A lipid transfer protein binds to a receptor involved in the control of plant defence responses.

Lipid transfer proteins (LTPs) and elicitins are both able to load and transfer lipidic molecules and share some structural and functional properties. While elicitins are known as elicitors of plant defence mechanisms, the biological function of LTP is still an enigma. We show that a wheat LTP1 binds with high affinity sites. Binding and in vivo competition experiments point out that these binding sites are common to LTP1 and elicitins and confirm that they are the biological receptors of elicitins. A mathematical analysis suggests that these receptors could be represented by an allosteric model corresponding to an oligomeric structure with four identical subunits.

Two novel plant cDNAs homologous to animal type-2 phosphatidate phosphatase are expressed in cowpea leaves and are differently regulated by water deficits.

Two cDNAs encoding putative phosphatidate phosphatases (PAPs) designated VuPAP-alpha and VuPAP-beta were cloned in cowpea (Vigna unguiculata L.) leaves. The predicted proteins have six membrane-spanning regions in common with animal type-2 PAPs. Unlike VuPAP-beta, VuPAP-alpha has an N-terminal transit peptide and is targeted in vitro to the chloroplasts. Gene expression of VuPAP-beta is stimulated by rapid air-desiccation of leaves and VuPAP-alpha transcripts increase during rehydration of plants exposed to drought-like conditions.

Comparison of lipid binding and transfer properties of two lipid transfer proteins from plants.

Plant lipid transfer proteins (LTPs) are soluble proteins which are characterized by their in vitro ability to transfer phospholipids between two membranes. We have compared the functional properties of two LTPs purified from maize and wheat seeds knowing that, despite a high degree of sequence identity, the two proteins exhibit structural differences. It was found that wheat LTP had a lower transfer activity than the maize LTP, consistent with a lower kinetics of fatty acid binding. The lower affinity for the fatty acids of the wheat LTP could be explained by a narrowing occurring in the middle part of the binding site, as revealed by comparing the fluorescence spectra of various anthroyloxy-labeled fatty acids associated with the two LTPs. The affinity for some natural fatty acids was studied by competition with fluorescent fatty acids toward binding to the protein. Again, wheat LTP had a lower affinity for those molecules. All together, these observations reveal the complexity of the LTP family in plants, probably reflecting the multiple roles played by these proteins.

A plastidial lysophosphatidic acid acyltransferase from oilseed rape.

The biosynthesis of phosphatidic acid, a key intermediate in the biosynthesis of lipids, is controlled by lysophosphatidic acid (LPA, or 1-acyl-glycerol-3-P) acyltransferase (LPAAT, EC 2.3.1.51). We have isolated a cDNA encoding a novel LPAAT by functional complementation of the Escherichia coli mutant plsC with an immature embryo cDNA library of oilseed rape (Brassica napus). Transformation of the acyltransferase-deficient E. coli strain JC201 with the cDNA sequence BAT2 alleviated the temperature-sensitive phenotype of the plsC mutant and conferred a palmitoyl-coenzyme A-preferring acyltransferase activity to membrane fractions. The BAT2 cDNA encoded a protein of 351 amino acids with a predicted molecular mass of 38 kD and an isoelectric point of 9.7. Chloroplast-import experiments showed processing of a BAT2 precursor protein to a mature protein of approximately 32 kD, which was localized in the membrane fraction. BAT2 is encoded by a minimum of two genes that may be expressed ubiquitously. These data are consistent with the identity of BAT2 as the plastidial enzyme of the prokaryotic glycerol-3-P pathway that uses a palmitoyl-ACP to produce phosphatidic acid with a prokaryotic-type acyl composition. The homologies between the deduced protein sequence of BAT2 with prokaryotic and eukaryotic microsomal LAP acytransferases suggest that seed microsomal forms may have evolved from the plastidial enzyme.

Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.

Lipid-transfer proteins from plants: structure and binding properties.

Plant cells contain lipid-transfer proteins (LTPs) able to transfer phospholipids between membranes in vitro. Plant LTPs share in common structural and functional features. Recent structural studies carried out by NMR and X-ray crystallography on an LTP isolated from maize seeds have showed that this protein involves four helices packed against a C-terminal region and stabilized by four disulfide bridges. A most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule and able to accommodate acyl chains. It was thus of interest to study the ability of maize LTP to bind hydrophobic ligands such as acyl chains or lysophosphatidylcholine and to determine the effect of this binding on phospholipid transfer. The binding abilities of maize LTP, presented in this paper, are discussed and compared to those of lipid-binding proteins from animal tissues.

Characterisation of acyl binding by a plant lipid-transfer protein.

Maize lipid-transfer protein (LTP) is a small soluble protein which is able to transfer in vitro phospholipids between membranes and to bind fatty acids or lysoderivatives. In the studies reported here, fluorescent-labelled fatty acids were used to characterise the nature of the binding site on LTP. A fluorescent analogue of 12 carbons with a pyrene moiety attached at the end, alone or in conjunction with an anthroyloxy analogue, indicated that LTP could bind two fatty acids although with a marked difference in affinity. The binding capacity was strongly affected after reduction of the protein by dithiothreitol, showing that the four S-S bonds of LTP are essential for its lipid binding property. Other analogues used were 16-carbon or 18-carbon fatty acids with an anthracene moiety attached at different points of the hydrocarbon chain. Emission maxima of these molecules varied with the analogue and suggested a motional constraint for the bound fatty acid which is more important around the middle of the chain than at its extremities. Binding displacement studies were carried out with a wide range of fatty acids or fatty acyl derivatives. Fatty acids of 16 to 19 carbons were found to be the preferred ligands. The presence of one double bond did not change appreciably the affinity of LTP, although the presence of two or three double bonds or of a hydroxyl moiety significantly reduced the affinity. Fatty acyl-CoA or lysoderivatives bound as well as the corresponding fatty acid.

Isolation of a cDNA from Arabidopsis thaliana that complements the sec14 mutant of yeast.

The SEC14 gene of Saccharomyces cerevisiae codes for a phosphatidylinositol-transfer protein (Sec14p(sc)) which is capable of transferring both phosphatidylinositol and phosphatidylcholine between membranes in vitro. Genetic and biochemical studies conducted in S. cerevisiae have shown that this protein acts as an inhibitor of phosphatidylcholine biosynthesis via the so-called Kennedy pathway only. This inhibition is controlled by the binding of phospholipids to the Sec14p(sc) protein. Here we describe the isolation of a cDNA from Arabidopsis thaliana by functional complementation of a sec14(ts) mutant of S. cerevisiae. This cDNA, designated AtSEC14, is capable of restoring the growth of the sec14(ts) mutant at the restrictive temperature of 37 degrees C. Extracellular invertase measurements indicated that the cDNA can partly restore protein secretion. In addition, the phosphatidylinositol-transfer activity measured in protein extracts is greatly enhanced in the complemented mutant strain when compared with the sec14(ts) mutant. The best sequence similarity at the amino acid level is found with the Sec14p protein of S. cerevisiae (36.5% similarity), and most of the amino acids that are thought to be involved in the binding of phospholipids in the yeast protein are conserved in the AtSEC14 gene product. Southern analysis suggests the presence of a single gene in the Arabidopsis genome, although the existence of distantly related sequences cannot be excluded. This gene is expressed in roots, leaves, flowers and siliques of Arabidopsis.

Rice lipid transfer protein (LTP) genes belong to a complex multigene family and are differentially regulated.

Several cDNA clones encoding three different lipid transfer proteins (LTPs) have been isolated from rice (Oryza sativa L.) in order to analyse the complexity, the evolution and the expression of the LTP gene family. The mature proteins deduced from three clones exhibited a molecular mass of 9 kDa, in agreement with the molecular mass of other LTPs from plants. The clones were shown to be homologous in the coding region, while the 3' non-coding regions diverged strongly between the clones. The occurrence of at least three small multigene families encoding these proteins in rice was confirmed by Southern blot analysis. When compared with each other and with LTPs from other plants, the cluster including rice LTPs and other cereal LTPs indicated that these genes duplicated rather recently and independently in the different plant phyla. The expression pattern of each gene family was also investigated. Northern blot experiments demonstrated that they are differentially regulated in the different tissues analysed. Components such as salt, salicylic acid and abscisic acid were shown to modulate Ltp gene expression, depending on tissues and gene classes, suggesting a complex regulation of these genes.

Solution structure and lipid binding of a nonspecific lipid transfer protein extracted from maize seeds.

The three-dimensional solution structure of a nonspecific lipid transfer protein extracted from maize seeds determined by 1H NMR spectroscopy is described. This cationic protein consists of 93 amino acid residues. Its structure was determined from 1,091 NOE-derived distance restraints, including 929 interresidue connectivities and 197 dihedral restraints (phi, psi, chi 1) derived from NOEs and 3J coupling constants. The global fold involving four helical fragments connected by three loops and a C-terminal tail without regular secondary structures is stabilized by four disulfide bridges. The most striking feature of this structure is the existence of an internal hydrophobic cavity running through the whole molecule. The global fold of this protein, very similar to that of a previously described lipid transfer protein extracted from wheat seeds (Gincel E et al., 1994, Eur J Biochem 226:413-422) constitutes a new architecture for alpha-class proteins. 1H NMR and fluorescence studies show that this protein forms well-defined complexes in aqueous solution with lysophosphatidylcholine. Dissociation constants, Kd, of 1.9 +/- 0.6 x 10(-6) M and > 10(-3) M were obtained with lyso-C16 and -C12, respectively. A structure model for a lipid-protein complex is proposed in which the aliphatic chain of the phospholipid is inserted in the internal cavity and the polar head interacts with the charged side chains located at one end of this cavity. Our model for the lipid-protein complex is qualitatively very similar to the recently published crystal structure (Shin DH et al., 1995, Structure 3:189-199).

Germination-specific lipid transfer protein cDNAs in Brassica napus L.

Three cDNA clones encoding lipid transfer proteins (LTPs) were isolated by applying the rapid amplification of cDNA ends (RACE) protocol to imbibed seeds and germinating seedlings Brassica napus. The deduced amino-acid sequences show a great degree of homology and they exhibit the common features shared by all LTPs. Their expression pattern indicates a strong developmental, hormonal, and environmental regulation. They are expressed only in cotyledons and hypocotyls of germinating seedlings and their levels of expression increase upon treatment with cis-abscisic acid and NaCl. Their distribution in the cotyledons of young seedlings is suggestive of a role related to the mobilization of lipid reserves.

A potent antimicrobial protein from onion seeds showing sequence homology to plant lipid transfer proteins.

An antimicrobial protein of about 10 kD, called Ace-AMP1, was isolated from onion (Allium cepa L.) seeds. Based on the near-complete amino acid sequence of this protein, oligonucleotides were designed for polymerase chain reaction-based cloning of the corresponding cDNA. The mature protein is homologous to plant nonspecific lipid transfer proteins (nsLTPs), but it shares only 76% of the residues that are conserved among all known plant nsLTPs and is unusually rich in arginine. Ace-AMP1 inhibits all 12 tested plant pathogenic fungi at concentrations below 10 micrograms mL-1. Its antifungal activity is either not at all or is weakly affected by the presence of different cations at concentrations approximating physiological ionic strength conditions. Ace-AMP1 is also active on two Gram-positive bacteria but is apparently not toxic for Gram-negative bacteria and cultured human cells. In contrast to nsLTPs such as those isolated from radish or maize seeds, Ace-AMP1 was unable to transfer phospholipids from liposomes to mitochondria. On the other hand, lipid transfer proteins from wheat and maize seeds showed little or no antimicrobial activity, whereas the radish lipid transfer protein displayed antifungal activity only in media with low cation concentrations. The relevance of these findings with regard to the function of nsLTPs is discussed.

Selective modifications of the phospholipid fatty acid composition in human platelet membranes using nonspecific and specific lipid transfer proteins.

In order to specifically modify the fatty acid composition of cell membrane phospholipids, we have developed an original method based on the transfer of pure phospholipid molecular species to membranes. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) subclasses containing 18:2n-6 and 22:6n-3 at the sn-2 position were incorporated into human platelet membranes using the endogenous phosphatidylinositol/PC transfer protein (PI/PC-TP) and the phospholipid transfer protein from maize (L-TP), respectively. PI/PC-TP was shown to catalyze a strict exchange of phospholipids between platelet membranes and unilamellar vesicles containing 1,2-diacylglycerophosphocholine (diacyl-GPC; 16:0/18:2-GPC, or 16:0/22:6-GPC). The proportions of 18:2n-6 and 22:6n-3 in diacyl-GPC of platelet membranes were gradually increased from 10.7 to 16.9% and from 0.8 to 10.1%, respectively, whereas the PE and PI fatty acid compositions were not changed. The diacyl-GPC enrichment in 22:6n-3 and 18:2n-6 did not induce changes in membrane fluidity parameters measured by electron-spin resonance of 5- and 16-nitroxy stearic acids. Similarly, 18:2n-6 and 22:6n-3 esterified in 1,2-diacylglycerophosphoethanolamine (diacyl-GPE) have been incorporated in platelet membranes by an apparent exchange process under conditions where donor vesicles had a phospholipid composition equivalent to that of platelet membranes. The proportions of 18:2n-6 and 22:6n-3 were selectively and progressively increased from 6.0 to 21.2% and from 2.2 to 17.2%, respectively, in diacyl-GPE of platelet membranes. Thus, the L-TP- and PI/PC-TP-catalyzed enrichment can be used for studying the modulation of membrane biological activities by defined changes of fatty acid composition of specific phospholipid classes or subclasses.

Compared selectivities of the phosphatidylinositol-synthase from maize coleoptiles either in microsomal membranes or after solubilization.

PI-synthase selectivity from etiolated maize coleptiles was studied either associated with the microsomal membranes or after solubilization by CHAPS and prepurification on a DEAE-trisacryl M column. When maize microsomes were incubated with [3H]inositol without any exogenous CPM-PA, the most heavily labelled molecular species were 16:0/18:2-PI (77%), 16:0/18:3-plus 18:2/18:2-PI (15%), 16:0/18:1-PI (4%) and 18:0/18:2-PI (4%). Addition to the incubation medium of up to 300 microM 16:0/16:0-CMP-PA unexpectedly resulted in the formation of very little labelled 16:0/16:0-PI. When the solubilized fraction from microsomes was incubated with [3H]inositol in absence of 16:0/16:0-CPM-PA, the same PI molecular species as above were synthesized. However, with increasing concentrations of 16:0/16:0-CMP-PA in the medium, increasing amounts of labelled 16:0/16:0-PI appeared as well. With prepurified PI-synthase eluted from a DEAE column, endogenous CMP-PA was poorly utilized for PI biosynthesis whereas the exogenous 16:0/16:0-CPM-PA was used actively. With time, the endogenous CMP-PA was utilized first and the exogenous substrate was utilized, albeit, much more slowly. The results demonstrate that the selectivity displayed by PI-synthase towards various molecular species of CMP-PA depends on the integration of the enzyme in the membrane structure. Solubilization of the enzyme, i.e., inclusion of the protein in micelles with detergents and lipids, results in an apparent loss of the selectivity for CMP-PA.

Effect of specific phospholipid molecular species incorporated in human platelet membranes on thromboxane A2/prostaglandin H2 receptors.

The incorporation of albumin-bound docosahexaenoic acid (22:6n-3), but not linoleic acid (18:2n-6), into cellular phospholipids inhibits platelet aggregation induced by the thromboxane analogue U46619. [3H]U46619 specific binding to thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors, as well as specific binding of the antagonist [3H]SQ29548 to these sites were also decreased in these modified cells (P. G., Swann et al. 1990. J. Biol. Chem. 265: 21692-21697). More than 80% of the 22:6n-3 incorporated in these cells was esterified in the various endogenous phospholipid classes and the remaining was found in neutral lipids and in the unesterified fatty acid pool. In this study, we determined whether the effects observed could be attributed to the esterification of 22:6n-3 in phospholipids and whether the 22:6n-3 biological activity might depend on its esterification in specific phospholipid classes. Therefore, pure phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecular species were transferred to platelet membranes, using lipid transfer proteins. PC and PE containing palmitate (16:0) and 22:6n-3 or 16:0 and 18:2n-6 at position sn-1 and sn-2, respectively, were incorporated into membranes only at the expense of the corresponding endogenous phospholipid class, by an apparent exchange process. When such modified membranes were tested for specific binding of U46619 and SQ29548, a significant decrease of the receptor site affinity was only observed in membranes highly enriched with 1-palmitoyl-2-docosahexaenoyl-glycerophosphocholine (16:0/22:6-GPC). Fluidity parameters measured by electron spin resonance of 5- and 16-nitroxy-stearic acids were not significantly different in membranes enriched with 16:0/22:6-GPC relative to those enriched with 16:0/18:2n-6-GPC, arguing against a generalized perturbation of the membrane due to 22:6n-3 incorporation. We conclude that molecular species of PC with 22:6n-3 at the sn-2 position can affect TXA2/PGH2 receptors. The selectivity of the inhibitory effect of PC containing 22:6n-3 is discussed.

Comparison of the Stereospecificity and Immunoreactivity of NADH-Ferricyanide Reductases in Plant Membranes.

The substrate stereospecificity of NADH-ferricyanide reductase activities in the inner mitochondrial membrane and peroxisomal membrane of potato (Solanum tuberosum L.) tubers, spinach (Spinacea oleracea L.) leaf plasma membrane, and red beetroot (Beta vulgaris L.) tonoplast were all specific for the [beta]-hydrogen of NADH, whereas the reductases in wheat root (Triticum aestivum L.) endoplasmic reticulum and potato tuber outer mitochondrial membrane were both [alpha]-hydrogen specific. In all isolated membrane fractions one or several polypeptides with an apparent size of 45 to 55 kD cross-reacted with antibodies raised against a microsomal NADH-ferricyanide reductase on western blots.

A pair of genes coding for lipid-transfer proteins in Sorghum vulgare.

Approximately five genes coding for lipid-transfer proteins (LTP) can be detected in Sorghum vulgare by DNA blots using a specific genomic probe. Two of these genes have been identified and sequenced. The two genes (ltp1 and ltp2) code for very similar (91.8% identity) proteins, they are separated by approx. 4 kb of DNA and their open reading frames may be read in the same direction. The gene (ltp1) located upstream has an intron placed in the same position already described for other ltp in maize and rice. Gene ltp2 has no intron. cDNAs corresponding to ltp1 have been identified in a 6-day-old plantlet library, but not for ltp2. The results of the comparison between the two sequences indicate the presence of a gap between the two genes in their promoter region. LTP seem to be coded for in plants by a small family of genes. At least in sorghum, two of its components are tightly clustered in the same genomic region.

Characterization of a rice gene coding for a lipid transfer protein.

The cloning and sequence analysis of a gene that encodes a lipid transfer protein (LTP) from rice is reported. A genomic DNA library from Oryza sativa was screened using a cDNA encoding a maize LTP. One genomic clone containing the gene (Ltp) was partially sequenced and analyzed. The open reading frame is interrupted by an 89-bp intron. From the results of Southern hybridizations, Ltp appears to be a member of a small multigenic family. Transcripts of the corresponding gene were detected in several tissues including coleoptile, leaf, endosperm, scutellum and root. The transcription start point was determined by primer extension. The deduced amino-acid sequence of the Ltp product is shown to be homologous to LTPs from other crops.

A phosphatidylinositol/phosphatidylcholine transfer protein is required for differentiation of the dimorphic yeast Yarrowia lipolytica from the yeast to the mycelial form.

The SEC14SC gene encodes the phosphatidylinositol/phosphatidylcholine transfer protein (PI/PC-TP) of Saccharomyces cerevisiae. The SEC14SC gene product (SEC14pSC) is associated with the Golgi complex as a peripheral membrane protein and plays an essential role in stimulating Golgi secretory function. We report the characterization of SEC14YL, the structural gene for the PI/PC-TP of the dimorphic yeast Yarrowia lipolytica. SEC14YL encodes a primary translation product (SEC14YL) that is predicted to be a 497-residue polypeptide of which the amino-terminal 300 residues are highly homologous to the entire SEC14pSC, and the carboxyl-terminal 197 residues define a dispensible domain that is not homologous to any known protein. In a manner analogous to the case for SEC14pSC, SEC14pYL localizes to punctate cytoplasmic structures in Y. lipolytica that likely represent Golgi bodies. However, SEC14pYL is neither required for the viability of Y. lipolytica nor is it required for secretory pathway function in this organism. This nonessentiality of SEC14pYL for growth and secretion is probably not the consequence of a second PI/PC-TP activity in Y. lipolytica as cell-free lysates prepared from delta sec14YL strains are devoid of measurable PI/PC-TP activity in vitro. Phenotypic analyses demonstrate that SEC14pYL dysfunction results in the inability of Y. lipolytica to undergo the characteristic dimorphic transition from the yeast to the mycelial form that typifies this species. Rather, delta sec14YL mutants form aberrant pseudomycelial structures as cells enter stationary growth phase. The collective data indicate a role for SEC14pYL in promoting the differentiation of Y. lipolytica cells from yeast to mycelia, and demonstrate that PI/PC-TP function is utilized in diverse ways by different organisms.