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.     

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.     

Arabidopsis cDNA encoding a membrane-associated protein with an acyl-CoA binding domain

Acyl-CoA binding proteins (ACBPs) are small (ca. 10 kDa) highly-conserved cytosolic proteins that bind long-chain acyl-CoAs. A novel cDNA encoding ACBP1, a predicted membrane protein of 24.1 kDa with an acyl-CoA binding protein domain at its carboxy terminus, was cloned from Arabidopsis thaliana. At this domain, ACBP1 showed 47% amino acid identity to Brassica ACBP and 35% to 40% amino acid identity to yeast, Drosophila, bovine and human ACBPs. Recombinant (His)6-ACBP1 fusion protein was expressed in Escherichia coli and was shown to bind 14[C]oleoyl-CoA. A hydrophobic domain, absent in the 10 kDa ACBPs, was located at the amino terminus of ACBP1. Using antipeptide polyclonal antibodies in western blot analysis, ACBP1 was shown to be a membrane-associated glycosylated protein with an apparent molecular mass of 33 kDa. The ACBP1 protein was also shown to accumulate predominantly in siliques and was localized to the seed within the silique. These results suggest that the biological role of ACBP1 is related to lipid metabolism in the seed, presumably in which acyl-CoA esters are involved. Northern blot analysis showed that the 1.4 kb ACBP1 mRNA was expressed in silique, root, stem, leaf and flower. Results from Southern blot analysis of genomic DNA suggest the presence of at least two genes encoding ACBPs in Arabidopsis.     

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.     

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.     

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.     

Tissue- and paralogue-specific functions of acyl-CoA-binding proteins in lipid metabolism in Caenorhabditis elegans

ACBP (acyl-CoA-binding protein) is a small primarily cytosolic protein that binds acyl-CoA esters with high specificity and affinity. ACBP has been identified in all eukaryotic species, indicating that it performs a basal cellular function. However, differential tissue expression and the existence of several ACBP paralogues in many eukaryotic species indicate that these proteins serve distinct functions. The nematode Caenorhabditis elegans expresses seven ACBPs: four basal forms and three ACBP domain proteins. We find that each of these paralogues is capable of complementing the growth of ACBP-deficient yeast cells, and that they exhibit distinct temporal and tissue expression patterns in C. elegans. We have obtained loss-of-function mutants for six of these forms. All single mutants display relatively subtle phenotypes; however, we find that functional loss of ACBP-1 leads to reduced triacylglycerol (triglyceride) levels and aberrant lipid droplet morphology and number in the intestine. We also show that worms lacking ACBP-2 show a severe decrease in the β-oxidation of unsaturated fatty acids. A quadruple mutant, lacking all basal ACBPs, is slightly developmentally delayed, displays abnormal intestinal lipid storage, and increased β-oxidation. Collectively, the present results suggest that each of the ACBP paralogues serves a distinct function in C. elegans.     

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.     

Binding site differences revealed by crystal structures of Plasmodium falciparum and bovine acyl-CoA binding protein

Acyl-CoA binding protein (ACBP) maintains a pool of fatty acyl-CoA molecules in the cell and plays a role in fatty acid metabolism. The biochemical properties of Plasmodium falciparum ACBP are described together with the 2.0 A resolution crystal structures of a P. falciparum ACBP-acyl-CoA complex and of bovine ACBP in two crystal forms. Overall, the bovine ACBP crystal structures are similar to the NMR structures published previously; however, the bovine and parasite ACBP structures are less similar. The parasite ACBP is shown to have a different ligand-binding pocket, leading to an acyl-CoA binding specificity different from that of bovine ACBP. Several non-conservative differences in residues that interact with the ligand were identified between the mammalian and parasite ACBPs. These, together with measured binding-specificity differences, suggest that there is a potential for the design of molecules that might selectively block the acyl-CoA binding site.     

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.     

Overexpression of Arabidopsis Acyl-CoA Binding Protein ACBP3 Promotes Starvation-Induced and Age-Dependent Leaf Senescence

In Arabidopsis thaliana, a family of six genes (ACBP1 to ACBP6) encodes acyl-CoA binding proteins (ACBPs). Investigations on ACBP3 reported here show its upregulation upon dark treatment and in senescing rosettes. Transgenic Arabidopsis overexpressing ACBP3 (ACBP3-OEs) displayed accelerated leaf senescence, whereas an acbp3 T-DNA insertional mutant and ACBP3 RNA interference transgenic Arabidopsis lines were delayed in dark-induced leaf senescence. Acyl-CoA and lipid profiling revealed that the overexpression of ACBP3 led to an increase in acyl-CoA and phosphatidylethanolamine (PE) levels, whereas ACBP3 downregulation reduced PE content. Moreover, significant losses in phosphatidylcholine (PC) and phosphatidylinositol, and gains in phosphatidic acid (PA), lysophospholipids, and oxylipin-containing galactolipids (arabidopsides) were evident in 3-week-old dark-treated and 6-week-old premature senescing ACBP3-OEs. Such accumulation of PA and arabidopsides (A, B, D, E, and G) resulting from lipid peroxidation in ACBP3-OEs likely promoted leaf senescence. The N-terminal signal sequence/transmembrane domain in ACBP3 was shown to be essential in ACBP3-green fluorescent protein targeting and in promoting senescence. Observations that recombinant ACBP3 binds PC, PE, and unsaturated acyl-CoAs in vitro and that ACBP3 overexpression enhances degradation of the autophagy (ATG)-related protein ATG8 and disrupts autophagosome formation suggest a role for ACBP3 as a phospholipid binding protein involved in the regulation of leaf senescence by modulating membrane phospholipid metabolism and ATG8 stability in Arabidopsis. Accelerated senescence in ACBP3-OEs is dependent on salicylic acid but not jasmonic acid signaling.     

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.     

The function of acyl-CoA-binding protein (ACBP)/Diazepam binding inhibitor (DBI)

Acyl-CoA-binding protein has been isolated independently by five different groups based on its ability to (1) displace diazepam from the GABA_A receptor, (2) affect cell growth, (3) induce medium-chain acyl-CoA-ester synthesis, (4) stimulate steroid hormone synthesis, and (5) affect glucose-induced insulin secretion. In this survey evidence is presented to show that ACBP is able to act as an intracellular acyl-CoA transporter and acyl-CoA pool former. The rat ACBP genomic gene consists of 4 exons and is actively expressed in all tissues tested with highest concentration being found in liver. ACBP consists of 86 amino acid residues and contains 4 -helices which are folded into a boomerang type of structure with -helices 1, 2 and 4 in the one arm and -helix 3 and an open loop in the other arm of the boomerang. ACBP is able to stimulate mitochondrial acyl-CoA synthetase by removing acyl-CoA esters from the enzyme. ACBP is also able to desorb acyl-CoA esters from immobilized membranes and transport and deliver these for mitochondrial -oxidation. ACBP efficiently protects acetyl-CoA carboxylase and the mitochondrial ADP/ATP translocase against acyl-CoA inhibition. Finally, ACBP is shown to be able to act as an intracellular acyl-CoA pool former by overexpression in yeast. The possible role of ACBP in lipid metabolism is discussed.     

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.