Membrane localization of Arabidopsis acyl-CoA binding protein ACBP2

Cytosolic acyl-CoA binding proteins bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and pool formers. Recently, we have characterized Arabidopsis thaliana cDNAs encoding novel forms of ACBP, designated ACBP1 and ACBP2, that contain a hydrophobic domain at the N-terminus and show conservation at the acyl-CoA binding domain to cytosolic ACBPs. We have previously demonstrated that ACBP1 is membrane-associated in Arabidopsis. Here, western blot analysis of anti-ACBP2 antibodies on A. thaliana protein showed that ACBP2 is located in the microsome-containing membrane fraction and in the subcellular fraction containing large particles (mitochondria, chloroplasts and peroxisomes), resembling the subcellular localization of ACBP1. To further investigate the subcellular localization of ACBP2, we fused ACBP2 translationally in-frame to GFP. By means of particle gene bombardment, ACBP2-GFP and ACBP1-GFP fusion proteins were observed transiently expressed at the plasma membrane and at the endoplasmic reticulum in onion epidermal cells. GFP fusions with deletion derivatives of ACBP1 or ACBP2 lacking the transmembrane domain were impaired in membrane targeting. Our investigations also showed that when the transmembrane domain of ACBP1 or that of ACBP2 was fused with GFP, the fusion protein was targeted to the plasma membrane, thereby establishing their role in membrane targeting. The localization of ACBP1-GFP is consistent with our previous observations using immunoelectron microscopy whereby ACBP1 was localized to the plasma membrane and vesicles. We conclude that ACBP2, like ACBP1, is a membrane protein that likely functions in membrane-associated acyl-CoA transfer/metabolism.     

Isolation and characterization of a cDNA encoding a membrane bound acyl-CoA binding protein from Agave americana L. epidermis.

A cDNA encoding an acyl-CoA binding protein (ACBP) homologue has been cloned from a cDNA library made from mRNA isolated from epidermis of young leaves of Agave americana L. The derived amino acid sequence reveals a protein corresponding to the membrane-associated form of ACBPs only previously described in Arabidopsis and rice. Northern blot analysis showed that the A. americana ACBP gene is mainly expressed in the epidermis of mature zone of the leaves. The epidermis of A. americana leaves have a well developed cuticle with the highest amounts of the cuticular components waxes, cutin and cutan suggesting a potential role of the protein in cuticle formation.     

Isolation of a gene encoding Arabidopsis membrane-associated acyl-CoA binding protein and immunolocalization of its gene product

Until recently, only cytosolic acyl-CoA binding proteins (ACBPs) have been characterized. The isolation of an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP that accumulates in developing seeds, designated ACBP1, has provided evidence for the existence of membrane-associated forms of ACBPs (Chye, 1998, Plant Mol. Biol. 38, 827-838). We now report on the isolation of its corresponding gene from an A. thaliana Columbia genomic library using the ACBP1 cDNA as a hybridization probe. Nucleotide sequence analysis of Arabidopsis ACBP1 showed that its promoter lacks a TATA box, resembling the promoters of rat, Drosophila and human genes encoding cytosolic ACBP and suggesting that it is a housekeeping gene. We show by Western blot analysis that ACBP1 expression in developing seeds coincides with lipid deposition and that homologues of membrane-associated ACBP1 exist in other plants. Using light microscopy, we show that ACBP1 is strongly expressed in the embryo at the cotyledons, hypocotyl, procambium of the axis and in most peripheral cells of the cotyledons and hypocotyl. Immunogold labelling localized ACBP1 to vesicles, to the plasma membrane especially at epidermal cells of heart, torpedo and cotyledonary stage embryos, and to the cell wall of the outer integument cells at the seed coat. Our results suggest that ACBP1 is involved in intermembrane lipid transport from the ER via vesicles to the plasma membrane where it could maintain a membrane-associated acyl pool; its immunolocalization to the cell wall of outer integument cells at the seed coat suggests a role in cuticle and cutin formation.     

Plant acyl-CoA-binding proteins: An emerging family involved in plant development and stress responses

Acyl-CoA-binding protein (ACBP) was first identified in mammals as a neuropeptide, and was demonstrated to belong to an important house-keeping protein family that extends across eukaryotes and some prokaryotes. In plants, the Arabidopsis ACBP family consists of six AtACBPs (AtACBP1 to AtACBP6), and has been investigated using gene knock-out mutants and overexpression lines. Herein, recent findings on the AtACBPs are examined to provide an insight on their functions in various plant developmental processes, such as embryo and seed development, seed dormancy and germination, seedling development and cuticle formation, as well as their roles under various environmental stresses. The significance of the AtACBPs in acyl-CoA/lipid metabolism, with focus on their interaction with long to very-long-chain (VLC) acyl-CoA esters and their potential role in the formation of lipid droplets in seeds and vegetative tissues are discussed. In addition, recent findings on the rice ACBP family are presented. The similarities and differences between ACBPs from Arabidopsis and rice, that represent eudicot and monocot model plants, respectively, are analyzed and the evolution of plant ACBPs by phylogenetic analysis reviewed. Finally, we propose potential uses of plant ACBPs in phytoremediation and in agriculture related to the improvement of environmental stress tolerance and seed oil production.     

New roles for acyl-CoA-binding proteins (ACBPs) in plant development, stress responses and lipid metabolism

ACBPs are implicated in acyl-CoA trafficking in many eukaryotes and some prokaryotes. Six genes encode proteins designated as AtACBP1-AtACBP6 in the Arabidopsis thaliana ACBP family. These ACBPs are conserved in the acyl-CoA-binding domain, but vary in size from 92 amino acids (10.4 kDa) to 668 amino acids (73.1 kDa), and are subcellularly localised to different compartments in plant cells. Results from in vitro binding assays show that their corresponding recombinant proteins exhibit differential binding affinities to acyl-CoA esters and phospholipids, implying that these ACBPs may have non-redundant biological functions in vivo. By using knockout/downregulated and overexpression lines of Arabidopsis ACBPs, recent investigations have revealed that in addition to their proposed roles in phospholipid metabolism, these ACBPs can influence plant development including early embryogenesis and leaf senescence, as well as plant stress responses including heavy metal resistance, oxidative stress, freezing tolerance and pathogen resistance. In this review, recent progress on the biochemical and functional analyses of Arabidopsis ACBPs, their links to metabolic/signalling pathways, and their potential applications in development of stress tolerance are discussed.     

Arabidopsis acyl-CoA-binding proteins ACBP4 and ACBP5 are subcellularly localized to the cytosol and ACBP4 depletion affects membrane lipid composition

In Arabidopsis thaliana, acyl-CoA-binding proteins (ACBPs) are encoded by six genes, and they display varying affinities for acyl-CoA esters. Recombinant ACBP4 and ACBP5 have been shown to bind oleoyl-CoA esters in vitro. In this study, the subcellular localizations of ACBP4 and ACBP5 were determined by biochemical fractionation followed by western blot analyses using anti-ACBP4 and anti-ACBP5 antibodies and immuno-electron microscopy. Confocal microscopy of autofluorescence-tagged ACBP4 and ACBP5, expressed transiently in onion epidermal cells and in transgenic Arabidopsis, confirmed their expression in the cytosol. Taken together, ACBP4 and ACBP5 are available in the cytosol to bind and transfer cytosolic oleoyl-CoA esters. Lipid profile analysis further revealed that an acbp4 knockout mutant showed decreases in membrane lipids (digalactosyldiacylglycerol, monogalactosyldiacylglycerol, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol) while acbp4-complemented lines attained levels similar to wild type, suggesting that ACBP4 plays a role in the biosynthesis of membrane lipids including galactolipids and phospholipids.     

Arabidopsis Acyl-CoA-Binding Protein ACBP2 Interacts With an Ethylene-Responsive Element-Binding Protein,AtEBP, via its Ankyrin Repeats

Cytosolic acyl-CoA-binding proteins (ACBP) bind long-chain acyl-CoAs and act as intracellular acyl-CoA transporters and maintain acyl-CoA pools. Arabidopsis thaliana ACBP2 shows conservation at the acyl-CoA-binding domain to cytosolic ACBPs but is distinct by the presence of an N-terminal transmembrane domain and C-terminal ankyrin repeats. The function of the acyl-CoA-binding domain in ACBP2 has been confirmed by site-directed mutagenesis and four conserved residues crucial for palmitoyl-CoA binding have been identified. Results from ACBP2:GFP fusions transiently expressed in onion epidermal cells have demonstrated that the transmembrane domain functions in plasma membrane targeting, suggesting that ACBP2 transfers acyl-CoA esters to this membrane. In this study, we investigated the significance of its ankyrin repeats in mediating protein-protein interactions by yeast two-hybrid analysis and in vitro protein-binding assays; we showed that ACBP2 interacts with the A. thaliana ethylene-responsive element-binding protein AtEBP via its ankyrin repeats. This interaction was lacking in yeast two-hybrid analysis upon removal of the ankyrin repeats. When the subcellular localizations of ACBP2 and AtEBP were further investigated using autofluorescent protein fusions in transient expression by agroinfiltration of tobacco leaves, the DsRed:ACBP2 fusion protein was localized to the plasma membrane while the GFP:AtEBP fusion protein was targeted to the nucleus and plasma membrane. Co-expression of DsRed:ACBP2 and GFP:AtEBP showed a common localization of both proteins at the plasma membrane, suggesting that ACBP2 likely interacts with AtEBP at the plasma membrane.     

Single amino acid substitutions at the acyl-CoA-binding domain interrupt 14[C]palmitoyl-CoA binding of ACBP2, an Arabidopsis acyl-CoA-binding protein with ankyrin repeats

Cytosolic acyl-CoA-binding proteins (ACBPs) are small proteins (ca. 10 kDa) that bind long-chain acyl-CoAs and are involved in the storage and intracellular transport of acyl-CoAs. Previously, we have characterized an Arabidopsis thaliana cDNA encoding a novel membrane-associated ACBP, designated ACBP1, demonstrating the existence of a new form of ACBP in plants (M.-L. Chye, Plant Mol. Biol. 38 (1998) 827–838). ACBP1 likely participates in intermembrane lipid transport from the ER to the plasma membrane, where it could maintain a membrane-associated acyl pool (Chye et al., Plant J. 18 (1999) 205–214). Here we report the isolation of cDNAs encoding ACBP2 (M _r 38 479) that shows conservation in the acyl-CoA-binding domain to previously reported ACBPs, and contains ankyrin repeats at its carboxy terminus. These repeats, which likely mediate protein-protein interactions, could constitute a potential docking site in ACBP2 for an enzyme that uses acyl-CoAs as substrate. In vitro binding assays on recombinant (His)₆-ACBP2 expressed in Escherichia coli show that it binds ¹⁴[C]palmitoyl-CoA preferentially to ¹⁴[C]oleoyl-CoA. Analysis of the acyl-CoA-binding domain in ACBP2 was carried out by in vitro mutagenesis. Mutant forms of recombinant (His)₆-ACBP2 with single amino acid substitutions at conserved residues within the acyl-CoA-binding domain were less effective in binding ¹⁴[C]palmitoyl-CoA. Northern blot analysis showed that the 1.6 kb ACBP2 mRNA, like that of ACBP1, is expressed in all plant organs. Analysis of the ACBP2 promoter revealed that, like the ACBP1 promoter, it lacks a TATA box suggesting the possibility of a housekeeping function for ACBP2 in plant lipid metabolism.     

Overexpression of membrane-associated acyl-CoA-binding protein ACBP1 enhances lead tolerance in Arabidopsis

In Arabidopsis thaliana , a family of six genes encodes acyl-CoA-binding proteins (ACBPs) that show conservation at the acyl-CoA-binding domain. They are the membrane-associated ACBP1 and ACBP2, extracellularly targeted ACBP3, kelch-motif-containing ACBP4 and ACBP5, and 10-kDa ACBP6. The acyl-CoA domain in each of ACBP1 to ACBP6 binds long-chain acyl-CoA esters in vitro , suggestive of possible roles in plant lipid metabolism. We addressed here the use of Arabidopsis ACBPs in conferring lead [Pb(II)] tolerance in transgenic plants because the 10-kDa human ACBP has been identified as a molecular target for Pb(II) in vivo . We investigated the effect of Pb(II) stress on the expression of genes encoding Arabidopsis ACBP1, ACBP2 and ACBP6. We showed that the expression of ACBP1 and ACBP2 , but not ACBP6 , in root is induced by Pb(II) nitrate treatment. In vitro Pb(II)-binding assays indicated that ACBP1 binds Pb(II) comparatively better, and ACBP1 was therefore selected for further investigations. When grown on Pb(II)-containing medium, transgenic Arabidopsis lines overexpressing ACBP1 were more tolerant to Pb(II)-induced stress than the wild type. Accumulation of Pb(II) in shoots of the ACBP1 -overepxressing plants was significantly higher than wild type. The acbp1 mutant showed enhanced sensitivity to Pb(II) when germinated and grown in the presence of Pb(II) nitrate and tolerance was restored upon complementation using an ACBP1 cDNA. Our results suggest that ACBP1 is involved in mediating Pb(II) tolerance in Arabidopsis with accumulation of Pb(II) in shoots. Such observations of Pb(II) accumulation, rather than Pb(II) extrusion, in the ACBP1 -overexpressing plants implicate possible use of ACBP1 in Pb(II) phytoremediation.     

Light-regulated Arabidopsis ACBP4 and ACBP5 encode cytosolic acyl-CoA-binding proteins that bind phosphatidylcholine and oleoyl-CoA ester

In Arabidopsis thaliana, six genes encode acyl-CoA-binding proteins (ACBPs) that show conservation of an acyl-CoA-binding domain. These ACBPs display varying affinities for acyl-CoA esters, suggesting of different cellular roles. We have recently reported that three members (ACBP4, ACBP5 and ACBP6) are subcellularly localized to the cytosol by biochemical fractionation, confocal microscopy of transgenic Arabidopsis expressing autofluorescence-tagged fusions and immuno-electron microscopy using ACBP-specific antibodies. In this study, we observed by Northern blot analysis that ACBP4 and ACBP5 mRNAs in rosettes were up-regulated by light and dampened-off in darkness, mimicking FAD7 which encodes omega-3-fatty acid desaturase, an enzyme involved in plastidial lipid metabolism. Results from in vitro binding assays indicate that recombinant ACBP4 and ACBP5 proteins bind [¹⁴C]oleoyl-CoA esters better than recombinant ACBP6, suggesting that light-regulated ACBP4 and ACBP5 encode cytosolic ACBPs that are potential candidates for the intracellular transport of oleoyl-CoA ester exported from the chloroplast to the endoplasmic reticulum for the biosynthesis of non-plastidial membrane lipids. Nonetheless, His-tagged ACBP4 and ACBP5 resemble ACBP6 in their ability to bind phosphatidylcholine suggesting that all three ACBPs are available for the intracellular transfer of phosphatidylcholine.     

Involvement of low molecular mass soluble acyl-CoA-binding protein in seed oil biosynthesis

Acyl-CoA-binding protein (ACBP), a low molecular mass (m) (～ 10 kDa) soluble protein ubiquitous in eukaryotes, plays an important housekeeping role in lipid metabolism by maintaining the intracellular acyl-CoA pool. ACBP is involved in lipid biosynthesis and transport, gene expression, and membrane biogenesis. In plants, low m ACBP and high m ACBPs participate in response mechanisms to biotic and abiotic factors, acyl-CoA transport in phloem, and biosynthesis of structural and storage lipids. In light of current research on the modification of seed oil, insight into mechanisms of substrate trafficking within lipid biosynthetic pathways is crucial for developing rational strategies for the production of specialty oils with the desired alterations in fatty acid composition. In this review, we summarize our knowledge of plant ACBPs with emphasis on the role of low m ACBP in seed oil biosynthesis, based on in vitro studies and analyses of transgenic plants. Future prospects and possible applications of low m ACBP in seed oil modification are discussed.     

Prediction of the most favorable configuration in the ACBP-membrane interaction based on electrostatic calculations

Acyl-CoA binding proteins (ACBPs) are highly conserved 10 kDa cytosolic proteins that bind medium- and long-chain acyl-CoA esters. They act as intracellular carriers of acyl-CoA and play a role in acyl-CoA metabolism, gene regulation, acyl-CoA-mediated cell signaling, transport-mediated lipid synthesis, membrane trafficking and also, ACBPs were indicated as a possible inhibitor of diazepam binding to the GABA-A receptor. To estimate the importance of the non-specific electrostatic energy in the ACBP-membrane interaction, we computationally modeled the interaction of HgACBP with both anionic and neutral membranes. To compute the Free Electrostatic Energy of Binding (dE), we used the Finite Difference Poisson Boltzmann Equation (FDPB) method as implemented in APBS. In the most energetically favorable orientation, ACBP brings charged residues Lys18 and Lys50 and hydrophobic residues Met46 and Leu47 into membrane surface proximity. This conformation suggests that these four ACBP amino acids are most likely to play a leading role in the ACBP-membrane interaction and ligand intake. Thus, we propose that long range electrostatic forces are the first step in the interaction mechanism between ACBP and membranes.     

Overexpression of the Arabidopsis 10-kilodalton acyl-coenzyme A-binding protein ACBP6 enhances freezing tolerance

Small 10-kD acyl-coenzyme A-binding proteins (ACBPs) are highly conserved proteins that are prevalent in eukaryotes. In Arabidopsis (Arabidopsis thaliana), other than the 10-kD ACBP homolog (designated Arabidopsis ACBP6), there are five larger forms of ACBPs ranging from 37.5 to 73.1 kD. In this study, the cytosolic subcellular localization of Arabidopsis ACBP6 was confirmed by analyses of transgenic Arabidopsis expressing autofluorescence-tagged ACBP6 and western-blot analysis of subcellular fractions using ACBP6-specific antibodies. The expression of Arabidopsis ACBP6 was noticeably induced at 48 h after 4 degrees C treatment by northern-blot analysis and western-blot analysis. Furthermore, an acbp6 T-DNA insertional mutant that lacked ACBP6 mRNA and protein displayed increased sensitivity to freezing temperature (-8 degrees C), while ACBP6-overexpressing transgenic Arabidopsis plants were conferred enhanced freezing tolerance. Northern-blot analysis indicated that ACBP6-associated freezing tolerance was not dependent on the induction of cold-regulated COLD-RESPONSIVE gene expression. Instead, ACBP6 overexpressors showed increased expression of mRNA encoding phospholipase Ddelta. Lipid profiling analyses of rosettes from cold-acclimated, freezing-treated (-8 degrees C) transgenic Arabidopsis plants overexpressing ACBP6 showed a decline in phosphatidylcholine (-36% and -46%) and an elevation of phosphatidic acid (73% and 67%) in comparison with wild-type plants. From our comparison, the gain in freezing tolerance in ACBP6 overexpressors that was accompanied by decreases in phosphatidylcholine and an accumulation of phosphatidic acid is consistent with previous findings on phospholipase Ddelta-overexpressing transgenic Arabidopsis. In vitro filter-binding assays indicating that histidine-tagged ACBP6 binds phosphatidylcholine, but not phosphatidic acid or lysophosphatidylcholine, further imply a role for ACBP6 in phospholipid metabolism in Arabidopsis, including the possibility of ACBP6 in the cytosolic trafficking of phosphatidylcholine.     

Acyl-CoA-binding protein in the armadillo Harderian gland: its primary structure and possible role in lipid secretion

Similar to those of other species, the Harderian glands of armadillo produce an abundant lipid secretion, most of which is composed of 1-alkyl-2,3-diacylglycerol. Biosynthesis of this component is apparently performed with the participation of one cytosolic pool of acyl-CoA and another of free fatty acids. The acyl-CoA-binding protein (ACBP) is present at a concentration at least 7-fold that of the heart-type fatty acid-binding protein (H-FABP), though lower than that in other armadillo organs such as liver and brain. The ACBP complete amino acid sequence was determined by Edman degradation of peptides generated by cleavage of the protein with cyanogen bromide, endopeptidase Glu-C, and trypsin. ACBP consists of 86 residues and has a calculated molecular mass of 9783 Da, taking into account that an acetyl group is blocking the N-terminus. Identity percentages between armadillo Harderian gland ACBP and other known ACBPs show that the protein belongs to the liver-specific ACBP isoform (L-ACBP). The fact that the ACBP concentration is higher than that of FABP suggests that the Harderian gland is able to store acyl-CoA amounts in ACBP larger than those of fatty acids in H-FABP for 1-alkyl-2,3-diacylglycerol synthesis.     

Molecular characterization of an Arabidopsis thaliana cDNA encoding a small GTP-binding protein, Rha1.

We have isolated a cDNA encoding a small GTP-binding protein from an Arabidopsis thaliana cDNA library using an oligonucleotide probe derived from the most conserved domain of the ras superfamily. The cDNA encodes a 21.8 kDa protein, designated Rha1, which shows high homology to members of the ras superfamily in the regions involved in GTP binding, GTPase activity, and membrane attachment. The amino acid sequence is 60% identical to the sequence of the mammalian Rab5 protein, a small GTP-binding protein which is believed to be involved in endocytosis. Several regions, including the putative effector domain are completely conserved. This high percentage of amino acid identity suggests that the Rha1 protein is the functional plant counterpart of the Rab5 protein. When expressed in E. coli, the Rha1 protein was shown to bind GTP. The rha1 gene is most highly expressed in root and callus tissue, weakly expressed in stems and inflorescences and virtually not expressed in leaves and seed pods. Genomic Southern analysis revealed that rha 1 is part of a small multigene family.