Lipid-modifying Enzymes Research Articles

Phospholipase D1 Production of Phosphatidic Acid at the Plasma Membrane Promotes Exocytosis of Large Dense-core Granules at a Late Stage

Substantial efforts have recently been made to demonstrate the importance of lipids and lipid-modifying enzymes in various membrane trafficking processes, including calcium-regulated exocytosis of hormones and neurotransmitters. Among bioactive lipids, phosphatidic acid (PA) is an attractive candidate to promote membrane fusion through its ability to change membrane topology. To date, however, the biosynthetic pathway, the dynamic location, and actual function of PA in secretory cells remain unknown. Using a short interference RNA strategy on chromaffin and PC12 cells, we demonstrate here that phospholipase D1 is activated in secretagogue-stimulated cells and that it produces PA at the plasma membrane at the secretory granule docking sites. We show that phospholipase D1 activation and PA production represent key events in the exocytotic progression. Membrane capacitance measurements indicate that reduction of endogenous PA impairs the formation of fusion-competent granules. Finally, we show that the PLD1 short interference RNA-mediated inhibition of exocytosis can be rescued by exogenous provision of a lipid that favors the transition of opposed bi-layer membranes to hemifused membranes having the outer leaflets fused. Our findings demonstrate that PA synthesis is required during exocytosis to facilitate a late event in the granule fusion pathway. We propose that the underlying mechanism is related to the ability of PA to alter membrane curvature and promote hemi-fusion.

Arabidopsis Phosphatidylinositol Phosphate Kinase 1 Binds F-actin and Recruits Phosphatidylinositol 4-Kinase β1 to the Actin Cytoskeleton

The actin cytoskeleton can be influenced by phospholipids and lipid-modifying enzymes. In animals the phosphatidylinositol phosphate kinases (PIPKs) are associated with the cytoskeleton through a scaffold of proteins; however, in plants such an interaction was not clear. Our approach was to determine which of the plant PIPKs interact with actin and determine whether the PIPK-actin interaction is direct. Our results indicate that AtPIPK1 interacts directly with actin and that the binding is mediated through a predicted linker region in the lipid kinase. AtPIPK1 also recruits AtPI4Kbeta1 to the cytoskeleton. Recruitment of AtPI4Kbeta1 to F-actin was dependent on the C-terminal catalytic domain of phosphatidylinositol-4-phosphate 5-kinase but did not require the presence of the N-terminal 251 amino acids, which includes 7 putative membrane occupation and recognition nexus motifs. In vivo studies confirm the interaction of plant lipid kinases with the cytoskeleton and suggest a role for actin in targeting PIPKs to the membrane.

Lipids

We have recently shown that an inhibition of sterol synthesis by fenpropimorph leads to an accumulation of sterol precursors, hydroxypalmitic acid-containing glucosylceramides and detergent resistant membranes in the Golgi bodies instead of the plasma membrane, suggesting that the individual molecules or the microdomains were blocked in the Golgi. These results and others from several eukaryotic models link lipid metabolism with membrane morphodynamics that are involved in membrane trafficking. Focus has been expanded to other lipid families, and numerous evidences are given showing lipids and lipid-modifying enzymes as key regulators of membrane homeostasis which can strongly regulate membrane morphodynamics and therefore trafficking. Beside protein-based machineries, lipid-based machineries are also shown as crucial regulatory forces involved in protein transport and sorting.

A common lipid links Mfn-mediated mitochondrial fusion and SNARE-regulated exocytosis

Fusion of vesicles into target membranes during many types of regulated exocytosis requires both SNARE-complex proteins and fusogenic lipids, such as phosphatidic acid. Mitochondrial fusion is less well understood but distinct, as it is mediated instead by the protein Mitofusin (Mfn). Here, we identify an ancestral member of the phospholipase D (PLD) superfamily of lipid-modifying enzymes that is required for mitochondrial fusion. Mitochondrial PLD (MitoPLD) targets to the external face of mitochondria and promotes trans-mitochondrial membrane adherence in a Mfn-dependent manner by hydrolysing cardiolipin to generate phosphatidic acid. These findings reveal that although mitochondrial fusion and regulated exocytic fusion are mediated by distinct sets of protein machinery, the underlying processes are unexpectedly linked by the generation of a common fusogenic lipid. Moreover, our findings suggest a novel basis for the mitochondrial fragmentation observed during apoptosis.

The unique role of apolipoprotein A-I in HDL remodeling and metabolism

To rationalize the distinctive biological behavior of apolipoprotein (apo)A-I and apoA-II in light of differences in their respective structures, properties, and physico-chemical behavior. The distinctive metabolic behavior of apoA-I compared with that of apoA-II, which are revealed as differences in their interactions with the HDL receptor, scavenger receptor class B type I, can be understood in terms of their physico-chemical properties. Detergent and chaotropic perturbation of HDL unmasks properties that distinguish apoA-I from apoA-II and emulate the secondary effects of lecithin: cholesterol acyltransferase, cholesteryl ester transfer protein, and phospholipid transfer protein - the key protein factors in HDL remodeling, that is, formation of lipid-free apoA-I but not apoA-II and particle fusion. Thus, of the two major HDL apolipoproteins, apoA-I is the more plastic and labile and this difference gives apoA-I a unique physiological role that has been verified in mouse models of HDL metabolism. The compositions, structures, and properties of HDL particles are important determinants of the mechanisms by which these antiatherogenic lipoproteins are metabolized. Although the plasma lipid transfer proteins and lipid-modifying enzymes are important determinants of HDL processing, the distinctive structures and properties of apoA-I and apoA-II, the two major HDL proteins, determine in different ways the thermodynamic stability of HDL - the former through its greater plasticity and the latter by its higher lipophilicity. These distinctions have been revealed by physico-chemical studies of HDL stability in the context of numerous studies of enzyme and lipid transfer activities and of the interaction of HDL with its hepatic scavenger receptor.

Gene expression profiling of the nitrogen starvation stress response in the mycorrhizal ascomycete Tuber borchii

The focus of this work is on the nitrogen starvation stress responses operating in a plant symbiotic fungus. A cDNA array profiling analysis was conducted on N-limited mycelia of the mycorrhizal ascomycete Tuber borchii. Fifty-one unique transcripts, out of 2062 redundant arrayed cDNAs, were differentially expressed by at least 1.5-fold in response to N deprivation. Only two N assimilation components—a nitrate transporter and a high-affinity ammonium transporter—were found among differentially expressed genes. All the other N status responsive genes code for as yet unidentified hypothetical proteins or components not directly involved in N assimilation or metabolism, especially carbohydrate binding proteins and oligosaccharide as well as lipid modifying enzymes. A subset of cDNA array data were confirmed and extended by Northern blot analysis, which showed that most of the latter components respond not only to nitrogen, but also to carbon source depletion.

A Role for ADP-ribosylation Factor 6 in the Processing of G-protein-coupled Receptors

After agonist-induced internalization, the vasopressin V2 receptor (V2R) does not recycle to the plasma membrane. The ADP-ribosylation factor (ARF) proteins initiate vesicular intracellular traffic by promoting the recruitment of adaptor proteins; thus, we sought to determine whether ARF6 could promote V2R recycling. Neither the agonist-induced internalization nor the recycling of the V2R was regulated by ARF6, but a constitutively active mutant of ARF6 reduced cell-surface V2Rs 10-fold in the absence of agonist treatment. Visualization of the ARF6 mutant-expressing cells revealed a vacuolar-staining pattern of the V2R instead of the normal plasma membrane expression. Analysis of V2R maturation revealed that reduced cell-surface expression was due to the diminished ability of the newly synthesized receptor to migrate from the endoplasmic reticulum to the Golgi network. The same mechanism affected processing of the V1aR and acetylcholine M2 receptors. Therefore, ARF6 controls the exit of the V2 and other receptors from the endoplasmic reticulum in addition to its established role in the trafficking of plasma-membrane-derived vesicles.

Temperature-sensitive paralytic mutants: insights into the synaptic vesicle cycle

Forward genetic screens have identified numerous proteins with critical roles in neurotransmission. One particularly fruitful screening target in Drosophila has been TS (temperature-sensitive) paralytic mutants, which have revealed proteins acutely required in neuronal signalling. In the present paper, we review recent insights and current questions from one recently cloned TS paralytic mutant, rbo (rolling blackout). The rbo mutant identifies a putative integral lipase of the pre-synaptic plasma membrane that is required for the SV (synaptic vesicle) cycle. Identification of this mutant adds to a growing body of evidence that lipid-modifying enzymes locally control specialized lipid microenvironments and lipid signalling pathways with key functions regulating neurotransmission strength. The RBO protein is absolutely required for phospholipase C signalling in phototransduction. We posit that RBO might be required to regulate the availability of fusogenic lipids such as phosphatidylinositol 4,5-bisphosphate and diacylglycerol that may directly modify membrane properties and/or activate lipid-binding fusogenic proteins mediating SV exocytosis.

Lipids, Lipases, and Lipid-Modifying Enzymes in Plant Disease Resistance

Lipids and lipid metabolites influence pathogenesis and resistance mechanisms associated with plant-microbe interactions. Some microorganisms sense their presence on a host by perceiving plant surface waxes, whereas others produce toxins that target plant lipid metabolism. In contrast, plants have evolved to recognize microbial lipopolysaccharides (LPSs), sphingolipids, and lipid-binding proteins as elicitors of defense response. Recent studies have demonstrated that the plasma membrane provides a surface on which some plant resistance (R) proteins perceive pathogen-derived effectors and thus confer race-specific resistance. Plant cell membranes also serve as reservoirs from which biologically active lipids and precursors of oxidized lipids are released. Some of these oxylipins, for example jasmonic acid (JA), are important signal molecules in plant defense. Arabidopsis thaliana is an excellent model plant to elucidate the biosynthesis and metabolism of lipids and lipid metabolites, and the characterization of signaling mechanisms involved in the modulation of plant defense responses by phytolipids. This review focuses on recent studies that highlight the involvement of lipids and lipid metabolites, and enzymes involved in lipid metabolism and modification in plant disease resistance.

Arf6: a new player in FcγRIIIA lymphocyte-mediated cytotoxicity

AbstractThe activation of phosphoinositide metabolism represents a critical step in the signaling pathways leading to the activation of cytolytic machinery, but its regulation is partially understood. We report here that the stimulation of the low-affinity receptor for immunoglobulin G (IgG) (FcγRIIIA, CD16) on primary human natural killer (NK) cells induces a phosphatidylinositol 3-kinase (PI3K)–dependent activation of the small G protein Arf6. We first demonstrate a functional role for Arf6-dependent signals in the activation of the antibody-dependent cellular cytotoxicity (ADCC) attributable to the control of secretion of lytic granule content. We also show that Arf6 couples CD16 to the lipid-modifying enzymes phosphatidylinositol4phosphate 5-kinase type I alpha (PI5KIα) and phospholipase D (PLD) that are involved in the control of granule secretion; Arf6, but not Rho family small G proteins RhoA and Rac1, is required for receptor-induced PI5KIα membrane targeting as well as for PI5KIα and PLD activation. Our findings suggest that Arf6 plays a crucial role in the generation of a phosphatidylinositol4,5-bisphosphate (PIP2) plasma membrane pool required for cytolytic granule-mediated target cell killing.

The Small G-protein Arf6GTP Recruits the AP-2 Adaptor Complex to Membranes

The small GTP-binding protein ADP-ribosylation factor 6 (Arf6) is involved in plasma membrane/endosomes trafficking. However, precisely how the activation of Arf6 regulates vesicular transport is still unclear. Here, we show that, in vitro, recombinant Arf6GTP recruits purified clathrin-adaptor complex AP-2 (but not AP-1) onto phospholipid liposomes in the absence of phosphoinositides. We also show that phosphoinositides and Arf6 tightly cooperate to translocate AP-2 to the membrane. In vivo, Arf6GTP (but not Arf6GDP) was found associated to AP-2. The expression of the GTP-locked mutant of Arf6 leads to the plasma membrane redistribution of AP-2 in Arf6GTP-enriched areas. Finally, we demonstrated that the expression of the GTP-locked mutant of Arf6 inhibits transferrin receptor internalization without affecting its recycling. Altogether, our results demonstrated that Arf6GTP interacts specifically with AP-2 and promotes its membrane recruitment. These findings strongly suggest that Arf6 plays a major role in clathrin-mediated endocytosis by directly controlling the assembly of the AP-2/clathrin coat.

Lipid regulation of the synaptic vesicle cycle

Membrane vesicle cycling is orchestrated through the combined actions of proteins and lipids. At neuronal synapses, this orchestration must meet the stringent demands of speed, fidelity and sustainability of the synaptic vesicle cycle that mediates neurotransmission. Historically, the lion's share of the attention has been focused on the proteins that are involved in this cycle; but, in recent years, it has become clear that the previously unheralded plasma membrane and vesicle lipids are also key regulators of this cycle. This article reviews recent insights into the roles of lipid-modifying enzymes and lipids in the acute modulation of neurotransmission.

Dynamics of GBF1, a Brefeldin A-Sensitive Arf1 Exchange Factor at the Golgi

Trafficking through the Golgi apparatus requires members of the Arf family of GTPases, whose activation is regulated by guanine nucleotide exchange factors (GEFs). Once activated, Arf-GTP recruits effectors such as coat complexes and lipid-modifying enzymes to specific membrane sites, creating a domain competent for cargo concentration and transport. GBF1 is a peripherally associated Arf GEF involved in both endoplasmic reticulum-Golgi and intra-Golgi transport. The mechanism of GBF1 binding to membranes is unknown. As a first step to understanding the mechanism of membrane association, we constructed a yellow fluorescent protein-tagged version of GBF1 and performed fluorescence recovery after photobleaching analysis to determine its residence time on Golgi membranes. We find that GBF1 molecules are not stably associated with the Golgi but rather cycle rapidly on and off membranes. The drug brefeldin A (BFA), an uncompetitive inhibitor of the exchange reaction that binds to an Arf-GDP-Arf GEF complex, stabilizes GBF1 on Golgi membranes. Using an in vivo assay to monitor Arf1-GTP levels, we show that GBF1 exchange activity on Arf1 is inhibited by BFA in mammalian cells. These results suggest that an Arf1-GBF1-BFA complex is formed and has a longer residence time on Golgi membranes than GBF1 or Arf1 alone.

Phospholipase A2 (PLA2) Enzymes in Membrane Trafficking: Mediators of Membrane Shape and Function

Since the mid-1990s, there have been tremendous advances in our understanding of the roles that lipid-modifying enzymes play in various intracellular membrane trafficking events. Phospholipases represent the largest group of lipid-modifying enzymes and accordingly display a wide range of functions. The largest class of phospholipases are the phospholipase A(2) (PLA2) enzymes, and these have been most extensively studied for their roles in the generation lipid signaling molecules, e.g. arachidonic acid. In recent years, however, cytoplasmic PLA2 enzymes have also become increasingly associated with various intracellular trafficking events, such as the formation of membrane tubules from the Golgi complex and endosomes, and membrane fusion events in the secretory and endocytic pathways. Moreover, the ability of cytoplasmic PLA2 enzymes to directly affect the structure and function of membranes by altering membrane curvature suggests novel functional roles for these enzymes. This review will focus on the role of cytoplasmic PLA2 enzymes in intracellular membrane trafficking and the mechanisms by which they influence membrane structure and function.

Separation of phospholipids in microfluidic chip device: application to high-throughput screening assays for lipid-modifying enzymes

Phospholipid molecules such as ceramide and phosphoinositides play crucial roles in signal transduction pathways. Lipid-modifying enzymes including sphingomyelinase and phosphoinositide kinases regulate the generation and degradation of these lipid-signaling molecules and are important therapeutic targets in drug discovery. We now report a sensitive and convenient method to separate these lipids using microfluidic chip-based technology. The method takes advantage of the high-separation power of the microchips that separate lipids based on micellar electrokinetic capillary chromatography (MEKC) and the high sensitivity of fluorescence detection. We further exploited the method to develop a homogenous assay to monitor activities of lipid-modifying enzymes. The assay format consists of two steps: an on-plate enzymatic reaction using fluorescently labeled substrates followed by an on-chip MEKC separation of the reaction products from the substrates. The utility of the assay format for high-throughput screening (HTS) is demonstrated using phospholipase A 2 on the Caliper 250 HTS system: throughput of 80 min per 384-well plate can be achieved with unattended running time of 5.4 h. This enabling technology for assaying lipid-modifying enzymes is ideal for HTS because it avoids the use of radioactive substrates and complicated separation/washing steps and detects both substrate and product simultaneously.

Molecular analysis of a bifunctional fatty acid conjugase/desaturase from tung. Implications for the evolution of plant fatty acid diversity.

The seed oil derived from the tung (Aleurites fordii Hemsl.) tree contains approximately 80% alpha-eleostearic acid (18:3delta(9cis,11trans,13trans)), an unusual conjugated fatty acid that imparts industrially important drying qualities to tung oil. Here, we describe the cloning and functional analysis of two closely related Delta(12) oleate desaturase-like enzymes that constitute consecutive steps in the biosynthetic pathway of eleostearic acid. Polymerase chain reaction screening of a tung seed cDNA library using degenerate oligonucleotide primers resulted in identification of two desaturases, FAD2 and FADX, that shared 73% amino acid identity. Both enzymes were localized to the endoplasmic reticulum of tobacco (Nicotiana tabacum cv Bright-Yellow 2) cells, and reverse transcriptase-polymerase chain reaction revealed that FADX was expressed exclusively within developing tung seeds. Expression of the cDNAs encoding these enzymes in yeast (Saccharomyces cerevisiae) revealed that FAD2 converted oleic acid (18:1delta(9cis)) into linoleic acid (18:2delta(9cis,12cis)) and that FADX converted linoleic acid into alpha-eleostearic acid. Additional characterization revealed that FADX exhibited remarkable enzymatic plasticity, capable of generating a variety of alternative conjugated and delta(12)-desaturated fatty acid products in yeast cells cultured in the presence of exogenously supplied fatty acid substrates. Unlike other desaturases reported to date, the double bond introduced by FADX during fatty acid desaturation was in the trans, rather than cis, configuration. Phylogenetic analysis revealed that tung FADX is grouped with delta(12) fatty acid desaturases and hydroxylases rather than conjugases, which is consistent with its desaturase activity. Comparison of FADX and other lipid-modifying enzymes (desaturase, hydroxylase, epoxygenase, acetylenase, and conjugase) revealed several amino acid positions near the active site that may be important determinants of enzymatic activity.

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses.

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.

Trans-Golgi network sorting.

The trans-Golgi network (TGN) is a major secretory pathway sorting station that directs newly synthesized proteins to different subcellular destinations. The TGN also receives extracellular materials and recycled molecules from endocytic compartments. In this review, we summarize recent progress on understanding TGN structure and the dynamics of trafficking to and from this compartment. Protein sorting into different transport vesicles requires specific interactions between sorting motifs on the cargo molecules and vesicle coat components that recognize these motifs. Current understanding of the various targeting signals and vesicle coat components that are involved in TGN sorting are discussed, as well as the molecules that participate in retrieval to this compartment in both yeast and mammalian cells. Besides proteins, lipids and lipid-modifying enzymes also participate actively in the formation of secretory vesicles. The possible mechanisms of action of these lipid hydrolases and lipid kinases are discussed. Finally, we summarize the fundamentally different apical and basolateral cell surface delivery mechanisms and the current facts and hypotheses on protein sorting from the TGN into the regulated secretory pathway in neuroendocrine cells.

The EH and SH3 domain Ese proteins regulate endocytosis by linking to dynamin and Eps15.

Clathrin-mediated endocytosis is a multistep process which requires interaction between a number of conserved proteins. We have cloned two mammalian genes which code for a number of endocytic adaptor proteins. Two of these proteins, termed Ese1 and Ese2, contain two N-terminal EH domains, a central coiled-coil domain and five C-terminal SH3 domains. Ese1 is constitutively associated with Eps15 proteins to form a complex with at least 14 protein-protein interaction surfaces. Yeast two-hybrid assays have revealed that Ese1 EH and SH3 domains bind epsin family proteins and dynamin, respectively. Overexpression of Ese1 is sufficient to block clathrin-mediated endocytosis in cultured cells, presumably through disruption of higher order protein complexes, which are assembled on the endogenous Ese1-Eps15 scaffold. The Ese1-Eps15 scaffold therefore links dynamin, epsin and other endocytic pathway components.

Apolipoprotein A-I deficiency. Biochemical and metabolic characteristics.

Familial HDL deficiencies are associated with variable susceptibility to premature coronary heart disease, but the mechanism underlying this association remains poorly understood. Three homozygotes with isolated complete apo A-I deficiency caused by an autosomal codominant apo A-I Q[-2]X mutation and one heterozygote developed coronary heart disease before age 40 years. We characterized the effects of this mutation on lipoprotein metabolism. LDL FC, phospholipid, and apo B were all significantly higher in homozygotes than in heterozygotes. The HDLs of the heterozygotes were apo A-I poor relative to apo A-II. Lecithin-cholesterol acyltransferase activity was 59% lower in homozygotes than in normal subjects or heterozygotes. Cholesteryl ester transfer activity was increased in a homozygote compared with a normolipidemic control subject. Postprandial lipid metabolism was studied in one homozygote and one heterozygote. Post-prandial TG response in the homozygote was significantly exaggerated, while residual plasma HDL level remained unaffected. The homozygote also had delayed clearance of retinyl ester, a marker of chylomicron remnant metabolism. Thus, homozygosity and heterozygosity for apo A-I Q[-2]X are associated with qualitative, as well as quantitative, disturbances in plasma HDLs, LDLs, lipid-modifying enzyme activities, and postprandial retinyl ester metabolism. The observed elevation of atherogenic lipoproteins and reduction in antiatherogenic lipoproteins in the affected members of the apo A-I Q[-2]X kindred are consistent with the primary deficiency in apo A-I having pleiotropic effects that markedly enhance susceptibility for coronary heart disease.