositionally complex membranes to phase separate, (ii) reflects the nanoscopic organization of live cell membranes, and (iii) provides a versatile platform for the investigation of the compositions and properties of the phases. Here, we show that the properties of coexisting phases in giant plasma membrane vesicles are dependent on isolation conditions-namely, the chemicals used to induce membrane blebbing. We observe strong correlations between the relative compositions and orders of the coexisting phases, and their resulting miscibility. Chemically unperturbed plasma membranes reflect these properties and validate the observations in chemically induced vesicles. Most importantly, we observe domains with a continuum of varying stabilities, orders, and compositions induced by relatively small differences in isolation conditions. These results show that, based on the principle of preferential association of raft lipids, domains of various properties can be produced in a membrane environment whose complexity is reflective of biological membranes. [ABSTRACT FROM AUTHOR]

Ten years ago, we wrote a Review on lipid rafts and signalling in the launch issue of Nature Reviews Molecular Cell Biology. At the time, this field was suffering from ambiguous methodology and imprecise nomenclature. Now, new techniques are deepening our insight into the dynamics of membrane organization. Here, we discuss how the field has matured and present an evolving model in which membranes are occupied by fluctuating nanoscale assemblies of sphingolipids, cholesterol and proteins that can be stabilized into platforms that are important in signalling, viral infection and membrane trafficking. [ABSTRACT FROM AUTHOR]

Abstract: In this review we describe the history of the development of the raft concept for membrane sub-compartmentalization. From its early beginnings as a mechanism for apical sorting in epithelial cells the concept has evolved to a general principle for membrane organisation. After a shaky start with crude methodology based on detergent extraction the field has become increasingly sophisticated, employing a host of different methods that support the existence of dynamic raft domains in membranes. These are composed of fluctuating nanoscale assemblies of sphingolipid, cholesterol and proteins that can be stabilized to coalesce, forming platforms that function in membrane signalling and trafficking. [Copyright &y& Elsevier]

Recently synthesized proteins are sorted at the trans-Golgi network into specialized routes for exocytosis. Surprisingly little is known about the underlying molecular machinery. Here, we present a visual screen to search for proteins involved in cargo sorting and vesicle formation. We expressed a GFP-tagged plasma membrane protein in the yeast deletion library and identified mutants with altered marker localization. This screen revealed a requirement of several enzymes regulating the synthesis of sphingolipids and ergosterol in the correct and efficient delivery of the marker protein to the cell surface. Additionally, we identified mutants regulating the actin cytoskeleton (Rvs161p and Vrp1p), known membrane traffic regulators (Kes1p and Chs5p), and several unknown genes. This visual screening method can now be used for different cargo proteins to search in a genome-wide fashion for machinery involved in post-Golgi sorting. [ABSTRACT FROM AUTHOR]

Views of how cell membranes are organized are presently changing. The lipid bilayer that constitutes these membranes is no longer understood to be a homogeneous fluid. Instead, lipid assemblies, termed rafts, have been introduced to provide fluid platforms that segregate membrane components and dynamically compartmentalize membranes. These assemblies are thought to be composed mainly of sphingolipids and cholesterol in the outer leaflet, somehow connected to domains of unknown composition in the inner leaflet. Specific classes of proteins are associated with the rafts. This review critically analyzes what is known of phase behavior and liquid-liquid immiscibility in model systems and compares these data with what is known of domain formation in cell membranes. [ABSTRACT FROM AUTHOR]

Sphingolipid-cholesterol rafts are microdomains in biological membranes with liquid-ordered phase properties which are implicated in membrane traffic and signalling events. We have used influenza virus haemagglutinin (HA) as a model protein to analyse the interaction of transmembrane proteins with these microdomains. Here we demonstrate that raft association is an intrinsic property encoded in the protein. Mutant HA molecules with foreign transmembrane domain (TMD) sequences lose their ability to associate with the lipid microdomains, and mutations in the HA TMD reveal a requirement for hydrophobic residues in contact with the exoplasmic leaflet of the membrane. We also provide experimental evidence that cholesterol is critically required for association of proteins with lipid rafts. Our data suggest that the binding to specific membrane domains can be encoded in transmembrane proteins and that this information will be used for polarized sorting and signal transduction processes. [ABSTRACT FROM AUTHOR]

Abstract: In eukaryotic cells, the trans-Golgi network serves as a sorting station for post-Golgi traffic. In addition to coat- and adaptor-mediated mechanisms, studies in mammalian epithelial cells and yeast have provided evidence for lipid-dependent protein sorting as a major delivery mechanism for cargo sorting to the cell surface. The mechanism for lipid-mediated sorting is the generation of raft platforms of sphingolipids, sterols and specific sets of cargo proteins by phase segregation in the TGN. Here, we review the evidence for such lipid-raft-based sorting at the TGN, as well as their involvement in the formation of TGN-to-PM transport carriers. This article is part of a Special Issue entitled Lipids and Vesicular Transport. [Copyright &y& Elsevier]

Increasing evidence suggests that biological membranes can be laterally subdivided into domains enriched in specific lipid and protein components and that these domains maybe involved in the regulation of a number of vital cellular processes. An example is membrane rafts, which are lipid-mediated domains dependent on preferential association between sterols and sphingolipids and inclusive of a specific subset of membrane proteins. While the lipid and protein composition of rafts has been extensively characterized, the structural details determining protein partitioning to these domains remain unresolved. Here, we review evidence suggesting that post-translation modification by saturated lipids recruits both peripheral and transmembrane proteins to rafts, while short, unsaturated, and/or branched hydrocarbon chains prevent raft association. The most widely studied group of raft-associated proteins are glycophosphatidylinositol- anchored proteins (GPI-AP), and we review a variety of evidence supporting raft-association of these saturated lipid-anchored extracellular peripheral proteins. For transmembrane and intracellular peripheral proteins, S-acylation with saturated fatty acids mediates raft partitioning, and the dynamic nature of this modification presents an exciting possibility of enzymatically regulated raft association. The other common lipid modifications, that is, prenylation and myristoylation, are discussed in light of their likely role in targeting proteins to nonraft membrane regions. Finally, although the association between raft affinity and lipid modification is well-characterized, we discuss several open questions regarding regulation and remodeling of these post-translational modifications as well as their role in transbilayer coupling of membrane domains. [ABSTRACT FROM AUTHOR]

Abstract: Several simplified membrane models featuring coexisting liquid disordered (Ld) and ordered (Lo) lipid phases have been developed to mimic the heterogeneous organization of cellular membranes, and thus, aid our understanding of the nature and functional role of ordered lipid–protein nanodomains, termed “rafts”. In spite of their greatly reduced complexity, quantitative characterization of local lipid environments using model membranes is not trivial, and the parallels that can be drawn to cellular membranes are not always evident. Similarly, various fluorescently labeled lipid analogs have been used to study membrane organization and function in vitro, although the biological activity of these probes in relation to their native counterparts often remains uncharacterized. This is particularly true for raft-preferring lipids (“raft lipids”, e.g. sphingolipids and sterols), whose domain preference is a strict function of their molecular architecture, and is thus susceptible to disruption by fluorescence labeling. Here, we analyze the phase partitioning of a multitude of fluorescent raft lipid analogs in synthetic Giant Unilamellar Vesicles (GUVs) and cell-derived Giant Plasma Membrane Vesicles (GPMVs). We observe complex partitioning behavior dependent on label size, polarity, charge and position, lipid headgroup, and membrane composition. Several of the raft lipid analogs partitioned into the ordered phase in GPMVs, in contrast to fully synthetic GUVs, in which most raft lipid analogs mis-partitioned to the disordered phase. This behavior correlates with the greatly enhanced order difference between coexisting phases in the synthetic system. In addition, not only partitioning, but also ligand binding of the lipids is perturbed upon labeling: while cholera toxin B binds unlabeled GM1 in the Lo phase, it binds fluorescently labeled GM1 exclusively in the Ld phase. Fluorescence correlation spectroscopy (FCS) by stimulated emission depletion (STED) nanoscopy on intact cellular plasma membranes consistently reveals a constant level of confined diffusion for raft lipid analogs that vary greatly in their partitioning behavior, suggesting different physicochemical bases for these phenomena. [Copyright &y& Elsevier]

The lipid raft concept proposes that biological membranes have the potential to form functional domains based on a selective interaction between sphingolipids and sterols. These domains seem to be involved in signal transduction and vesicular sorting of proteins and lipids. Although there is biochemical evidence for lipid raft-dependent protein and lipid sorting in the yeast Saccharomyces cerevisiae, direct evidence for an interaction between yeast sphingolipids and the yeast sterol ergosterol, resulting in membrane domain formation, is lacking. Here we show that model membranes formed from yeast total lipid extracts possess an inherent self-organization potential resulting in liquid-disordered-liquid-ordered phase coexistence at physiologically relevant temperature. Analyses of lipid extracts from mutants defective in sphingolipid metabolism as well as reconstitution of purified yeast lipids in model membranes of defined composition suggest that membrane domain formation depends on specific interactions between yeast sphingolipids and ergosterol. Taken together, these results provide a mechanistic explanation for lipid raft-dependent lipid and protein sorting in yeast. [ABSTRACT FROM AUTHOR]

The β-secretase, BACE, is a membrane spanning aspartic protease, which cleaves the amyloid precursor protein (APP) in the first step of proteolytic processing leading to the formation of the neurotoxic β-amyloid peptide (Aβ). Previous results have suggested that the regulation of β-secretase and BACE access to APP is lipid dependent, and involves lipid rafts. Using the baculovirus expression system, we have expressed recombinant human full-length BACE in insect cells and purified milligram amounts to homogeneity. We have studied partitioning of fluorophor-conjugated BACE between the liquid ordered and disordered phases in giant (10–150 μm) unilamellar vesicles, and found ∼20% to associate with the raft-like, liquid-ordered phase; the fraction associated with liquid-ordered phase increased upon cross-linking of raft lipids. To examine involvement of individual lipid species in modulating BACE activity, we have reconstituted the purified BACE in large (∼100 nm) unilamellar vesicles, and determined its specific activity in vesicles of various lipid compositions. We have identified 3 groups of lipids that stimulate proteolytic activity of BACE: 1) neutral glycosphingolipids (cerebrosides), 2) anionic glycerophospholipids, and 3) sterols (cholesterol). [ABSTRACT FROM AUTHOR]