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.     

Arabidopsis ACBP3 is an extracellularly targeted acyl-CoA-binding protein

Cytosolic 10-kDa acyl-CoA-binding proteins (ACBPs) function in the storage and intracellular transport of acyl-CoA esters in eukaryotes. Fatty acids synthesized de novo in plant chloroplasts are exported as oleoyl-CoA and palmitoyl-CoA esters. In Arabidopsis, other than the 10-kDa ACBP, there exists five larger ACBPs (ACBP1 to ACBP5) of which homologues have not been characterized in other organisms. To investigate the significance of this gene family, we have attempted to subcellularly localize them and compare their acyl-CoA-binding affinities. We have previously shown that Arabidopsis ACBP1 and ACBP2 are membrane-associated proteins while ACBP4 and ACBP5 contain kelch motifs. Here, to localize ACBP3, we have expressed ACBP3-red fluorescent protein (DsRed2) from the CaMV 35S promoter. ACBP3-DsRed was localized extracellularly in transiently expressed tobacco BY-2 cells and onion epidermal cells. The function of the acyl-CoA-binding domain in ACBP3 was investigated by in vitro binding assays using (His)₆-ACBP3, which was observed to bind [¹⁴C]arachidonyl-CoA with high affinity in comparison to [¹⁴C]palmitoyl-CoA and [¹⁴C]oleoyl-CoA. To identify the residues functional in binding, five mutants with single amino acid substitutions in the acyl-CoA-binding domain of (His)₆-ACBP3 and (His)₆-ACBP1 (which also binds [¹⁴C]arachidonyl-CoA) were generated by site-directed mutagenesis. Binding assays with arachidonyl-CoA revealed that replacement of a conserved R residue (R150A in ACBP1 and R284A in ACBP3), disrupted binding. In contrast, other substitutions in ACBP1 (Y126A, K130A, K152A and Y171A) and in ACBP3 (F260A, K264A, K286A and Y305A) did not affect arachidonyl-CoA binding, unlike their equivalents in (His)₆-ACBP2, (His)₆-ACBP4 and (His)₆-ACBP5, which had altered binding to palmitoyl-CoA or oleoyl-CoA.     

Arabidopsis cytosolic acyl-CoA-binding proteins ACBP4, ACBP5 and ACBP6 have overlapping but distinct roles in seed development

Eukaryotic cytosolic ACBPs (acyl-CoA-binding proteins) bind acyl-CoA esters and maintain a cytosolic acyl-CoA pool, but the thermodynamics of their protein–lipid interactions and physiological relevance in plants are not well understood. Arabidopsis has three cytosolic ACBPs which have been identified as AtACBP4, AtACBP5 and AtACBP6, and microarray data indicated that all of them are expressed in seeds; AtACBP4 is expressed in early embryogenesis, whereas AtACBP5 is expressed later. ITC (isothermal titration calorimetry) in combination with transgenic Arabidopsis lines were used to investigate the roles of these three ACBPs from Arabidopsis thaliana. The dissociation constants, stoichiometry and enthalpy change of AtACBP interactions with various acyl-CoA esters were determined using ITC. Strong binding of recombinant (r) AtACBP6 with long-chain acyl-CoA (C16- to C18-CoA) esters was observed with dissociation constants in the nanomolar range. However, the affinity of rAtACBP4 and rAtACBP5 to these acyl-CoA esters was much weaker (dissociation constants in the micromolar range), suggesting that they interact with acyl-CoA esters differently from rAtACBP6. When transgenic Arabidopsis expressing AtACBP6pro::GUS was generated, strong GUS (β-glucuronidase) expression in cotyledonary-staged embryos and seedlings prompted us to measure the acyl-CoA contents of the acbp6 mutant. This mutant accumulated higher levels of C18:1-CoA and C18:1- and C18:2-CoAs in cotyledonary-staged embryos and seedlings, respectively, in comparison with the wild type. The acbp4acbp5acbp6 mutant showed the lightest seed weight and highest sensitivity to abscisic acid during germination, suggesting their physiological functions in seeds.     

Arabidopsis acyl-CoA-binding protein ACBP6 localizes in the phloem and affects jasmonate composition

Arabidopsis thaliana ACYL-COA-BINDING PROTEIN6 (AtACBP6) encodes a cytosolic 10-kDa AtACBP. It confers freezing tolerance in transgenic Arabidopsis, possibly by its interaction with lipids as indicated by the binding of acyl-CoA esters and phosphatidylcholine to recombinant AtACBP6. Herein, transgenic Arabidopsis transformed with an AtACBP6 promoter-driven β-glucuronidase (GUS) construct exhibited strong GUS activity in the vascular tissues. Immunoelectron microscopy using anti-AtACBP6 antibodies showed AtACBP6 localization in the phloem especially in the companion cells and sieve elements. Also, the presence of gold grains in the plasmodesmata indicated its potential role in systemic trafficking. The AtACBP6 protein, but not its mRNA, was found in phloem exudate of wild-type Arabidopsis. Fatty acid profiling using gas chromatography-mass spectrometry revealed an increase in the jasmonic acid (JA) precursor, 12-oxo-cis,cis-10,15-phytodienoic acid (cis-OPDA), and a reduction in JA and/or its derivatives in acbp6 phloem exudates in comparison to the wild type. Quantitative real-time PCR showed down-regulation of COMATOSE (CTS) in acbp6 rosettes suggesting that AtACBP6 affects CTS function. AtACBP6 appeared to affect the content of JA and/or its derivatives in the sieve tubes, which is consistent with its role in pathogen-defense and in its wound-inducibility of AtACBP6pro::GUS. Taken together, our results suggest the involvement of AtACBP6 in JA-biosynthesis in Arabidopsis phloem tissues.     

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.     

固醇调节元件结合蛋白与脂质代谢

固醇调节元件结合蛋白(sterol regulatory element-binding proteins,SREBPs)是脊椎动物细胞脂质稳态的转录调节物,可直接激活多个参与胆固醇、脂肪酸、甘油三酯、磷脂合成和摄取,以及辅助因子NADPH等基因的表达,从而调控胆固醇及脂肪酸等脂类的代谢过程。本文综述了SREBPs转运和活化的过程,以及调节细胞脂质稳态功能的分子机制,并探讨了其在脂代谢紊乱相关疾病发生中的重要作用。     

Novel type aquaporin SIPs are mainly localized to the ER membrane and show cell-specific expression in Arabidopsis thaliana

We investigated the fourth subgroup of Arabidopsis aquaporin, small and basic intrinsic proteins (SIPs). When they were expressed in yeast, SIP1;1 and SIP1;2, but not SIP2;1, gave water-channel activity. The transient expression of SIPs linked with green fluorescent protein in Arabidopsis cells and the subcellular fractionation of the tissue homogenate showed their ER localization. The SIP proteins were detected in all of the tissues, except for dry seeds. Histochemical analysis of promoter-beta-glucuronidase fusions revealed the cell-specific expression of SIPs. SIP1;1 and SIP1;2 may function as water channels in the ER, while SIP2;1 might act as an ER channel for other small molecules or ions.     

Galectin-3 interacts with membrane lipids and penetrates the lipid bilayer

The precise mechanism by which galectin-3 and other cytosolic proteins that lack signal peptides are secreted is yet to be elucidated. In the present analyses, we determined that galectin-3, a beta-galactoside binding protein, can interact directly with membrane lipids in solid phase binding assays. More interestingly, we determined by spectrophotometric methods that it can spontaneously penetrate the lipid bilayer of liposomes in either direction. These findings suggest that galectin-3 on its own has the capacity to traverse the lipid bilayer. Whereas the situation is rather simplified in liposomes, the interaction of galectin-3 with the plasma membrane may involve cholesterol-rich membrane domains where galectin-3 can be concentrated and form multimers or interact covalently with other proteins.     

Effects of membrane composition and lipid structure on the photopolymerization of lipid diacetylenes in bilayer membranes

Molecules analogous to biological and synthetic lipids have been prepared with conjugated diacetylene moieties in the long alkyl chain. These lipid diacetylenes form bilayer structures when suspended in aqueous buffers. Ultraviolet light (254 nm) exposure initiates the polymerization of the diacetylenes in the lipid bilayer to give a fully conjugated, highly colored product. The reaction is topotactic, and its efficiency depends on the correct alignment of the monomeric units. Thus, the lipid diacetylenes are photopolymerizable if the hydrocarbon chains are in a regular lattice found at temperatures below the lipid transition temperature; polymerization is inhibited above this transition. The photopolymerization of a diacetylenic glycerophosphocholine in lipid bilayer membranes was observed in two-component mixtures with a nonpolymerizable lipid, either dioleoylphosphatidylcholine or distearoylphosphatidylcholine. The photochemical and thermochemical characteristics suggest that the diacetylenic glycerophosphocholine exists largely in separate domains in the mixed bilayers. Lipid diacetylenes analogous to a dialkyldimethylammonium salt and to a dialkyl phosphate have a plane of symmetry, which suggests that both chains penetrate equally into the bilayer. The photopolymerization of these symmetrical synthetic species is more than 10³-times more efficient than that of the diacetylenic glycerophosphocholine. These differences are interpretable in terms of the expected conformational preference of the lipid molecules.     

Neuregulin-1 proteins in rat brain and transfected cells are localized to lipid rafts

Neuregulin-1 proteins and their receptors, which are members of the ErbB subfamily of receptor tyrosine kinases, play essential roles in the development of the nervous system and heart. Most neuregulin-1 isoforms are synthesized as transmembrane proproteins that are proteolytically processed to yield an N-terminal fragment containing the bioactive EGF-like domain. In this study we investigated whether neuregulins are found in lipid rafts, membrane microdomains hypothesized to have important roles in signal transduction, protein trafficking, and proteolytic processing. We found that 45% of a 140-kDa neuregulin protein in rat brain synaptosomal plasma membrane fractions was insoluble in 1% Triton X-100. Flotation gradient analysis demonstrated the presence of the brain 140 kDa neuregulin protein in low-density fractions enriched in PSD-95, a known lipid raft protein. In transfected cells expressing the neuregulin I-beta 1a or the III-beta 1a isoform, most of the neuregulin proprotein was insoluble in 1% Triton X-100, and neuregulin proproteins and C-terminal fragments were detected in lipid raft fractions. In contrast, the III-beta 1a N-terminal fragment was detected only in the detergent-soluble fraction. These results suggest that localization of neuregulins to lipid rafts may play a role in neuregulin signaling within the nervous system.     

Cancer cachexia modifies the zonal distribution of lipid metabolism-related proteins in rat liver

Cancer cachexia is a syndrome that causes profound metabolic disruption. Lipid metabolism in the liver is markedly affected. We investigated the effect of cachexia upon liver-acinus lipid-metabolism zonation in Walker 245 carcinosarcoma-bearing rats (TB). The expression of protein (by Western blotting) and mRNA (by semi-quantitative polymerase chain reaction) of the enzymes of the carnitine palmitoyltransferase system (CPT I and CPT II) and of liver fatty-acid-binding protein (L-FABP) was studied. Although no changes were found for these parameters, the maximal activities (by radioassay) of CPT I and II were reduced (P<0.05) in TB compared with controls. CPT II activity in the perivenous (PV) region was higher in TB compared with controls. The distribution of CPT II and L-FABP (by immunohistochemistry) within the acinus was modified by cachexia: whereas CPT II positivity was restricted to the PV zone, L-FABP labelling shifted from periportal (control) to perivenous (TB) zone. These changes in metabolic zonation, together with decreased CPT II activity, may contribute to the aggravation of cachexia. The authors thank FAPESP for financial support (00/03761-2) and for the scholarship awarded to M. Kazantzis.     

Tgl4p and Tgl5p, two triacylglycerol lipases of the yeast Saccharomyces cerevisiae are localized to lipid particles

Triacylglycerol (TAG) lipases are required for mobilization of TAG stored in lipid particles. Recently, Tgl3p was identified as a major TAG lipase of the yeast Saccharomyces cerevisiae (Athenstaedt, K., and Daum, G. (2003) J. Biol. Chem. 278, 23317-23323). Here, we report the identification of Tgl4p and Tgl5p as additional TAG lipases of the yeast. Both polypeptides, encoded by open reading frames YKR089c/TGL4 and YOR081c/TGL5, share 30 and 26% homology, respectively, to Tgl3p. Cell fractionation experiments and microscopic inspection of strains bearing Tgl4p-GFP and Tgl5p-GFP hybrids demonstrated that both proteins are localized to lipid particles similar to Tgl3p. A 1.7-fold increased amount of TAG enriched in myristic and palmitic acids and the reduced mobilization rate of TAG from tgl4Delta in the presence of the fatty acid synthesis inhibitor cerulenin demonstrated the lipolytic function of Tgl4p in vivo. In contrast, neither the total amount of TAG nor the TAG mobilization rate after addition of cerulenin was affected in tgl5Delta cells. However, the enrichment of C26:0 esterified to TAG of tgl5Delta, an additional increase of TAG in the tgl4Deltatgl5Delta double deletion mutant compared with tgl4Delta, and the impairment of TAG mobilization in the tgl4Deltatgl5Delta strain in the presence of cerulenin suggested that also Tgl5p functions as a TAG lipase in vivo. Most importantly, the purified His(6)-tagged Tgl4p and Tgl5p hybrids exhibited TAG lipase activity demonstrating their function in vitro. In summary, our data obtained by biochemical, molecular, and cell biological analyses unambiguously identified Tgl4p and Tgl5p as novel TAG lipases of yeast lipid particles with certain enzymatic specificities.     

The tale of tail-anchored proteins: coming from the cytosol and looking for a membrane

A group of integral membrane proteins, known as C-tail anchored, is defined by the presence of a cytosolic NH2-terminal domain that is anchored to the phospholipid bilayer by a single segment of hydrophobic amino acids close to the COOH terminus. The mode of insertion into membranes of these proteins, many of which play key roles in fundamental intracellular processes, is obligatorily posttranslational, is highly specific, and may be subject to regulatory processes that modulate the protein's function. Although recent work has elucidated structural features in the tail region that determine selection of the correct target membrane, the molecular machinery involved in interpreting this information, and in modulating tail-anchored protein localization, has not been identified yet.     

BAR domains, amphipathic helices and membrane-anchored proteins use the same mechanism to sense membrane curvature

The internal membranes of eukaryotic cells are all twists and bends characterized by high curvature. During recent years it has become clear that specific proteins sustain these curvatures while others simply recognize membrane shape and use it as “molecular information” to organize cellular processes in space and time. Here we discuss this new important recognition process termed membrane curvature sensing (MCS). First, we review a new fluorescence-based experimental method that allows characterization of MCS using measurements on single vesicles and compare it to sensing assays that use bulk/ensemble liposome samples of different mean diameter. Next, we describe two different MCS protein motifs (amphipathic helices and BAR domains) and suggest that in both cases curvature sensitive membrane binding results from asymmetric insertion of hydrophobic amino acids in the lipid membrane. This mechanism can be extended to include the insertion of alkyl chain in the lipid membrane and consequently palmitoylated and myristoylated proteins are predicted to display similar curvature sensitive binding. Surprisingly, in all the aforementioned cases, MCS is predominantly mediated by a higher density of binding sites on curved membranes instead of higher affinity as assumed so far. Finally, we integrate these new insights into the debate about which motifs are involved in sensing versus induction of membrane curvature and what role MCS proteins may play in biology.     

Antibodies to lipopolysaccharide and outer membrane proteins of Salmonella enteritidis PT4 are not involved in protection from experimental infection

BALB/c and Schofield mice were inoculated with formalin-killed bacteria prepared from strains of Salmonella enteritidis belonging to phage type (PT) 4 and carrying a 38 MDa plasmid and expressing long-chain lipopolysaccharide, or strains without a 38 MDa plasmid or lacking the ability to express lipopolysaccharide. Vaccinated mice were challenged with viable bacteria belonging to a virulent strain of S. enteritidis (PT4). Mice surviving this viable challenge were examined for a humoral antibody response to membrane antigens of S. enteritidis (PT4) that might relate to the possession of a given virulence property. BALB/c mice immunized with any of the test antigens were found to be immune to S. enteritidis (PT4), and this immunity was protective. Serum antibodies, of the IgG class, were detected to OmpA and a minor outer membrane protein (OMP) of 31 kDa. Schofield mice also raised IgG antibodies to these outer membrane proteins; however, non-immunized mice of this strain were resistant to infection. The virulence of S. enteritidis (PT4) was also tested using mice belonging to strains B10D2 (new), Biozzi (high), Biozzi (low), C3HeJ, B10ITYR and C57/L.     

Larval nutrition affects lipid storage and growth, but not protein or carbohydrate storage in newly eclosed adults of the grasshopper Schistocerca americana

Nitrogen availability from dietary protein can have profound effects on the physiology and evolutionary ecology of insect herbivores. While many studies consider the effects of nutrition on consumption and gross body composition of protein and other important nutrients, few consider partitioning to storage for future use. I used chemically defined artificial diets to quantitatively manipulate the amount of dietary carbohydrates and proteins available to growing larvae of the grasshopper Schistocerca americana to determine how larval nutrient availability affects growth and all three classes of stored nutrients (proteins, lipids, and carbohydrates) carried over from larval feeding into adulthood. Larvae on poor diets increased consumption, but could not compensate for diet quality, eclosing small and containing no significant nutrient stores at adulthood. Individuals fed intermediate to high nutrient content diets as larvae were significantly larger and contained a significantly greater proportion of lipid stores at adult eclosion, but not protein or carbohydrate stores than individuals fed low nutrient content diets. This suggests that larvally derived lipid stores may be more important to adult fitness than carbohydrate or protein stores. This result is contrary to previous studies performed on the role of larval nutrition and allocation to protein stores, and this difference is likely due to variation in the relative availability of protein in adult diets across species.     

The relationship between plasma membrane lipid composition and physical-chemical properties. I. Fluorescence polarization studies of fatty acid-altered EL4 tumor cell membranes

EL4 cells were cultured with exogenous fatty acids under conditions that resulted in their incorporation into membrane phospholipids. The behavior of the fluorescent lipid probes diphenylhexatriene and perylene was monitored in intact EL4 cells and in isolated EL4 plasma membranes. In whole cells substituted with unsaturated fatty acids, there was always a marked decrease in the $\text{P}$ value of both probes compared to the $\text{P}$ value of the probes in unsubstituted cells. In whole cells substituted with saturated fatty acids, on the other hand, $\text{P}$ values for both probes were unchanged compared to unsubstituted cells. In plasma membrane isolated from EL4 cells, no difference in $\text{P}$ values for either probe was observed among membranes from unsubstituted, saturated fatty acid substituted or unsaturated fatty acid substituted cells, even when the degree of fatty acid substitution was quite substantial. Most of the fluorescent signal for both probes in whole cells appeared to come from cytoplasmic lipid droplets. The value of techniques such as fluorescent polarization for monitoring physical properties of membranes (such as ‘fluidity’) is discussed.     

Cadmium leads to stimulated expression of the lipid transfer protein genes in barley: implications for the involvement of lipid transfer proteins in wax assembly

In order to investigate the nature of genes expressed in leaf epidermal cells of higher plants, we have identified the nucleotide sequence of a cDNA designated ltp 7a2b encoding a novel nonspecific lipid transfer protein of barley (Hordeum vulgare L. cv. Gerbel). The cDNA of 755 basepairs contains an open reading frame of 366 nucleotides coding for a 12.3-kDa polypeptide. The first 29 amino acids constitute the putative signal peptide, characteristic for targeting to the secretory pathway. Analysis of mRNA levels by Northern blotting indicated that ltp 7a2b is preferentially expressed in the leaf epidermis. Levels of mRNA decreased during ageing of leaf tissue. Expression of ltp 7a2b was stimulated by a factor of 2–3 when the seedlings were grown in the presence of cadmium (10–1600 μM). Concomitantly, the primary leaves of Cd-exposed seedlings contained elevated levels of abscisic acid and a thicker wax layer of the cuticle. At 100 μM Cd in the hydroponic medium, the wax cover was increased by 50%. The increase in abscisic acid content, ltp 7a2b mRNA and wax coverage was either not seen, or seen much less, in Ni- and Zn-stressed seedlings. The data add circumstantial evidence to the recently proposed hypothesis that nonspecific lipid transfer proteins function in transfer of cutin and/or wax monomers from the site of synthesis in the cell to the cuticle. Received: 1 April 1996 / Accepted: 11 February 1997