Membrane Lipids In Eukaryotes Research Articles

Crossing the lipid divide

Archaeal membrane lipids are structurally different from bacterial and eukaryotic membrane lipids, but little is known about the enzymes involved in their synthesis. In a recent study, Exterkate et al. identified and characterized a cardiolipin synthase from the archaeon Methanospirillum hungatei. This enzyme can synthesize archaeal, bacterial, and mixed archaeal/bacterial cardiolipin species from a wide variety of substrates, some of which are not even naturally occurring. This discovery could revolutionize synthetic lipid biology, being used to construct a variety of lipids with nonnatural head groups and mixed archaeal/bacterial hydrophobic chains.

Structural insights into the regulation of human serine palmitoyltransferase complexes

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase (SPT) complex. The SPT complex consists of SPTLC1 and SPTLC2 as catalytic components, and ssSPTa and ORMDL3 as regulatory components. To understand the assembly, substrate processing and regulation of the complex, we determined cryo-electron microscopy structures of the human SPT complex in various functional states at resolutions of 2.6–3.4 Å. The structures elucidate how catalytic components recognize the substrate, as well as how regulatory components modulate the substrate-binding tunnel to control enzyme activity. These findings reveal the molecular mechanism of sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

Structural insights into the regulation of human serine palmitoyltransferase complexes.

Sphingolipids are essential lipids in eukaryotic membranes. In humans, the first and rate-limiting step of sphingolipid synthesis is catalyzed by the serine palmitoyltransferase holocomplex, which consists of catalytic components (SPTLC1 and SPTLC2) and regulatory components (ssSPTa and ORMDL3). However, the assembly, substrate processing and regulation of the complex are unclear. Here, we present 8 cryo-electron microscopy structures of the human serine palmitoyltransferase holocomplex in various functional states at resolutions of 2.6-3.4 Å. The structures reveal not only how catalytic components recognize the substrate, but also how regulatory components modulate the substrate-binding tunnel to control enzyme activity: ssSPTa engages SPTLC2 and shapes the tunnel to determine substrate specificity. ORMDL3 blocks the tunnel and competes with substrate binding through its amino terminus. These findings provide mechanistic insights into sphingolipid biogenesis governed by the serine palmitoyltransferase complex.

Combining Colistin and Fluconazole Synergistically Increases Fungal Membrane Permeability and Antifungal Cidality.

The increasing emergence of drug-resistant fungal pathogens, together with the limited number of available antifungal drugs, presents serious clinical challenges to treating systemic, life-threatening infections. Repurposing existing drugs to augment the antifungal activity of well-tolerated antifungals is a promising antifungal strategy with the potential to be implemented rapidly. Here, we explored the mechanism by which colistin, a positively charged lipopeptide antibiotic, enhances the antifungal activity of fluconazole, the most widely used orally available antifungal. In a range of susceptible and drug-resistant isolates and species, colistin was primarily effective at reducing fluconazole tolerance, a property of subpopulations of cells that grow slowly in the presence of a drug and may promote the emergence of persistent infections and resistance. Clinically relevant concentrations of colistin synergized with fluconazole, reducing fluconazole minimum inhibitory concentration 4-fold. Combining fluconazole and colistin also increased survival in a C. albicans Galleria mellonella infection, especially for a highly fluconazole-tolerant isolate. Mechanistically, colistin increased permeability to fluorescent antifungal azole probes and to intracellular dyes, accompanied by an increase in cell death that was dependent upon pharmacological or genetic inhibition of the ergosterol biosynthesis pathway. The positive charge of colistin is critical to its antifungal, and antibacterial, activity: colistin directly binds to several eukaryotic membrane lipids (i.e., l-α-phosphatidylinositol, l-α-phosphatidyl-l-serine, and l-α-phosphatidylethanolamine) that are enriched in the membranes of ergosterol-depleted cells. These results support the idea that colistin binds to fungal membrane lipids and permeabilizes fungal cells in a manner that depends upon the degree of ergosterol depletion.

Chemical diversity and mode of action of natural products targeting lipids in the eukaryotic cell membrane.

Covering: up to 2019Nature furnishes bioactive compounds (natural products) with complex chemical structures, yet with simple, sophisticated molecular mechanisms. When natural products exhibit their activities in cells or bodies, they first have to bind or react with a target molecule in/on the cell. The cell membrane is a major target for bioactive compounds. Recently, our understanding of the molecular mechanism of interactions between natural products and membrane lipids progressed with the aid of newly-developed analytical methods. New technology reconnects old compounds with membrane lipids, while new membrane-targeting molecules are being discovered through the screening for antimicrobial potential of natural products. This review article focuses on natural products that bind to eukaryotic membrane lipids, and includes clinically important molecules and key research tools. The chemical diversity of membrane-targeting natural products and the molecular basis of lipid recognition are described. The history of how their mechanism was unveiled, and how these natural products are used in research are also mentioned.

Improved Cation Binding to Lipid Bilayers with Negatively Charged POPS by Effective Inclusion of Electronic Polarization.

Phosphatidylserine (PS) lipids are important signaling molecules and the most common negatively charged lipids in eukaryotic membranes. The signaling can be often regulated by calcium, but its interactions with PS headgroups are not fully understood. Classical molecular dynamics (MD) simulations can potentially give detailed description of lipid-ion interactions, but the results strongly depend on the used force field. Here, we apply the electronic continuum correction (ECC) to the Amber Lipid17 parameters of 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) lipid to improve its interactions with K+, Na+, and Ca2+ ions. The partial charges of the headgroup, glycerol backbone, and carbonyls of POPS, bearing a unit negative charge, were scaled with a factor of 0.75, derived for monovalent ions, and the Lennard-Jones σ parameters of the same segments were scaled with a factor of 0.89. The resulting ECC-POPS model gives more realistic interactions with Na+ and Ca2+ cations than the original Amber Lipid17 parameters when validated using headgroup order parameters and the "electrometer concept". In ECC-lipids simulations, populations of complexes of Ca2+ cations with more than two PS lipids are negligible, and interactions of Ca2+ cations with only carboxylate groups are twice more likely than with only phosphate groups, while interactions with carbonyls almost entirely involve other groups as well. Our results pave the way for more realistic MD simulations of biomolecular systems with anionic membranes, allowing signaling processes involving PS and Ca2+ to be elucidated.

Mycobacterium tuberculosis (green) uses a surface‐exposed sphingomyelinase (ribbon diagram) to cleave the eukaryotic membrane lipid sphingomyelin (space filling model). This activity lyses erythrocytes and enhances replication of Mtb in macrophages (red/blue). For details, see the article by Speer et al. on pp. 881–897 of this issue.

<i>Mycobacterium tuberculosis </i>(green) uses a surface‐exposed sphingomyelinase (ribbon diagram) to cleave the eukaryotic membrane lipid sphingomyelin (space filling model). This activity lyses erythrocytes and enhances replication of <i>Mtb </i>in macrophages (red/blue). For details, see the article by Speer <i>et al. </i>on pp. 881–897 of this issue.

Editorial: "Agrobacterium biology and its application to transgenic plant production".

Editorial: "Agrobacterium biology and its application to transgenic plant production".

Autophagic flux and autophagosome morphogenesis require the participation of sphingolipids.

Apoptosis and autophagy are two evolutionary conserved processes that exert a critical role in the maintenance of tissue homeostasis. While apoptosis is a tightly regulated cell program implicated in the removal of damaged or unwanted cells, autophagy is a cellular catabolic pathway that is involved in the lysosomal degradation and recycling of proteins and organelles, and is thereby considered an important cytoprotection mechanism. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including lipid rafts and caveolae, and contribute to a number of cellular functions such as cell proliferation, apoptosis and, as suggested more recently, autophagy. For instance, SLs are hypothesized to be involved in several intracellular processes, including organelle membrane scrambling, whilst at the plasma membrane lipid rafts, acting as catalytic domains, strongly contribute to the ignition of critical signaling pathways determining cell fate. In particular, by targeting several shared regulators, ceramide, sphingosine-1-phosphate, dihydroceramide, sphingomyelin and gangliosides seem able to differentially regulate the autophagic pathway and/or contribute to the autophagosome formation. This review illustrates recent studies on this matter, particularly lipid rafts, briefly underscoring the possible implication of SLs and their alterations in the autophagy disturbances and in the pathogenesis of some human diseases.

Novel reactions in acyl editing of phosphatidylcholine by lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities in microsomal preparations of plant tissues

Main conclusion Plants have lysophosphatidylcholine transacylase (LPCT) and acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activities. The combined action of LPCT and GPCAT provides a novel route of PC re-synthesis after its deacylation. Phosphatidylcholine (PC) is the major lipid in eukaryotic membranes and has a central role in overall plant lipid metabolism. It is also the site of production of polyunsaturated fatty acids in plants. The recently discovered acyl-CoA:glycerophosphocholine acyltransferase (GPCAT) activity in yeast provides a novel route of re-synthesising PC via lysophosphatidylcholine (LPC) after its deacylation. This route does not require the degradation of the glycerophosphocholine (GPC) into free choline, the activation of choline to CDP-choline, nor the utilization of CDP-choline by the CDP-choline:diacylglycerol cholinephosphotransferase. We show here that GPCAT activities also are present in membrane preparations from developing oil seeds of safflower and other species as well as in membrane preparations of roots and leaves of Arabidopsis, indicating that GPCAT activity plays a ubiquitous role in plant lipid metabolism. The last step in formation of GPC, the substrate for GPCAT, is the deacylation of LPC. Microsomal membranes of developing safflower seeds utilized LPC in LPC:LPC transacylation reactions (LPCT activities) creating PC and GPC. The results demonstrate that safflower membranes have LPCT and GPCAT activities that represent novel reactions for PC acyl editing. The physiological relevance of these reactions probably has to await identification of the enzymes catalysing these reactions.

The pleiotropic roles of sphingolipid signaling in autophagy.

The autophagic process involves encompassing damaged proteins and organelles within double- or multi-membraned structures and delivering these molecules to the lytic compartments of vacuoles. Sphingolipids (SLs), which are ubiquitous membrane lipids in eukaryotes, participate in the generation of various membrane structures, including rafts, caveolae, and cytosolic vesicles. SLs are a complex family of molecules that have a growing number of members, including ceramide, sphingosine-1-phosphate, and dihydroceramide, which have been associated with the essential cellular process of autophagy. This review highlights recent studies focusing on the regulation and function of SL-associated autophagy and its role in cell fate, diseases, and therapeutic interventions.

The widespread plant‐colonizing bacterial species Pseudomonas syringae detects and exploits an extracellular pool of choline in hosts

The quaternary ammonium compound (QAC) choline is a major component of membrane lipids in eukaryotes and, if available to microbial colonists of plants, could provide benefits for growth and protection from stress. Free choline is found in homogenized plant tissues, but its subcellular location and availability to plant microbes are not known. Whole-cell bacterial bioreporters of the phytopathogen Pseudomonas syringae were constructed that couple a QAC-responsive transcriptional fusion with well-characterized bacterial QAC transporters. These bioreporters demonstrated the presence of abundant free choline compounds released from germinating seeds and seedlings of the bean Phaseolus vulgaris, and a smaller but consistently detectable amount of QACs, probably choline, from leaves. The localization of P.syringae bioreporter cells to the surface and intercellular sites of plant tissues demonstrated the extracellular location of these QAC pools. Moreover, P.syringae mutants that were deficient in the uptake of choline compounds exhibited reduced fitness on leaves, highlighting the importance of extracellular choline to P.syringae on leaves. Our data support a model in which this choline pool is derived from the phospholipid phosphatidylcholine through plant-encoded phospholipases that release choline into the intercellular spaces of plant tissues, such as for membrane lipid recycling. The consequent extracellular release of choline compounds enables their interception and exploitation by plant-associated microbes, and thus provides a selective advantage for microbes such as P.syringae that are adapted to maximally exploit choline.

Functional convergence of hopanoids and sterols in membrane ordering

Liquid-ordered phases are one of two biochemically active membrane states, which until now were thought to be a unique consequence of the interactions between eukaryotic membrane lipids. The formation of a liquid-ordered phase depends crucially on the ordering properties of sterols. However, it is not known whether this capacity exists in organisms that lack sterols, such as bacteria. We show that diplopterol, the simplest bacterial hopanoid, has similar properties and that hopanoids are bacterial "sterol surrogates" with the ability to order saturated lipids and to form a liquid-ordered phase in model membranes. These observations suggest that the evolution of an ordered biochemically active liquid membrane could have evolved before the oxygenation of Earth's surface and the emergence of sterols.

Molecular Determinants of Sphingomyelin Specificity of a Eukaryotic Pore-forming Toxin

Sphingomyelin (SM) is abundant in the outer leaflet of the cell plasma membrane, with the ability to concentrate in so-called lipid rafts. These specialized cholesterol-rich microdomains not only are associated with many physiological processes but also are exploited as cell entry points by pathogens and protein toxins. SM binding is thus a widespread and important biochemical function, and here we reveal the molecular basis of SM recognition by the membrane-binding eukaryotic cytolysin equinatoxin II (EqtII). The presence of SM in membranes drastically improves the binding and permeabilizing activity of EqtII. Direct binding assays showed that EqtII specifically binds SM, but not other lipids and, curiously, not even phosphatidylcholine, which presents the same phosphorylcholine headgroup. Analysis of the EqtII interfacial binding site predicts that electrostatic interactions do not play an important role in the membrane interaction and that the two most important residues for sphingomyelin recognition are Trp(112) and Tyr(113) exposed on a large loop. Experiments using site-directed mutagenesis, surface plasmon resonance, lipid monolayer, and liposome permeabilization assays clearly showed that the discrimination between sphingomyelin and phosphatidylcholine occurs in the region directly below the phosphorylcholine headgroup. Because the characteristic features of SM chemistry lie in this subinterfacial region, the recognition mechanism may be generic for all SM-specific proteins.

Multiple functions of ergosterol in the fission yeast Schizosaccharomyces pombe

Sterols are a major class of membrane lipids in eukaryotes. In Schizosaccharomyces pombe, sterol 24-C-methyltransferase (Erg6p), C-8 sterol isomerase (Erg2p), C-5 sterol desaturase (Erg31p, Erg32p), C-22 sterol desaturase (Erg5p) and C-24 (28) sterol reductase (Sts1p/Erg4p) have been predicted, but not yet determined, to catalyse a sequence of reactions from zymosterol to ergosterol. Disruption mutants of these genes were unable to synthesize ergosterol, and most were tolerant to the polyene drugs amphotericin B and nystatin. Disruption of erg31(+) or erg32(+) did not cause ergosterol deficiency or tolerance to polyene drugs, indicating that the two C-5 sterol desaturases have overlapping functions. GFP-tagged DRM (detergent-resistant membrane)-associated protein Pma1p localized to the plasma membrane in ergDelta mutants. DRM fractionation revealed that the association between Pma1-GFP and DRM was weakened in erg6Delta but not in other erg mutants. Several GFP-tagged plasma membrane proteins were tested, and an amino acid permease homologue, SPBC359.03c, was found to mislocalize to intracellular punctate structures in the ergDelta mutants. These results indicate that these proteins are responsible for ergosterol biosynthesis in fission yeast, similar to the situation in Saccharomyces cerevisiae. Furthermore, in fission yeast, ergosterol is important for plasma membrane structure and function and for localization of plasma membrane proteins.

Glycolipid transfer protein: Clear structure and activity, but enigmatic function

Glycosphingolipids comprize a small (typically 5–10% by weight) but vital fraction of membrane lipids in eukaryotes (Holthuis et al., 2001). They provide the plasma membrane with chemical and mechanical stabilities and take part in fundamental biological processes including differentiation, cell–cell interaction, and transmembrane signaling. For these lipids, as for most lipids in general, local metabolism and selective transport are important determinants (Sprong et al., 2001). Little is known about these processes, but it is clear that several key players in the organization and control of sphingolipid composition remain to be identified. A protein purified from bovine spleen cytosol specifically transferring glycolipids was already described more than 20 years ago ([Metz and Radin, 1980] and [Radin and Metz, 1982]). An absolute specificity was found for glycolipids containing a β-linked sugar to the hydrophobic backbone (Yamada et al., 1986). Glycolipid transfer proteins (GLTPs, EC number not assigned) are water-soluble proteins of average size of 24 kDa that have been studied extensively in vitro ([Abe and Sasaki, 1985], [Abe et al., 1982], [Brown et al., 1990], [Gammon et al., 1987] and [Metz and Radin, 1982]). Very recently, their structural and in vitro functional properties were reviewed in detail (Brown and Mattjus, 2007). These proteins have been isolated from different sources ranging from spinach chloroplasts to mammalian brain, liver and kidney ([Brown et al., 1990], [Sasaki, 1985] and [Sasaki, 1990]). GLTP orthologs in fungi and plants were found to be involved in programmed cell death ([Brodersen et al., 2002], [Mattjus et al., 2003] and [Saupe et al., 1994]) but their function in mammals is largely unknown. The activity of GLTP is preserved after expression in E. coli ([Godi et al., 2004], Lin et al., 2000 X. Lin, P. Mattjus, H.M. Pike, A.J. Windebank and R.E. Brown, Cloning and expression of glycolipid transfer protein from bovine and porcine brain, J Biol Chem 275 (2000), pp. 5104–5110. Full Text via CrossRef | View Record in Scopus | Cited By in Scopus (32)[Lin et al., 2000], [Malinina et al., 2004] and [Rao et al., 2004]) allowing for detailed structure–function studies in vitro.

Virulence of Agrobacterium tumefaciens requires phosphatidylcholine in the bacterial membrane

Phosphatidylcholine (PC, lecithin) has long been considered a solely eukaryotic membrane lipid. Only a minority of all bacteria is able to synthesize PC. The plant-transforming bacterium Agrobacterium tumefaciens encodes two potential PC forming enzymes, a phospholipid N-methyltransferase (PmtA) and a PC synthase (Pcs). We show that PC biosynthesis and tumour formation on Kalanchoë plants was impaired in the double mutant. The virulence defect was due to a complete lack of the type IV secretion machinery in the Agrobacterium PC mutant. Our results strongly suggest that PC in bacterial membranes is an important determinant for the establishment of host-microbe interactions.

Abnormal sterols in cholesterol-deficiency diseases cause secretory granule malformation and decreased membrane curvature

Cholesterol is an abundant lipid in eukaryotic membranes, implicated in numerous structural and functional capacities. Here, we have investigated the mechanism by which cholesterol affects secretory granule biogenesis in vivo using Dhcr7(-/-) and Sc5d(-/-) mouse models of the human diseases, Smith-Lemli-Opitz syndrome (SLOS) and lathosterolosis. These homozygous-recessive multiple-malformation disorders are characterized by the functional absence of one of the last two enzymes in the cholesterol biosynthetic pathway, resulting in the accumulation of precursors. Cholesterol-deficient mice exhibit a significant decrease in the numbers of secretory granules in the pancreas, pituitary and adrenal glands. Moreover, there was an increase in morphologically aberrant granules in the exocrine pancreas of Dhcr7(-/-) acinar cells. Regulated secretory pathway function was also severely diminished in these cells, but could be restored with exogenous cholesterol. Sterol precursors incorporated in artificial membranes resulted in decreased bending rigidity and intrinsic curvature compared with cholesterol, thus providing a cholesterol-mediated mechanism for normal granule budding, and an explanation for granule malformation in SLOS and lathosterolosis.

Enterotoxigenic Escherichia coli vesicles target toxin delivery into mammalian cells

Enterotoxigenic Escherichia coli (ETEC) is a prevalent cause of traveler's diarrhea and infant mortality in third-world countries. Heat-labile enterotoxin (LT) is secreted from ETEC via vesicles composed of outer membrane and periplasm. We investigated the role of ETEC vesicles in pathogenesis by analyzing vesicle association and entry into eukaryotic cells. Fluorescently labeled vesicles from LT-producing and LT-nonproducing strains were compared in their ability to bind adrenal and intestinal epithelial cells. ETEC-derived vesicles, but not control nonpathogen-derived vesicles, associated with cells in a time-, temperature-, and receptor-dependent manner. Vesicles were visualized on the cell surface at 4 degrees C and detected intracellularly at 37 degrees C. ETEC vesicle endocytosis depended on cholesterol-rich lipid rafts. Entering vesicles partially colocalized with caveolin, and the internalized vesicles accumulated in a nonacidified compartment. We conclude that ETEC vesicles serve as specifically targeted transport vehicles that mediate entry of active enterotoxin and other bacterial envelope components into host cells. These data demonstrate a role in virulence for ETEC vesicles.

Interaction of triclosan with eukaryotic membrane lipids.

The possibility that triclosan and PVM/MA (polyvinylmethyl ether/maleic acid) copolymer, additives to dentrifrices, could interact with eukaryotic membrane lipids was studied by two methods: first, by determining the pressure/molecular area isotherms at 37 degrees C of glycerophospholipid monolayers, using the Langmuir technique; and second, by phase-transition parameters in liposomes of the same lipids, using differential scanning calorimetry (DSC). Triclosan interacted, in a concentration-independent manner, with monolayers of saturated phosphatidylcholines (PC; i.e. markers of the outer membrane leaflet of eukaryotic cells). Triclosan and PVM/MA copolymer mixtures were shown to clearly interact in a concentration-dependent manner with PC. Triclosan was found to interact with liposomes of saturated and unsaturated phosphatidylcholines and phosphatidylserines (PS; i.e. markers of the inner membrane leaflet of eukaryotic cells), and saturated ethanolamines (PE; i.e. markers of the inner membrane leaflet of eukaryotic cells), resulting in a decrease of the lipid melting temperature (Tm). PVM/MA copolymer changed the Tm of PS, PC, and PE in different manners. By adding PVM/MA or triclosan-PVM/MA copolymer mixtures to 1-stearoyl-2-oleoyl-sn-glycero-3-phosphoserine (SOPS) no lipid transitions were detected. A biphasic change of the PC transition temperature resulted when triclosan or triclosan PVM/MA copolymer mixtures were added, indicating domain formation and change of the lipid polymorphism.