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1.  Phosphatidic acid modulation of Kv channel voltage sensor function 
eLife  2014;3:e04366.
Membrane phospholipids can function as potent regulators of ion channel function. This study uncovers and investigates the effect of phosphatidic acid on Kv channel gating. Using the method of reconstitution into planar lipid bilayers, in which protein and lipid components are defined and controlled, we characterize two effects of phosphatidic acid. The first is a non-specific electrostatic influence on activation mediated by electric charge density on the extracellular and intracellular membrane surfaces. The second is specific to the presence of a primary phosphate group, acts only through the intracellular membrane leaflet and depends on the presence of a particular arginine residue in the voltage sensor. Intracellular phosphatidic acid accounts for a nearly 50 mV shift in the midpoint of the activation curve in a direction consistent with stabilization of the voltage sensor's closed conformation. These findings support a novel mechanism of voltage sensor regulation by the signaling lipid phosphatidic acid.
eLife digest
The electrical signals that carry information through the nervous system rely on positively charged potassium ions moving in and out of neurons. These ions move through proteins called voltage-gated potassium channels that are embedded in the plasma membrane that surrounds the neurons. The potassium channels contain pores that can be opened and closed to control the movement of the potassium ions.
The main factor that controls the opening and closing of these channels—a process known as ‘gating’—is the voltage across the membrane. However, the channels can also be controlled by proteins, or by other molecules.
The plasma membrane is made of several different types of molecules called phospholipids. Some of these phospholipids are known to be involved in gating potassium channels, but the roles of other phospholipids remain unclear.
To investigate the role of a phospholipid called phosphatidic acid, Hite et al. placed potassium ion channels in artificial plasma membranes. These experiments revealed that phosphatidic acid alters the gating of potassium ion channels in two ways. The first way is generic: the negative charge in phosphatidic acid shifts the membrane voltage. The second way is specific to phosphatidic acid: the end of the molecule with the negative charge interacts with the part of the potassium channel that senses changes in voltage to keep the pore closed. The next challenge is to understand how neurons shift their phosphatidic acid levels to regulate their electrical activity.
PMCID: PMC4212207  PMID: 25285449
ion channels; electrophysiology; voltage-gated potassium channels; rat
2.  Inactivation of the Host Lipin Gene Accelerates RNA Virus Replication through Viral Exploitation of the Expanded Endoplasmic Reticulum Membrane 
PLoS Pathogens  2014;10(2):e1003944.
RNA viruses take advantage of cellular resources, such as membranes and lipids, to assemble viral replicase complexes (VRCs) that drive viral replication. The host lipins (phosphatidate phosphatases) are particularly interesting because these proteins play key roles in cellular decisions about membrane biogenesis versus lipid storage. Therefore, we examined the relationship between host lipins and tombusviruses, based on yeast model host. We show that deletion of PAH1 (phosphatidic acid phosphohydrolase), which is the single yeast homolog of the lipin gene family of phosphatidate phosphatases, whose inactivation is responsible for proliferation and expansion of the endoplasmic reticulum (ER) membrane, facilitates robust RNA virus replication in yeast. We document increased tombusvirus replicase activity in pah1Δ yeast due to the efficient assembly of VRCs. We show that the ER membranes generated in pah1Δ yeast is efficiently subverted by this RNA virus, thus emphasizing the connection between host lipins and RNA viruses. Thus, instead of utilizing the peroxisomal membranes as observed in wt yeast and plants, TBSV readily switches to the vastly expanded ER membranes in lipin-deficient cells to build VRCs and support increased level of viral replication. Over-expression of the Arabidopsis Pah2p in Nicotiana benthamiana decreased tombusvirus accumulation, validating that our findings are also relevant in a plant host. Over-expression of AtPah2p also inhibited the ER-based replication of another plant RNA virus, suggesting that the role of lipins in RNA virus replication might include several more eukaryotic viruses.
Author Summary
Genetic diseases alter cellular pathways and they likely influence pathogen-host interactions as well. To test the relationship between a key cellular gene, whose mutation causes genetic diseases, and a pathogen, the authors have chosen the cellular lipins. Lipins are involved in a key cellular decision on using lipids for membrane biogenesis or for storage. Spontaneous mutations in the LIPIN1 gene in mammals, which cause impaired lipin-1 function, contribute to common metabolic dysregulation and several major diseases, such as obesity, hyperinsulinemia, type 2 diabetes, fatty liver distrophy and hypertension. In this work, the authors tested if tomato bushy stunt virus (TBSV), which, similar to many (+)RNA viruses, depends on host membrane biogenesis, is affected by deletion of the single lipin gene (PAH1) in yeast model host. They show that pah1Δ yeast supports increased replication of TBSV. They demonstrate that TBSV takes advantage of the expanded ER membranes in lipin-deficient yeast to efficiently assemble viral replicase complexes. Their findings suggest possible positive effect of a genetic disease caused by mutation on the replication of an infectious agent.
PMCID: PMC3930575  PMID: 24586157
3.  Metabolism and Regulation of Glycerolipids in the Yeast Saccharomyces cerevisiae 
Genetics  2012;190(2):317-349.
Due to its genetic tractability and increasing wealth of accessible data, the yeast Saccharomyces cerevisiae is a model system of choice for the study of the genetics, biochemistry, and cell biology of eukaryotic lipid metabolism. Glycerolipids (e.g., phospholipids and triacylglycerol) and their precursors are synthesized and metabolized by enzymes associated with the cytosol and membranous organelles, including endoplasmic reticulum, mitochondria, and lipid droplets. Genetic and biochemical analyses have revealed that glycerolipids play important roles in cell signaling, membrane trafficking, and anchoring of membrane proteins in addition to membrane structure. The expression of glycerolipid enzymes is controlled by a variety of conditions including growth stage and nutrient availability. Much of this regulation occurs at the transcriptional level and involves the Ino2–Ino4 activation complex and the Opi1 repressor, which interacts with Ino2 to attenuate transcriptional activation of UASINO-containing glycerolipid biosynthetic genes. Cellular levels of phosphatidic acid, precursor to all membrane phospholipids and the storage lipid triacylglycerol, regulates transcription of UASINO-containing genes by tethering Opi1 to the nuclear/endoplasmic reticulum membrane and controlling its translocation into the nucleus, a mechanism largely controlled by inositol availability. The transcriptional activator Zap1 controls the expression of some phospholipid synthesis genes in response to zinc availability. Regulatory mechanisms also include control of catalytic activity of glycerolipid enzymes by water-soluble precursors, products and lipids, and covalent modification of phosphorylation, while in vivo function of some enzymes is governed by their subcellular location. Genome-wide genetic analysis indicates coordinate regulation between glycerolipid metabolism and a broad spectrum of metabolic pathways.
PMCID: PMC3276621  PMID: 22345606
4.  Biosynthesis of phosphatidic acid in lipid particles and endoplasmic reticulum of Saccharomyces cerevisiae. 
Journal of Bacteriology  1997;179(24):7611-7616.
Lipid particles of the yeast Saccharomyces cerevisiae harbor two enzymes that stepwise acylate glycerol-3-phosphate to phosphatidic acid, a key intermediate in lipid biosynthesis. In lipid particles of the s1c1 disruptant YMN5 (M. M. Nagiec et al., J. Biol. Chem. 268:22156-22163, 1993) acylation stops after the first step, resulting in the accumulation of lysophosphatidic acid. Two-dimensional gel electrophoresis confirmed that S1c1p is a component of lipid particles. Lipid particles of a second mutant strain, TTA1 (T. S. Tillman and R. M. Bell, J. Biol. Chem. 261:9144-9149, 1986), which harbors a point mutation in the GAT gene, are essentially devoid of glycerol-3-phosphate acyltransferase activity in vitro. Synthesis of phosphatidic acid is reconstituted by combining lipid particles from YMN5 and TTA1. These results indicate that two distinct enzymes are necessary for phosphatidic acid synthesis in lipid particles: the first step, acylation of glycerol-3-phosphate, is catalyzed by a putative Gat1p; the second step, acylation of lysophosphatidic acid, requires S1c1p. Surprisingly, YMN5 and TTA1 mutants grow like the corresponding wild types because the endoplasmic reticulum of both mutants has the capacity to form a reduced but significant amount of phosphatidic acid. As a consequence, an s1c1 gat1 double mutant is also viable. Lipid particles from this double mutant fail completely to acylate glycerol-3-phosphate, whereas endoplasmic reticulum membranes harbor residual enzyme activities to synthesize phosphatidic acid. Thus, yeast contains at least two independent systems of phosphatidic acid biosynthesis.
PMCID: PMC179720  PMID: 9401016
5.  Lipidome and proteome of lipid droplets from the methylotrophic yeast Pichia pastoris 
Biochimica et Biophysica Acta  2013;1831(2):282-290.
Lipid droplets (LD) are the main depot of non-polar lipids in all eukaryotic cells. In the present study we describe isolation and characterization of LD from the industrial yeast Pichia pastoris. We designed and adapted an isolation procedure which allowed us to obtain this subcellular fraction at high purity as judged by quality control using appropriate marker proteins. Components of P. pastoris LD were characterized by conventional biochemical methods of lipid and protein analysis, but also by a lipidome and proteome approach. Our results show several distinct features of LD from P. pastoris especially in comparison to Saccharomyces cerevisiae. P. pastoris LD are characterized by their high preponderance of triacylglycerols over steryl esters in the core of the organelle, the high degree of fatty acid (poly)unsaturation and the high amount of ergosterol precursors. The high phosphatidylinositol to phosphatidylserine of ~ 7.5 ratio on the surface membrane of LD is noteworthy. Proteome analysis revealed equipment of the organelle with a small but typical set of proteins which includes enzymes of sterol biosynthesis, fatty acid activation, phosphatidic acid synthesis and non-polar lipid hydrolysis. These results are the basis for a better understanding of P. pastoris lipid metabolism and lipid storage and may be helpful for manipulating cell biological and/or biotechnological processes in this yeast.
► We isolated and characterized lipid droplets from P. pastoris for the first time. ► Lipidome and proteome analysis of P. pastoris lipid droplets were performed. ► Lipid droplets from P. pastoris are different from S. cerevisiae lipid droplets. ► P. pastoris lipid droplets contain much triacylglycerols but little steryl esters. ► A large number of P. pastoris lipid droplet proteins are involved in lipid metabolism.
PMCID: PMC3787741  PMID: 23041514
TG, triacylglycerols; SE, steryl esters; LD, lipid droplets; PA, phosphatidic acid; LP, lysophospholipids; PI, phosphatidylinositol; PS, phosphatidylserine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; DMPE, dimethyl-PE; CL, cardiolipin; DMCD, 4,14-dimethyl-cholesta-8,24-dienol; MS, mass spectrometry; GFP, green fluorescent protein; WT, wild type; Lipid droplet; Triacylglycerol; Steryl ester; Lipidome; Proteome; Pichia pastoris
6.  Identification and physiological characterization of phosphatidic acid phosphatase enzymes involved in triacylglycerol biosynthesis in Streptomyces coelicolor 
Phosphatidic acid phosphatase (PAP, EC catalyzes the dephosphorylation of phosphatidate yielding diacylglycerol (DAG), the lipid precursor for triacylglycerol (TAG) biosynthesis. Despite the importance of PAP activity in TAG producing bacteria, studies to establish its role in lipid metabolism have been so far restricted only to eukaryotes. Considering the increasing interest of bacterial TAG as a potential source of raw material for biofuel production, we have focused our studies on the identification and physiological characterization of the putative PAP present in the TAG producing bacterium Streptomyces coelicolor.
We have identified two S. coelicolor genes, named lppα (SCO1102) and lppβ (SCO1753), encoding for functional PAP proteins. Both enzymes mediate, at least in part, the formation of DAG for neutral lipid biosynthesis. Heterologous expression of lppα and lppβ genes in E. coli resulted in enhanced PAP activity in the membrane fractions of the recombinant strains and concomitantly in higher levels of DAG. In addition, the expression of these genes in yeast complemented the temperature-sensitive growth phenotype of the PAP deficient strain GHY58 (dpp1lpp1pah1). In S. coelicolor, disruption of either lppα or lppβ had no effect on TAG accumulation; however, the simultaneous mutation of both genes provoked a drastic reduction in de novo TAG biosynthesis as well as in total TAG content. Consistently, overexpression of Lppα and Lppβ in the wild type strain of S. coelicolor led to a significant increase in TAG production.
The present study describes the identification of PAP enzymes in bacteria and provides further insights on the genetic basis for prokaryotic oiliness. Furthermore, this finding completes the whole set of enzymes required for de novo TAG biosynthesis pathway in S. coelicolor. Remarkably, the overexpression of these PAPs in Streptomyces bacteria contributes to a higher productivity of this single cell oil. Altogether, these results provide new elements and tools for future cell engineering for next-generation biofuels production.
PMCID: PMC3599759  PMID: 23356794
PAP; Triacylglycerol; Oleaginous bacteria; Lipid metabolism
7.  Lipins, Lipinopathies, and the Modulation of Cellular Lipid Storage and Signaling 
Progress in lipid research  2013;52(3):10.1016/j.plipres.2013.04.001.
Members of the lipin protein family are phosphatidate phosphatase (PAP) enzymes, which catalyze the dephosphorylation of phosphatidic acid to diacylglycerol, the penultimate step in TAG synthesis. Lipins are unique among the glycerolipid biosynthetic enzymes in that they also promote fatty acid oxidation through their activity as co-regulators of gene expression by DNA-bound transcription factors. Lipin function has been evolutionarily conserved from a single ortholog in yeast to the mammalian family of three lipin proteins—lipin-1, lipin-2, and lipin-3. In mice and humans, the levels of lipin activity are a determinant of TAG storage in diverse cell types, and humans with deficiency in lipin-1 or lipin-2 have severe metabolic diseases. Recent work has highlighted the complex physiological interactions between members of the lipin protein family, which exhibit both overlapping and unique functions in specific tissues. The analysis of “lipinopathies” in mouse models and in humans has revealed an important role for lipin activity in the regulation of lipid intermediates (phosphatidate and diacylglycerol), which influence fundamental cellular processes including adipocyte and nerve cell differentiation, adipocyte lipolysis, and hepatic insulin signaling. The elucidation of lipin molecular and physiological functions could lead to novel approaches to modulate cellular lipid storage and metabolic disease.
PMCID: PMC3830937  PMID: 23603613
8.  Transcription Factor Reb1p Regulates DGK1-encoded Diacylglycerol Kinase and Lipid Metabolism in Saccharomyces cerevisiae* 
The Journal of Biological Chemistry  2013;288(40):29124-29133.
Background: Diacylglycerol kinase produces phosphatidate, a major precursor for the synthesis of membrane phospholipids.
Results: The expression of diacylglycerol kinase is induced by the Reb1p transcription factor, and the resulting activity increase is essential for the enzyme function in phospholipid synthesis.
Conclusion: The Reb1p-mediated transcriptional activation regulates the expression of diacylglycerol kinase activity.
Significance: Diacylglycerol kinase is regulated at the level of transcription.
In the yeast Saccharomyces cerevisiae, the DGK1-encoded diacylglycerol kinase catalyzes the CTP-dependent phosphorylation of diacylglycerol to form phosphatidate. This enzyme, in conjunction with PAH1-encoded phosphatidate phosphatase, controls the levels of phosphatidate and diacylglycerol for phospholipid synthesis, membrane growth, and lipid droplet formation. In this work, we showed that a functional level of diacylglycerol kinase is regulated by the Reb1p transcription factor. In the electrophoretic mobility shift assay, purified recombinant Reb1p was shown to specifically bind its consensus recognition sequence (CGGGTAA, −166 to −160) in the DGK1 promoter. Analysis of cells expressing the PDGK1-lacZ reporter gene showed that mutations (GT→TG) in the Reb1p-binding sequence caused an 8.6-fold reduction in β-galactosidase activity. The expression of DGK1(reb1), a DGK1 allele containing the Reb1p-binding site mutation, was greatly lower than that of the wild type allele, as indicated by analyses of DGK1 mRNA, Dgk1p, and diacylglycerol kinase activity. In the presence of cerulenin, an inhibitor of de novo fatty acid synthesis, the dgk1Δ mutant expressing DGK1(reb1) exhibited a significant defect in growth as well as in the synthesis of phospholipids from triacylglycerol mobilization. Unlike DGK1, the DGK1(reb1) expressed in the dgk1Δ pah1Δ mutant did not result in the nuclear/endoplasmic reticulum membrane expansion, which occurs in cells lacking phosphatidate phosphatase activity. Taken together, these results indicate that the Reb1p-mediated regulation of diacylglycerol kinase plays a major role in its in vivo functions in lipid metabolism.
PMCID: PMC3790011  PMID: 23970552
Diacylglycerol; Glycerolipid; Phosphatidate; Phospholipid; Phospholipid Metabolism; Phospholipid Turnover; Diacylglycerol Kinase
9.  Evidence that biosynthesis of phosphatidylethanolamine, phosphatidylcholine, and triacylglycerol occurs on the cytoplasmic side of microsomal vesicles 
The Journal of Cell Biology  1978;76(1):245-253.
Experiments were performed to localize the hepatic microsomal enzymes of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol biosynthesis to the cytoplasmic or lumenal surface of microsomal vesicles. Greater than 90 percent of the activities of fatty acid-CoA ligase (EC, sn-glycerol 3-phosphate acyltransferase (EC, lysophosphatidic acid acyltransferase, diacylglycerol acyltransferase (EC, diacylglycerol cholinephosphotransferase (EC, and diacylglycerol ethanolaminephosphotransferase (EC was inactivated by proteolysis of intact microsomal vesicles. The phosphatidic acid phosphatase (EC was not inactivated by any of the protease tested. Under conditions employed, <5 percent of the luminal mannose-6-phosphatase (EC activity was lost. After microsomal integrity was disrupted with detergents, protease treatment resulted in a loss of >74 percent of the mannose-6-phosphatase activity. The latency of the mannose-6-phosphatase activity was not affected by protease treatment. Mannose-6-phosphatase latency was not decreased by the presence of the assay components of several of the lipid biosynthetic activities, indicating that those components did not disrupt the microsomal vesicles. None of the lipid biosynthetic activities appeared latent. The presence of a protease-sensitive component of these biosynthetic activities on the cytoplasmic surface of microsomal vesicles, and the absence of latency for any of these biosynthetic activities suggest that the biosynthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol occurs asymmetrically on the cytoplasmic surface of the endoplasmic reticulum. The location of biosynthetic activities within the transverse plane of the endoplasmic reticulum is of particular interest for enzymes whose products may be either secreted or retained within the cell. Phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol account for the vast majority of hepatic glycerolipid biosynthesis. The phospholipids are utilized for hepatic membrane biogenesis and for the formation of lipoproteins, and the triacylglycerols are incorporated into lipoproteins or accumulate within the hepatocyte in certain disease states (14). The enzymes responsible for the biosynthesis of these glycerolipids (Scheme I) from fatty acids and glycerol-3P have all been localized to the microsomal subcellular fraction (12, 16, 29, 30). Microsomes are derived from the endoplasmic reticulum and are sealed vesicles which maintain proper sidedness. (11, 22). The external surface of these vesicles corresponds to the cytoplasmic surface of the endoplasmic reticulum. Macromolecules destined for secretion must pass into the lumen of the endoplasmic reticulum (5, 23). Uncharged molecules of up to approximately 600 daltons are able to enter the lumen of rat liver microsomes, but macromolecules and charged molecules of low molecular weight do not cross the vesicle membrane (10, 11). Because proteases neither cross the microsomal membrane nor destroy the permeability barrier of the microsomal vesicles, only the enzymes and proteins located on the cytoplasmic surface of microsomal vesicles are susceptible to proteolysis unless membrane integrity is disrupted (10, 11). By use of this approach, several enzymes and proteins have been localized in the transverse plane of microsomal membranes (11). With the possible exception of cytochrome P 450, all of the enzymes and proteins investigated were localized asymmetrically by the proteolysis technique (11). By studies of this type, as well as by product localization, glucose-6-phosphate (EC has been localized to the luminal surface of microsomal vesicles (11) and of the endoplasmic reticulum (18, 19). All microsomal vesicles contain glucose-6-phosphatase (18, 19) which can effectively utilize mannose-6-P as a substrate, provided the permeability barrier of the vesicles has been disrupted to allow the substrate access to the active site located on the lumenal surface (4). An exact correspondence between mannose- 6-phosphate activity and membrane permeability to EDTA has been established (4). The latency of mannose-6-phosphatase activity provides a quantitative index of microsomal integrity (4.) Few of the microsomal enzymes in the synthesis of phosphatidylcholine, phosphatidylethanolamine, and triacylglycerol have been solubilized and/or purified, and little is known about the topography of these enzymes in the transverse or lateral planes of the endoplasmic reticulum. An asymmetric location of these biosynthetic enzymes on the cytoplasmic or lumenal surface of microsomal vesicles may provide a mechanism for regulation of the glycerolipids to be retained or secreted by the cell, and for the biogenesis of asymmetric phospholipid bilayers. In this paper, we report investigations on the localization of all seven microsomal enzymes (Scheme I) in the biosynthesis of triacylglycerol, phosphatidylcholine, and phosphatidylethanolamine, using the protease technique with mannose-6-phosphatase serving as luminal control activity. The latency of these lipid biosynthetic enzymes was also investigated, using the latency of mannose-6-phosphatase as an index of microsomal integrity.
PMCID: PMC2109952  PMID: 618895
10.  The discovery of the fat-regulating phosphatidic acid phosphatase gene 
Frontiers in biology  2011;6(3):172-176.
Phosphatidic acid phosphatase is a fat-regulating enzyme that plays a major role in controlling the balance of phosphatidic acid (substrate) and diacylglycerol (product), which are lipid precursors used for the synthesis of membrane phospholipids and triacylglycerol. Phosphatidic acid is also a signaling molecule that triggers phospholipid synthesis gene expression, membrane expansion, secretion, and endocytosis. While this important enzyme has been known for several decades, its gene was only identified recently from yeast. This discovery showed the importance of phosphatidic acid phosphatase in lipid metabolism in yeast as well as in higher eukaryotes including humans.
PMCID: PMC3139499  PMID: 21785579
phosphatidic acid phosphatase; yeast; lipin; phospholipid; triacylglycerol
11.  Enigmatic MYC Conducts an Unfolding Systems Biology Symphony 
Genes & cancer  2010;1(6):526-531.
The enigmatic MYC oncogene, which participates broadly in cancers, revealed itself recently as the maestro of an unfolding symphony of cell growth, proliferation, death, and metabolism. The study of MYC is arguably most challenging to its students but at the same time exhilarating when MYC reveals its deeply held secrets. It is the excitement of our richer understanding of MYC that is captured in each review of this special issue of Genes & Cancer. Collectively, our deeper understanding of MYC reveals that it is a symphony conductor, controlling a large orchestra of target genes. Although MYC controls many orchestra sections, which are necessary but not sufficient for Myc function, ribosome biogenesis stands out to reveal Myc’s primordial function particularly in fruit flies. Because ribosome biogenesis and the associated translational machinery are bioenergetically demanding, Myc’s other target genes involved in energy metabolism must be coupled with energy demand to ensure that cells can replicate their genome and produce daughter cells. Normal cells have feedback loops that diminish MYC expression when nutrients are scarce. On the other hand, when deregulated Myc transforms cells, their constitutive bioenergetic demand can trigger cell death when energy is unavailable. This special issue captures the unfolding symphony of MYC-mediated tumorigenesis through reviews that span from a timeline of MYC research, fundamental understanding of how the MYC gene itself is regulated, the study of Myc in model organisms, Myc function, and target genes to translational research in search of new therapeutic modalities for the treatment of cancer.
PMCID: PMC3017351  PMID: 21218193
oncogene; transcription; cancer; targeted therapy
12.  Enigmatic MYC Conducts an Unfolding Systems Biology Symphony 
Genes & Cancer  2010;1(6):526-531.
The enigmatic MYC oncogene, which participates broadly in cancers, revealed itself recently as the maestro of an unfolding symphony of cell growth, proliferation, death, and metabolism. The study of MYC is arguably most challenging to its students but at the same time exhilarating when MYC reveals its deeply held secrets. It is the excitement of our richer understanding of MYC that is captured in each review of this special issue of Genes & Cancer. Collectively, our deeper understanding of MYC reveals that it is a symphony conductor, controlling a large orchestra of target genes. Although MYC controls many orchestra sections, which are necessary but not sufficient for Myc function, ribosome biogenesis stands out to reveal Myc’s primordial function particularly in fruit flies. Because ribosome biogenesis and the associated translational machinery are bioenergetically demanding, Myc’s other target genes involved in energy metabolism must be coupled with energy demand to ensure that cells can replicate their genome and produce daughter cells. Normal cells have feedback loops that diminish MYC expression when nutrients are scarce. On the other hand, when deregulated Myc transforms cells, their constitutive bioenergetic demand can trigger cell death when energy is unavailable. This special issue captures the unfolding symphony of MYC-mediated tumorigenesis through reviews that span from a timeline of MYC research, fundamental understanding of how the MYC gene itself is regulated, the study of Myc in model organisms, Myc function, and target genes to translational research in search of new therapeutic modalities for the treatment of cancer.
PMCID: PMC3017351  PMID: 21218193
oncogene; transcription; cancer; targeted therapy
13.  Interaction of the Spo20 Membrane-Sensor Motif with Phosphatidic Acid and Other Anionic Lipids, and Influence of the Membrane Environment 
PLoS ONE  2014;9(11):e113484.
The yeast protein Spo20 contains a regulatory amphipathic motif that has been suggested to recognize phosphatidic acid, a lipid involved in signal transduction, lipid metabolism and membrane fusion. We have investigated the interaction of the Spo20 amphipathic motif with lipid membranes using a bioprobe strategy that consists in appending this motif to the end of a long coiled-coil, which can be coupled to a GFP reporter for visualization in cells. The resulting construct is amenable to in vitro and in vivo experiments and allows unbiased comparison between amphipathic helices of different chemistry. In vitro, the Spo20 bioprobe responded to small variations in the amount of phosphatidic acid. However, this response was not specific. The membrane binding of the probe depended on the presence of phosphatidylethanolamine and also integrated the contribution of other anionic lipids, including phosphatidylserine and phosphatidyl-inositol-(4,5)bisphosphate. Inverting the sequence of the Spo20 motif neither affected the ability of the probe to interact with anionic liposomes nor did it modify its cellular localization, making a stereo-specific mode of phosphatidic acid recognition unlikely. Nevertheless, the lipid binding properties and the cellular localization of the Spo20 alpha-helix differed markedly from that of another amphipathic motif, Amphipathic Lipid Packing Sensor (ALPS), suggesting that even in the absence of stereo specific interactions, amphipathic helices can act as subcellular membrane targeting determinants in a cellular context.
PMCID: PMC4245137  PMID: 25426975
14.  Involvement of phosphatidate phosphatase in the biosynthesis of triacylglycerols in Chlamydomonas reinhardtii * #  
Lipid biosynthesis is essential for eukaryotic cells, but the mechanisms of the process in microalgae remain poorly understood. Phosphatidic acid phosphohydrolase or 3-sn-phosphatidate phosphohydrolase (PAP) catalyzes the dephosphorylation of phosphatidic acid to form diacylglycerols and inorganic orthophosphates. This reaction is integral in the synthesis of triacylglycerols. In this study, the mRNA level of the PAP isoform CrPAP2 in a species of Chlamydomonas was found to increase in nitrogen-free conditions. Silencing of the CrPAP2 gene using RNA interference resulted in the decline of lipid content by 2.4%–17.4%. By contrast, over-expression of the CrPAP2 gene resulted in an increase in lipid content by 7.5%–21.8%. These observations indicate that regulation of the CrPAP2 gene can control the lipid content of the algal cells. In vitro CrPAP2 enzyme activity assay indicated that the cloned CrPAP2 gene exhibited biological activities.
PMCID: PMC3863370  PMID: 24302712
Phosphatidate phosphohydrolase 2; Triacylglycerol biosynthesis; RNAi; Chlamydomonas reinhardtii; Nitrogen deprivation; Over-expression
15.  Redundant Systems of Phosphatidic Acid Biosynthesis via Acylation of Glycerol-3-Phosphate or Dihydroxyacetone Phosphate in the Yeast Saccharomyces cerevisiae 
Journal of Bacteriology  1999;181(5):1458-1463.
In the yeast Saccharomyces cerevisiae lipid particles harbor two acyltransferases, Gat1p and Slc1p, which catalyze subsequent steps of acylation required for the formation of phosphatidic acid. Both enzymes are also components of the endoplasmic reticulum, but this compartment contains additional acyltransferase(s) involved in the biosynthesis of phosphatidic acid (K. Athenstaedt and G. Daum, J. Bacteriol. 179:7611–7616, 1997). Using the gat1 mutant strain TTA1, we show here that Gat1p present in both subcellular fractions accepts glycerol-3-phosphate and dihydroxyacetone phosphate as a substrate. Similarly, the additional acyltransferase(s) present in the endoplasmic reticulum can acylate both precursors. In contrast, yeast mitochondria harbor an enzyme(s) that significantly prefers dihydroxyacetone phosphate as a substrate for acylation, suggesting that at least one additional independent acyltransferase is present in this organelle. Surprisingly, enzymatic activity of 1-acyldihydroxyacetone phosphate reductase, which is required for the conversion of 1-acyldihydroxyacetone phosphate to 1-acylglycerol-3-phosphate (lysophosphatidic acid), is detectable only in lipid particles and the endoplasmic reticulum and not in mitochondria. In vivo labeling of wild-type cells with [2-3H, U-14C]glycerol revealed that both glycerol-3-phosphate and dihydroxyacetone phosphate can be incorporated as a backbone of glycerolipids. In the gat1 mutant and the 1-acylglycerol-3-phosphate acyltransferase slc1 mutant, the dihydroxyacetone phosphate pathway of phosphatidic acid biosynthesis is slightly preferred as compared to the wild type. Thus, mutations of the major acyltransferases Gat1p and Slc1p lead to an increased contribution of mitochondrial acyltransferase(s) to glycerolipid synthesis due to their substrate preference for dihydroxyacetone phosphate.
PMCID: PMC93534  PMID: 10049376
16.  Identification of Two Legionella pneumophila Effectors that Manipulate Host Phospholipids Biosynthesis 
PLoS Pathogens  2012;8(11):e1002988.
The intracellular pathogen Legionella pneumophila translocates a large number of effector proteins into host cells via the Icm/Dot type-IVB secretion system. Some of these effectors were shown to cause lethal effect on yeast growth. Here we characterized one such effector (LecE) and identified yeast suppressors that reduced its lethal effect. The LecE lethal effect was found to be suppressed by the over expression of the yeast protein Dgk1 a diacylglycerol (DAG) kinase enzyme and by a deletion of the gene encoding for Pah1 a phosphatidic acid (PA) phosphatase that counteracts the activity of Dgk1. Genetic analysis using yeast deletion mutants, strains expressing relevant yeast genes and point mutations constructed in the Dgk1 and Pah1 conserved domains indicated that LecE functions similarly to the Nem1-Spo7 phosphatase complex that activates Pah1 in yeast. In addition, by using relevant yeast genetic backgrounds we examined several L. pneumophila effectors expected to be involved in phospholipids biosynthesis and identified an effector (LpdA) that contains a phospholipase-D (PLD) domain which caused lethal effect only in a dgk1 deletion mutant of yeast. Additionally, LpdA was found to enhance the lethal effect of LecE in yeast cells, a phenomenon which was found to be dependent on its PLD activity. Furthermore, to determine whether LecE and LpdA affect the levels or distribution of DAG and PA in-vivo in mammalian cells, we utilized fluorescent DAG and PA biosensors and validated the notion that LecE and LpdA affect the in-vivo levels and distribution of DAG and PA, respectively. Finally, we examined the intracellular localization of both LecE and LpdA in human macrophages during L. pneumophila infection and found that both effectors are localized to the bacterial phagosome. Our results suggest that L. pneumophila utilize at least two effectors to manipulate important steps in phospholipids biosynthesis.
Author Summary
Legionella pneumophila is an intracellular pathogen that causes a severe pneumonia known as Legionnaires' disease. Following infection, the bacteria use a Type-IVB secretion system to translocate multiple effector proteins into macrophages and generate the Legionella-containing vacuole (LCV). The formation of the LCV involves the recruitment of specific bacterial effectors and host cell factors to the LCV as well as changes in its lipids composition. By screening L. pneumophila effectors for yeast growth inhibition, we have identified an effector, named LecE, that strongly inhibits yeast growth. By using yeast genetic tools, we found that LecE activates the yeast lipin homolog – Pah1, an enzyme that catalyzes the conversion of diacylglycerol to phosphatidic acid, these two molecules function as bioactive lipid signaling molecules in eukaryotic cells. In addition, by using yeast deletion mutants in genes relevant to lipids biosynthesis, we have identified another effector, named LpdA, which function as a phospholipase-D enzyme. Both effectors were found to be localized to the LCV during infection. Our results reveal a possible mechanism by which an intravacuolar pathogen might change the lipid composition of the vacuole in which it resides, a process that might lead to the recruitment of specific bacterial and host cell factors to the vacoule.
PMCID: PMC3486869  PMID: 23133385
17.  CDP-Diacylglycerol Synthetase Coordinates Cell Growth and Fat Storage through Phosphatidylinositol Metabolism and the Insulin Pathway 
PLoS Genetics  2014;10(3):e1004172.
During development, animals usually undergo a rapid growth phase followed by a homeostatic stage when growth has ceased. The increase in cell size and number during the growth phase requires a large amount of lipids; while in the static state, excess lipids are usually stored in adipose tissues in preparation for nutrient-limited conditions. How cells coordinate growth and fat storage is not fully understood. Through a genetic screen we identified Drosophila melanogaster CDP-diacylglycerol synthetase (CDS/CdsA), which diverts phosphatidic acid from triacylglycerol synthesis to phosphatidylinositol (PI) synthesis and coordinates cell growth and fat storage. Loss of CdsA function causes significant accumulation of neutral lipids in many tissues along with reduced cell/organ size. These phenotypes can be traced back to reduced PI levels and, subsequently, low insulin pathway activity. Overexpressing CdsA rescues the fat storage and cell growth phenotypes of insulin pathway mutants, suggesting that CdsA coordinates cell/tissue growth and lipid storage through the insulin pathway. We also revealed that a DAG-to-PE route mediated by the choline/ethanolamine phosphotransferase Bbc may contribute to the growth of fat cells in CdsA RNAi.
Author Summary
During development, animals undergo a rapid increase in cell size and number, which requires large amounts of lipids, in the form of phospholipids, for the expansion of cell membranes. Once the growth phase ends, excess lipids are usually stored as body fat, in the form of triacylglycerol (TAG), for use when nutrients are limited. How cells coordinate growth and fat storage is not fully understood. By screening for genes that affect lipid storage in the fruitfly Drosophila we discovered that the enzyme CDP-diacylglycerol synthetase (CdsA) coordinates cell growth and fat storage. Phospholipids and TAG have a common precursor, phosphatidic acid, which is diverted by CdsA from TAG synthesis to synthesis of the phospholipid phosphatidylinositol (PI). We also uncovered a link between CdsA and the insulin signaling pathway, which plays a major role in regulating cell and tissue growth. CdsA regulates the level of PI, which modulates insulin pathway activity; insulin pathway activity, in turn, influences the level of CdsA. The lipid metabolism pathways and the insulin signaling pathway are conserved in other animals including humans. Our findings may therefore provide further insights into clinically important imbalances in fat storage such as obesity.
PMCID: PMC3945474  PMID: 24603715
18.  A Role for Phosphatidic Acid in the Formation of “Supersized” Lipid Droplets 
PLoS Genetics  2011;7(7):e1002201.
Lipid droplets (LDs) are important cellular organelles that govern the storage and turnover of lipids. Little is known about how the size of LDs is controlled, although LDs of diverse sizes have been observed in different tissues and under different (patho)physiological conditions. Recent studies have indicated that the size of LDs may influence adipogenesis, the rate of lipolysis and the oxidation of fatty acids. Here, a genome-wide screen identifies ten yeast mutants producing “supersized” LDs that are up to 50 times the volume of those in wild-type cells. The mutated genes include: FLD1, which encodes a homologue of mammalian seipin; five genes (CDS1, INO2, INO4, CHO2, and OPI3) that are known to regulate phospholipid metabolism; two genes (CKB1 and CKB2) encoding subunits of the casein kinase 2; and two genes (MRPS35 and RTC2) of unknown function. Biochemical and genetic analyses reveal that a common feature of these mutants is an increase in the level of cellular phosphatidic acid (PA). Results from in vivo and in vitro analyses indicate that PA may facilitate the coalescence of contacting LDs, resulting in the formation of “supersized” LDs. In summary, our results provide important insights into how the size of LDs is determined and identify novel gene products that regulate phospholipid metabolism.
Author Summary
Lipid droplets (LD) are primary lipid storage structures that also function in membrane and lipid trafficking, protein turnover, and the reproduction of deadly viruses. Increased LD accumulation in liver, skeletal muscle, and adipose tissue is a hallmark of the metabolic syndrome. Enlarged LDs are often found in these tissues under disease conditions. However, little is known about how the size of LDs is controlled in eukaryotic cells. In this study, we use genetic and biochemical methods to identify important gene products that regulate the size of the LDs. Notably, a common feature among these mutants with “supersized” LDs is an increased level of phosphatidic acid (PA). We also show that a small amount of PA can increase the size of artificial LDs in vitro. Overall, our study identifies important lipids and proteins in determining LD size. These results provide valuable insights into how human cells/tissues handle abnormal influx of lipids in today's obesogenic environment.
PMCID: PMC3145623  PMID: 21829381
19.  Srf1 Is a Novel Regulator of Phospholipase D Activity and Is Essential to Buffer the Toxic Effects of C16:0 Platelet Activating Factor 
PLoS Genetics  2011;7(2):e1001299.
During Alzheimer's Disease, sustained exposure to amyloid-β42 oligomers perturbs metabolism of ether-linked glycerophospholipids defined by a saturated 16 carbon chain at the sn-1 position. The intraneuronal accumulation of 1-O-hexadecyl-2-acetyl-sn-glycerophosphocholine (C16:0 PAF), but not its immediate precursor 1-O-hexadecyl-sn-glycerophosphocholine (C16:0 lyso-PAF), participates in signaling tau hyperphosphorylation and compromises neuronal viability. As C16:0 PAF is a naturally occurring lipid involved in cellular signaling, it is likely that mechanisms exist to protect cells against its toxic effects. Here, we utilized a chemical genomic approach to identify key processes specific for regulating the sensitivity of Saccharomyces cerevisiae to alkyacylglycerophosphocholines elevated in Alzheimer's Disease. We identified ten deletion mutants that were hypersensitive to C16:0 PAF and five deletion mutants that were hypersensitive to C16:0 lyso-PAF. Deletion of YDL133w, a previously uncharacterized gene which we have renamed SRF1 (Spo14 Regulatory Factor 1), resulted in the greatest differential sensitivity to C16:0 PAF over C16:0 lyso-PAF. We demonstrate that Srf1 physically interacts with Spo14, yeast phospholipase D (PLD), and is essential for PLD catalytic activity in mitotic cells. Though C16:0 PAF treatment does not impact hydrolysis of phosphatidylcholine in yeast, C16:0 PAF does promote delocalization of GFP-Spo14 and phosphatidic acid from the cell periphery. Furthermore, we demonstrate that, similar to yeast cells, PLD activity is required to protect mammalian neural cells from C16:0 PAF. Together, these findings implicate PLD as a potential neuroprotective target capable of ameliorating disruptions in lipid metabolism in response to accumulating oligomeric amyloid-β42.
Author Summary
Accelerated cognitive decline in Alzheimer's patients is associated with accumulation of choline-containing lipids. One of these lipids, C16:0 platelet activating factor (PAF), is specifically elevated in brains of Alzheimer's patients. As elevated exposure to C16:0 PAF ultimately leads to neuronal death, it is crucial to identify underlying mechanisms that mitigate the toxic effects of this lipid. In this study we exploit the conserved biology between humans and baker's yeast to identify key genes that are essential to buffer the toxic effects of C16:0 PAF. We found that Srf1, or Spo14 Regulatory Factor 1, the previously uncharacterized protein Ydl133w, is essential for mitigating the toxic effects of C16:0 PAF in yeast. We determine that Srf1 interacts with yeast phospholipase D (PLD) Spo14 and is required for PLD activity in mitotic cells. Hence we discovered a novel regulator of PLD in yeast. Further, we extend our studies to higher eukaryotes demonstrating that PLD is required to buffer the neurotoxic effect of C16:0 PAF. Our study suggests that therapeutic strategies modulating PLD activity may be effective in ameliorating Alzheimer's Disease pathology associated with disruptions in lipid metabolism.
PMCID: PMC3037409  PMID: 21347278
20.  OpenCyto: An Open Source Infrastructure for Scalable, Robust, Reproducible, and Automated, End-to-End Flow Cytometry Data Analysis 
PLoS Computational Biology  2014;10(8):e1003806.
Flow cytometry is used increasingly in clinical research for cancer, immunology and vaccines. Technological advances in cytometry instrumentation are increasing the size and dimensionality of data sets, posing a challenge for traditional data management and analysis. Automated analysis methods, despite a general consensus of their importance to the future of the field, have been slow to gain widespread adoption. Here we present OpenCyto, a new BioConductor infrastructure and data analysis framework designed to lower the barrier of entry to automated flow data analysis algorithms by addressing key areas that we believe have held back wider adoption of automated approaches. OpenCyto supports end-to-end data analysis that is robust and reproducible while generating results that are easy to interpret. We have improved the existing, widely used core BioConductor flow cytometry infrastructure by allowing analysis to scale in a memory efficient manner to the large flow data sets that arise in clinical trials, and integrating domain-specific knowledge as part of the pipeline through the hierarchical relationships among cell populations. Pipelines are defined through a text-based csv file, limiting the need to write data-specific code, and are data agnostic to simplify repetitive analysis for core facilities. We demonstrate how to analyze two large cytometry data sets: an intracellular cytokine staining (ICS) data set from a published HIV vaccine trial focused on detecting rare, antigen-specific T-cell populations, where we identify a new subset of CD8 T-cells with a vaccine-regimen specific response that could not be identified through manual analysis, and a CyTOF T-cell phenotyping data set where a large staining panel and many cell populations are a challenge for traditional analysis. The substantial improvements to the core BioConductor flow cytometry packages give OpenCyto the potential for wide adoption. It can rapidly leverage new developments in computational cytometry and facilitate reproducible analysis in a unified environment.
PMCID: PMC4148203  PMID: 25167361
21.  Phosphatidic Acid Synthesis in Bacteria 
Biochimica et biophysica acta  2012;1831(3):495-502.
Membrane phospholipid synthesis is a vital facet of bacterial physiology. Although the spectrum of phospholipid headgroup structures produced by bacteria is large, the key precursor to all of these molecules is phosphatidic acid (PtdOH). Glycerol-3-phosphate derived from the glycolysis via glycerol-phosphate synthase is the universal source for the glycerol backbone of PtdOH. There are two distinct families of enzymes responsible for the acylation of the 1-position of glycerol-3-phosphate. The PlsB acyltransferase was discovered in Escherichia coli, and homologs are present in many eukaryotes. This protein family primarily uses acyl-acyl carrier protein (ACP) endproducts of fatty acid synthesis as acyl donors, but may also use acyl-CoA derived from exogenous fatty acids. The second protein family, PlsY, is more widely distributed in bacteria and utilizes the unique acyl donor, acyl-phosphate, which is produced from acyl-ACP by the enzyme PlsX. The acylation of the 2-position is carried out by members of the PlsC protein family. All PlsCs use acyl-ACP as the acyl donor, although the PlsCs of the γ-proteobacteria also may use acyl-CoA. Phospholipid headgroups are precursors in the biosynthesis of other membrane-associated molecules and the diacylglycerol product of these reactions is converted to PtdOH by one of two distinct families of lipid kinases. The central importance of the de novo and recycling pathways to PtdOH in cell physiology suggest these enzymes are suitable targets for the development of antibacterial therapeutics in Gram-positive pathogens. This article is part of a Special Issue entitled Phospholipids and Phospholipid Metabolism.
PMCID: PMC3548993  PMID: 22981714
bacteria; acyltransferase; phosphatidic acid; glycerol-phosphate; acyl carrier protein; coenzyme A; diacylglycerol
22.  Phosphatidic Acid and Lipid Sensing by mTOR 
Mammalian target of rapamycin (mTOR) has been implicated as a sensor of nutrient sufficiency for dividing cells and is activated by essential amino acids and glucose. However, cells also require lipids for membrane biosynthesis. A central metabolite in the synthesis of membrane phospholipids is phosphatidic acid (PA), which is required for the stability and activity of mTOR complexes. While PA is commonly generated by the phospholipase D-catalyzed hydrolysis of phosphatidylcholine, PA is also generated by diacylglycerol kinases and lysophosphatidic acid acyltransferases, which are at the center of phospholipid biosynthesis. It is proposed that the responsiveness of mTOR/TOR to PA evolved as a means for sensing lipid precursors for membrane biosynthesis prior to doubling the mass of a cell and dividing.
PMCID: PMC3669661  PMID: 23507202
mTOR; phosphatidic acid; DG kinase; LPAAT; phospholipase D
23.  An Unconventional Diacylglycerol Kinase That Regulates Phospholipid Synthesis and Nuclear Membrane Growth*♦ 
The Journal of Biological Chemistry  2008;283(29):20433-20442.
Changes in nuclear size and shape during the cell cycle or during development require coordinated nuclear membrane remodeling, but the underlying molecular events are largely unknown. We have shown previously that the activity of the conserved phosphatidate phosphatase Pah1p/Smp2p regulates nuclear structure in yeast by controlling phospholipid synthesis and membrane biogenesis at the nuclear envelope. Two screens for novel regulators of phosphatidate led to the identification of DGK1. We show that Dgk1p is a unique diacylglycerol kinase that uses CTP, instead of ATP, to generate phosphatidate. DGK1 counteracts the activity of PAH1 at the nuclear envelope by controlling phosphatidate levels. Overexpression of DGK1 causes the appearance of phosphatidate-enriched membranes around the nucleus and leads to its expansion, without proliferating the cortical endoplasmic reticulum membrane. Mutations that decrease phosphatidate levels decrease nuclear membrane growth in pah1Δ cells. We propose that phosphatidate metabolism is a critical factor determining nuclear structure by regulating nuclear membrane biogenesis.
PMCID: PMC2459266  PMID: 18458075
24.  Interactions between the PDZ domains of Bazooka (Par-3) and phosphatidic acid: in vitro characterization and role in epithelial development 
Molecular Biology of the Cell  2012;23(18):3743-3753.
The polarity regulator Bazooka (Par-3) is shown to directly bind the signaling lipid phosphatidic acid via its PDZ domains in vitro. In vivo, the interaction appears to support Bazooka functions important for Drosophila embryonic epithelial structure. Thus Bazooka has a closer connection to membrane lipids than previously recognized.
Bazooka (Par-3) is a conserved polarity regulator that organizes molecular networks in a wide range of cell types. In epithelia, it functions as a plasma membrane landmark to organize the apical domain. Bazooka is a scaffold protein that interacts with proteins through its three PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains and other regions. In addition, Bazooka has been shown to interact with phosphoinositides. Here we show that the Bazooka PDZ domains interact with the negatively charged phospholipid phosphatidic acid immobilized on solid substrates or in liposomes. The interaction requires multiple PDZ domains, and conserved patches of positively charged amino acid residues appear to mediate the interaction. Increasing or decreasing levels of diacylglycerol kinase or phospholipase D—enzymes that produce phosphatidic acid—reveal a role for phosphatidic acid in Bazooka embryonic epithelial activity but not its localization. Mutating residues implicated in phosphatidic acid binding revealed a possible role in Bazooka localization and function. These data implicate a closer connection between Bazooka and membrane lipids than previously recognized. Bazooka polarity landmarks may be conglomerates of proteins and plasma membrane lipids that modify each other's activities for an integrated effect on cell polarity.
PMCID: PMC3442420  PMID: 22833561
25.  A systematic screen for protein–lipid interactions in Saccharomyces cerevisiae 
Lipids are important cellular metabolites, with a wide range of structural and functional diversity. Many operate as signaling molecules. Lipids though have rarely been studied in large-scale interaction screen; they are poorly represented in current biological networks.Here, we describe the use of miniaturized lipid–arrays for the large-scale study of protein–lipid interactions. In yeast, we show general feasibility with a systematic screen implying 172 proteins. We report 530 protein–lipid associations, the majority is novel and several were validated using other techniques.The screen uncovers numerous insights into lipid function in yeast and equivalent systems in humans. It revealed (i) previously undetected cryptic lipid-binding domains, (ii) series of new cellular targets for sphingolipids and (iii) new ligands for some PH domains that can cooperatively bind additional lipids and work as coincidence sensor to integrate both phosphatidylinositol phosphates and sphingolipid signaling pathways.The significant number of biological insights uncovered shows that even major classes of metabolites have been insufficiently studied. This illustrates the general relevance of such systematic screens and calls for further system-wide analyses.
Deciphering the molecular mechanisms behind cellular processes requires the systematic charting of the multitude of interactions between all cellular components. While protein–protein and protein–DNA networks have been the subject of many systematic surveys, other critically important cellular components, such as lipids, have to date rarely been studied in large-scale interaction screens. Growing numbers of lipids are known to operate as signaling molecules. The importance of protein–lipid interactions is evident from the variety of protein domains that have evolved to bind particular lipids (Lemmon, 2008 #392) and from the large list of disorders, such as cancer and bipolar disorder, arising from altered protein–lipid interactions. The current understanding of protein–lipid recognition comes from the study of a limited number of lipids, principally PtdInsPs (Zhu et al, 2001 #16), and lipid-binding domains (LBDs) in isolation (Dowler et al, 2000 #81; Yu and Lemmon, 2001 #396; Yu et al, 2004 #31). For other signaling lipids, such as sphingolipids, intracellular targets and molecular mechanisms are only partially understood (Hannun and Obeid, 2008 #397). The importance of lipids in biological processes and their under-representation in current biological networks suggest the need for systematic, unbiased biochemical screens.
To systematically study protein–lipid interactions, we developed miniaturized arrays that contained sets of 56 lipids covering the main lipid classes in yeast. We used the arrays to determine the binding profiles of 172 soluble proteins. The selection included proteins that contained one or several predicted LBD that were lipid regulated or enzymes involved in lipid metabolism (Figure 1). We obtained 530 protein–lipid interactions (accuracy and coverage: 61 and 60%, respectively). More than half were supported by additional experimental evidences obtained from a large validation effort using a variety of biochemical and cell biology approaches, and the integration of a data set of genetic interactions (Figure 1). As a substantial fraction (45%) of the analyzed proteins were conserved in humans, the protein–lipid data set will have functional implications for higher eukaryotes and thus for human biology.
Overall, 68% of all interactions were novel or unexpected from either protein sequences or known LBDs specificities. We discovered cryptic LBDs that were previously undetected in Ecm25 (a RhoGAP) and Ira2 (a RasGAP). We also identified a set of proteins that bound sphingolipids, a class of bioactive lipids that play important signaling functions in yeast and higher eukaryotes. The exact mode of action for these lipids remains elusive and the data set points to series of new cellular targets. We identified 63 proteins, involved in endocytosis, cell polarity and lipid metabolism that interacted with sphingoid long-chain bases (LCBs), ceramides or phosphorylated LCBs (Figure 5).
Despite the importance of sphingolipids in signaling processes, only a few domains, such as START or Saposins, have been reported to specifically bind these lipids in higher eukaryotes, and none of them have been found in yeast. Interestingly, almost 60% of proteins binding to phosphorylated LCBs in our assay also contained a pleckstrin homology (PH) domain and bound PtdInsPs (Figure 5). This suggests some PH domains might have unanticipated ligands and also have a function in sphingolipid recognition. We showed, using a variety of biochemical and cell-based assays, that the PH domain of Slm1, a component of the TORC2 signaling pathway (Fadri et al, 2005 #429), can bind PtdIns(4,5)P2 and sphingolipid cooperatively. The structure of Slm1-PH, which we solved by X-ray crystallography at 2 Å resolution, suggests the presence of two positively charged binding pockets for anionic lipids. These results indicate that the PH domain of Slm1 might work as a coincidence sensor to integrate both PtdInsP and sphingolipid signaling pathways. This reinforces the emerging notion that cooperative mechanisms have important functions in PH domains functioning (Maffucci and Falasca, 2001 #528). These mechanisms initially described between PtdInsPs and proteins can now be extended to new lipid classes, illustrating the benefit of unbiased and systematic analyses.
This work shows the feasibility and benefits of large-scale analyses combining biochemical arrays and live-cell imaging for charting protein–lipid interactions. Accurate representations of biological processes require systematic charting of the physical and functional links between all cellular components. There is a clear need to expand molecular interaction space from proteome- to metabolome-wide efforts and of systematic classifications of bioactive molecules based on their binding profiles. The data provided here represents an excellent resource to enhance the understanding of lipids function in eukaryotic systems.
Protein–metabolite networks are central to biological systems, but are incompletely understood. Here, we report a screen to catalog protein–lipid interactions in yeast. We used arrays of 56 metabolites to measure lipid-binding fingerprints of 172 proteins, including 91 with predicted lipid-binding domains. We identified 530 protein–lipid associations, the majority of which are novel. To show the data set's biological value, we studied further several novel interactions with sphingolipids, a class of conserved bioactive lipids with an elusive mode of action. Integration of live-cell imaging suggests new cellular targets for these molecules, including several with pleckstrin homology (PH) domains. Validated interactions with Slm1, a regulator of actin polarization, show that PH domains can have unexpected lipid-binding specificities and can act as coincidence sensors for both phosphatidylinositol phosphates and phosphorylated sphingolipids.
PMCID: PMC3010107  PMID: 21119626
interactome; lipid–array; network; pleckstrin homology domains; sphingolipids

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