Cardiolipin synthase of Arabidopsis thaliana

Functional expression studies in microorganisms showed that the Arabidopsis thaliana gene At4g04870 represents the cardiolipin synthase (CLS) gene encoding a hydrophobic preprotein of 38 kDa with a cleavable signal peptide for the import into mitochondria. CLS of Arabidopsis over-expressed in Escherichia coli has an alkaline pH optimum, a strict requirement for divalent cations and a distinctly lower K(m) for cytidinediphosphate-diacylglycerol than for phosphatidylglycerol. It displayed a preference for both its substrates esterified with unsaturated acyl groups. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of cardiolipin, to acquire its catalytically active conformation.     

Lipids and topological rules governing membrane protein assembly

Membrane protein folding and topogenesis are tuned to a given lipid profile since lipids and proteins have co-evolved to follow a set of interdependent rules governing final protein topological organization. Transmembrane domain (TMD) topology is determined via a dynamic process in which topogenic signals in the nascent protein are recognized and interpreted initially by the translocon followed by a given lipid profile in accordance with the Positive Inside Rule. The net zero charged phospholipid phosphatidylethanolamine and other neutral lipids dampen the translocation potential of negatively charged residues in favor of the cytoplasmic retention potential of positively charged residues (Charge Balance Rule). This explains why positively charged residues are more potent topological signals than negatively charged residues. Dynamic changes in orientation of TMDs during or after membrane insertion are attributed to non-sequential cooperative and collective lipid–protein charge interactions as well as long-term interactions within a protein. The proportion of dual topological conformers of a membrane protein varies in a dose responsive manner with changes in the membrane lipid composition not only in vivo but also in vitro and therefore is determined by the membrane lipid composition. Switching between two opposite TMD topologies can occur in either direction in vivo and also in liposomes (designated as fliposomes) independent of any other cellular factors. Such lipid-dependent post-insertional reversibility of TMD orientation indicates a thermodynamically driven process that can occur at any time and in any cell membrane driven by changes in the lipid composition. This dynamic view of protein topological organization influenced by the lipid environment reveals previously unrecognized possibilities for cellular regulation and understanding of disease states resulting from mis-folded proteins. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

The diacylglycerol forming pathways differ among floral organs of Petunia hybrida

The origin of diacylglycerol, a substrate for membrane lipid biosynthesis, is not fully understood. Here, we report that Petunia hybrida floral organs contain large amounts of diacylglycerol. Our data suggest that in stamens and pistils diacylglycerol is supplied both from phosphatidylcholine by non-specific phospholipase C activity and de novo via the Kennedy pathway and phosphatidic acid phosphatase, whereas in petals the two-step pathway catalyzed by phospholipase D and phosphatidic acid phosphatase predominates. Therefore, the pathways that supply diacylglycerol differ among floral reproductive organs, although large amounts of diacylglycerol are commonly accumulated in these organs.     

Lipid analysis of mitochondrial membranes from the yeast Pichia pastoris

Here we describe for the first time isolation and biochemical characterization of highly purified mitochondrial inner and outer membranes from Pichia pastoris and systematic lipid analysis of submitochondrial fractions. Mitochondria of this yeast are best developed during growth on glycerol or sorbitol, but also on methanol or fatty acids. To obtain organelle membranes at high quality, methods of isolation and subfractionation of mitochondria originally developed for Saccharomyces cerevisiae were adapted and employed. A characteristic feature of the outer mitochondrial membrane of P. pastoris is the higher phospholipid to protein ratio and the lower ergosterol to phospholipid ratio compared to the inner membrane. Another marked difference between the two mitochondrial membranes is the phospholipid composition. Phosphatidylcholine and phosphatidylethanolamine are major phospholipids of both membranes, but the inner membrane is enriched in cardiolipin, whereas the outer membrane contains a high amount of phosphatidylinositol. The fatty acid composition of both mitochondrial membranes is similar. Variation of the carbon source, however, leads to marked changes of the fatty acid pattern both in total and mitochondrial membranes. In summary, our data are the first step to understand the P. pastoris lipidome which will be prerequisite to manipulate membrane components of this yeast for biotechnological purposes.     

Absence of diglyceride kinase activity in the photoreceptor cells of Drosophila mutants

Diglyceride kinase (DG) activity was found to be almost absent in the photoreceptor cells of Drosophila vision mutants, norpA (no receptor potential A). Other enzymes related to the phosphatidic acid (PA) metabolism, such as monoacyl-glycerophosphate acyltransferase and PA phosphatase, were normal in the mutant. The deficiency in PA content in the retinular cells of norpA, demonstrated by 32P incorporation in vivo is, therefore, due to abnormal reduction in DG kinase activity. The DG kinase was found to be localized in the retinular cell by assaying its activity in the mutants rdgA (receptor cells degenerated) and sine oculis (compound eyes absent).     

Phosphatidylcholine biosynthesis and function in bacteria

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and is estimated to be present in about 15% of the domain Bacteria. Usually, PC can be synthesized in bacteria by either of two pathways, the phospholipid N-methylation (Pmt) pathway or the phosphatidylcholine synthase (Pcs) pathway. The three subsequent enzymatic methylations of phosphatidylethanolamine are performed by a single phospholipid N-methyltransferase in some bacteria whereas other bacteria possess multiple phospholipid N-methyltransferases each one performing one or several distinct methylation steps. Phosphatidylcholine synthase condenses choline directly with CDP-diacylglycerol to form CMP and PC. Like in eukaryotes, bacterial PC also functions as a biosynthetic intermediate during the formation of other biomolecules such as choline, diacylglycerol, or diacylglycerol-based phosphorus-free membrane lipids. Bacterial PC may serve as a specific recognition molecule but it affects the physicochemical properties of bacterial membranes as well. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

The Arabidopsis thaliana lysophospholipid acyltransferase At1g78690p acylates a variety of lysophospholipids including bis(monoacylglycero)phosphate

When the lysoglycerophospholipid (GPL) acyltransferase At1g78690 from Arabidopsis thaliana is over-expressed in Escherichiacoli a headgroup acylated GPL, acyl phosphatidylglycerol (PG), accumulates despite that in vitro this enzyme catalyzes the transfer of an acyl chain from acyl-CoA to the sn-2 position of 1-acyl phosphatidylethanolamine (PE) or 1-acyl PG to form the sn-1, sn-2, di acyl PE and PG respectively; it does not acylate PG to form acyl PG. To begin to understand why the overexpression of a lyso GPL acyltransferase leads to the accumulation of a headgroup acylated GPL in E. coli we investigated the headgroup specificity of At1g78690. Using membranes prepared from E. coli overexpressing At1g78690, we assessed the ability of At1g78690 to catalyze the transfer of acyl chains from acyl-coenzyme A to a variety of lyso GPL acyl acceptors including lyso-phosphatidic acid (PA), -phosphatidylcholine (PC), -phosphatidylserine (PC), -phosphatidylinositol (PI) and three stereoisoforms of bis(monoacylglycero)phosphate (BMP). The predicted products were formed when lyso PI and lyso PC were used as the acyl acceptor but not with lyso PC or lyso PA. In addition, At1g78690 robustly acylates two BMP isoforms with sn-2 and/or sn-2′ hydroxyls in the R-stereoconfiguration, but not the BMP isoform with the sn-2 and sn-2′ hydroxyls in the S-stereoconfiguration. This strongly suggests that At1g78690 is stereoselective for hydroxyls with R-stereochemistry. In addition, this robust acylation of BMPs by At1g78690, which yields acyl PG like molecules, may explain the mechanism by which At1g78690 so strikingly alters the lipid composition of E. coli.     

Modulation of the in vitro activity of lysosomal phospholipase A1 by membrane lipids

Lysosomal phospholipases play a critical role for degradation of cellular membranes after their lysosomal segregation. We investigated the regulation of lysosomal phospholipase A1 by cholesterol, phosphatidylethanolamine, and negatively-charged lipids in correlation with changes of biophysical properties of the membranes induced by these lipids. Lysosomal phospholipase A1 activity was determined towards phosphatidylcholine included in liposomes of variable composition using a whole-soluble lysosomal fraction of rat liver as enzymatic source. Phospholipase A1 activity was then related to membrane fluidity, lipid phase organization and membrane potential as determined by fluorescence depolarization of DPH, 31P NMR and capillary electrophoresis. Phospholipase A1 activity was markedly enhanced when the amount of negatively-charged lipids included in the vesicles was increased from 10 to around 30% of total phospholipids and the intensity of this effect depended on the nature of the acidic lipids used (ganglioside GM1相似文献   

Metabolism, function and mass spectrometric analysis of bis(monoacylglycero)phosphate and cardiolipin

Polyglycerophospholipids (PGPLs) such as bismonoacylglycerophosphate (BMP) and cardiolipin are important membrane phospholipid species for the maintenance of membrane integrity. While BMP serves as membrane curvature regulator in multivesicular bodies for efficient lysosomal enzyme function, cardiolipin stabilizes the electron transfer complex in the inner mitochondrial membrane, which is crucial for physiological ATP production. Beside their membrane modulatory functions PGPLs play an important role in various signaling events. Although a number of disease associations were found for PGPL species, detailed information about their molecular role still remains unknown. This article reviews the known biological functions of PGPLs and the existing mass spectrometric methods. We discuss the different analytical strategies and how ESI–MS/MS can expand our understanding of PGPL homeostasis.     

Identification of membrane O-acyltransferase family motifs

Cellular membranes contain several classes of glycerophospholipids, which have numerous structural and functional roles in cells. Membrane diversity and asymmetry are important for membrane fluidity, curvature, and storage of lipid mediator precursors. Using acyl-CoAs, glycerophospholipids are first formed in the de novo pathway (Kennedy pathway), and then modified in the remodeling pathway (Lands’ cycle) to generate mature membrane. Recently, several lysophospholipid acyltransferases (LPLATs) from two families, the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) family and the membrane bound O-acyltransferase (MBOAT) family, were shown to function in the remodeling pathway. The MBOAT family possesses either LPLAT activity or protein O-acyltransferase activity. While the motifs of the AGPAT family have been well characterized, the MBOAT motifs remain unclear. In this study, we identified four MBOAT motifs essential for LPLAT activities by extensive site-directed mutagenesis. These findings further our understanding of the enzyme reaction mechanisms and will contribute to structure predictions for the MBOAT family enzymes.     

Fragmentation of mitochondrial cardiolipin by copper ions in the Atp7b-/- mouse model of Wilson's disease

Cellular copper overload as found in Wilson's disease may disturb mitochondrial function and integrity. Atp7b^−/− mice accumulate copper in the liver and serve as an animal model for this inherited disease. The molecular mechanism of copper toxicity in hepatocytes is poorly understood. Total mitochondrial lipids from liver of wild-type mice were subjected to oxidative stress by the Cu²⁺/H₂O₂/ascorbate system. Phosphatidic acid (PA) and phosphatidylhydroxyacetone (PHA) were detected as cardiolipin fragmentation products by thin-layer chromatography combined with MALDI-TOF mass spectrometry in oxidized samples, but not in unperturbed ones. The formation of PA and PHA in copper-treated model membrane correlated well with the decrease of cardiolipin. Mitochondrial lipids from Atp7b^−/− mice of different age were analyzed for the presence of PA. While 32-weeks old wild-type (control) and Atp7b^−/− mice did not show any PA, there was a steady increase in the amount of this lipid in Atp7b^−/− mice in contrast to control with increasing age. Hepatocytes from elder Atp7b^−/−mice contained morphologically changed mitochondria unlike cells from wild-type animals of the same age. We concluded that free-radical fragmentation of cardiolipin with the formation of PA is a likely mechanism that damages mitochondria under conditions of oxidative stress due to copper overload. Our findings are relevant for better understanding of molecular mechanisms for liver damage found in Wilson's disease.     

The lipid profile of brown adipose tissue is sex-specific in mice

Brown adipose tissue (BAT) is a thermogenic organ with a vital function in small mammals and potential as metabolic drug target in humans. By using high-resolution LC-tandem-mass spectrometry, we quantified 329 lipid species from 17 (sub)classes and identified the fatty acid composition of all phospholipids from BAT and subcutaneous and gonadal white adipose tissue (WAT) from female and male mice. Phospholipids and free fatty acids were higher in BAT, while DAG and TAG levels were higher in WAT. A set of phospholipids dominated by the residue docosahexaenoic acid, which influences membrane fluidity, showed the highest specificity for BAT. We additionally detected major sex-specific differences between the BAT lipid profiles, while samples from the different WAT depots were comparatively similar. Female BAT contained less triacylglycerol and more phospholipids rich in arachidonic and stearic acid whereas another set of fatty acid residues that included linoleic and palmitic acid prevailed in males. These differences in phospholipid fatty acid composition could greatly affect mitochondrial membranes and other cellular organelles and thereby regulate the function of BAT in a sex-specific manner.     

Surfactant phospholipid metabolism

Pulmonary surfactant is essential for life and is composed of a complex lipoprotein-like mixture that lines the inner surface of the lung to prevent alveolar collapse at the end of expiration. The molecular composition of surfactant depends on highly integrated and regulated processes involving its biosynthesis, remodeling, degradation, and intracellular trafficking. Despite its multicomponent composition, the study of surfactant phospholipid metabolism has focused on two predominant components, disaturated phosphatidylcholine that confers surface-tension lowering activities, and phosphatidylglycerol, recently implicated in innate immune defense. Future studies providing a better understanding of the molecular control and physiological relevance of minor surfactant lipid components are needed. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.     

Glycerolipid synthesis in Chlorella kessleri 11h: I. Existence of a eukaryotic pathway

The fatty acid distributions at the sn-1 and sn-2 positions in major chloroplast lipids of Chlorella kessleri 11h, monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG), were determined to show the coexistence of both C₁₆ and C₁₈ acids at the sn-2 position, i.e. of prokaryotic and eukaryotic types in these galactolipids. For investigation of the biosynthetic pathway for glycerolipids in C. kessleri 11h, cells were fed with [¹⁴C]acetate for 30 min, and then the distribution of the radioactivity among glycerolipids and their constituent fatty acids during the subsequent chase period was determined. MGDG and DGDG were labeled predominantly as the sn-1-C₁₈-sn-2-C₁₆ (C₁₈/C₁₆) species as early as by the start of the chase, which suggested the synthesis of these lipids within chloroplasts via a prokaryotic pathway. On the other hand, the sn-1-C₁₈-sn-2-C₁₈ (C₁₈/C₁₈) species of these galactolipids gradually gained radioactivity at later times, concomitant with a decrease in the radioactivity of the C₁₈/C₁₈ species of phosphatidylcholine (PC). The change at later times can be explained by the conversion of the C₁₈/C₁₈ species of PC into galactolipids through a eukaryotic pathway. The results showed that C. kessleri 11h, distinct from most of other green algal species that were postulated mainly to use a prokaryotic pathway for the synthesis of chloroplast lipids, is similar to a group of higher plants designated as 16:3 plants in terms of the cooperation of prokaryotic and eukaryotic pathways to synthesize chloroplast lipids. We propose that the physiological function of the eukaryotic pathway in C. kessleri 11h is to supply chloroplast membranes with 18:3/18:3-MGDG for their functioning, and that the acquisition of a eukaryotic pathway by green algae was favorable for evolution into land plants.     

Increasing levels of cardiolipin differentially influence packing of phospholipids found in the mitochondrial inner membrane

It is essential to understand the role of cardiolipin (CL) in mitochondrial membrane organization given that changes in CL levels contribute to mitochondrial dysfunction in type II diabetes, ischemia–reperfusion injury, heart failure, breast cancer, and aging. Specifically, there are contradictory data on how CL influences the molecular packing of membrane phospholipids. Therefore, we determined how increasing levels of heart CL impacted molecular packing in large unilamellar vesicles, modeling heterogeneous lipid mixtures found within the mitochondrial inner membrane, using merocyanine (MC540) fluorescence. We broadly categorized lipid vesicles of equal mass as loosely packed, intermediate, and highly packed based on peak MC540 fluorescence intensity. CL had opposite effects on loosely versus highly packed vesicles. Exposure of loosely packed vesicles to increasing levels of CL dose-dependently increased membrane packing. In contrast, increasing amounts of CL in highly packed vesicles decreased the packing in a dose-dependent manner. In vesicles that were categorized as intermediate packing, CL had either no effect or decreased packing at select doses in a dose-independent manner. Altogether, the results aid in resolving some of the discrepant data by demonstrating that CL displays differential effects on membrane packing depending on the composition of the lipid environment. This has implications for mitochondrial protein activity in response to changing CL levels in microdomains of varying composition.     

Lipid Replacement Therapy: A natural medicine approach to replacing damaged lipids in cellular membranes and organelles and restoring function

Lipid Replacement Therapy, the use of functional oral supplements containing cell membrane phospholipids and antioxidants, has been used to replace damaged, usually oxidized, membrane glycerophospholipids that accumulate during aging and in various clinical conditions in order to restore cellular function. This approach differs from other dietary and intravenous phospholipid interventions in the composition of phospholipids and their defense against oxidation during storage, ingestion, digestion and uptake as well as the use of protective molecules that noncovalently complex with phospholipid micelles and prevent their enzymatic and bile disruption. Once the phospholipids have been taken in by transport processes, they are protected by several natural mechanisms involving lipid receptors, transport and carrier molecules and circulating cells and lipoproteins until their delivery to tissues and cells where they can again be transferred to intracellular membranes by specific and nonspecific transport systems. Once delivered to membrane sites, they naturally replace and stimulate removal of damaged membrane lipids. Various chronic clinical conditions are characterized by membrane damage, mainly oxidative but also enzymatic, resulting in loss of cellular function. This is readily apparent in mitochondrial inner membranes where oxidative damage to phospholipids like cardiolipin and other molecules results in loss of trans-membrane potential, electron transport function and generation of high-energy molecules. Recent clinical trials have shown the benefits of Lipid Replacement Therapy in restoring mitochondrial function and reducing fatigue in aged subjects and patients with a variety of clinical diagnoses that are characterized by loss of mitochondrial function and include fatigue as a major symptom. This Article is Part of a Special Issue Entitled: Membrane Structure and Function: Relevance in the Cell's Physiology, Pathology and Therapy.     

Lipids of mitochondria

A unique organelle for studying membrane biochemistry is the mitochondrion whose functionality depends on a coordinated supply of proteins and lipids. Mitochondria are capable of synthesizing several lipids autonomously such as phosphatidylglycerol, cardiolipin and in part phosphatidylethanolamine, phosphatidic acid and CDP-diacylglycerol. Other mitochondrial membrane lipids such as phosphatidylcholine, phosphatidylserine, phosphatidylinositol, sterols and sphingolipids have to be imported. The mitochondrial lipid composition, the biosynthesis and the import of mitochondrial lipids as well as the regulation of these processes will be main issues of this review article. Furthermore, interactions of lipids and mitochondrial proteins which are highly important for various mitochondrial processes will be discussed. Malfunction or loss of enzymes involved in mitochondrial phospholipid biosynthesis lead to dysfunction of cell respiration, affect the assembly and stability of the mitochondrial protein import machinery and cause abnormal mitochondrial morphology or even lethality. Molecular aspects of these processes as well as diseases related to defects in the formation of mitochondrial membranes will be described.