Association of NMT2 with the acyl-CoA carrier ACBD6 protects the N-myristoyltransferase reaction from palmitoyl-CoA

The covalent attachment of a 14-carbon aliphatic tail on a glycine residue of nascent translated peptide chains is catalyzed in human cells by two N-myristoyltransferase (NMT) enzymes using the rare myristoyl-CoA (C₁₄-CoA) molecule as fatty acid donor. Although, NMT enzymes can only transfer a myristate group, they lack specificity for C₁₄-CoA and can also bind the far more abundant palmitoyl-CoA (C₁₆-CoA) molecule. We determined that the acyl-CoA binding protein, acyl-CoA binding domain (ACBD)6, stimulated the NMT reaction of NMT2. This stimulatory effect required interaction between ACBD6 and NMT2, and was enhanced by binding of ACBD6 to its ligand, C_18:2-CoA. ACBD6 also interacted with the second human NMT enzyme, NMT1. The presence of ACBD6 prevented competition of the NMT reaction by C₁₆-CoA. Mutants of ACBD6 that were either deficient in ligand binding to the N-terminal ACBD or unable to interact with NMT2 did not stimulate activity of NMT2, nor could they protect the enzyme from utilizing the competitor C₁₆-CoA. These results indicate that ACBD6 can locally sequester C₁₆-CoA and prevent its access to the enzyme binding site via interaction with NMT2. Thus, the ligand binding properties of the NMT/ACBD6 complex can explain how the NMT reaction can proceed in the presence of the very abundant competitive substrate, C₁₆-CoA.     

Characterization of an acyl-coenzyme A binding protein predominantly expressed in human primitive progenitor cells

Human acyl-coenzyme A binding domain-containing member 6 (ACBD6) is a modular protein that carries an acyl-CoA binding domain at its N terminus and two ankyrin motifs at its C terminus. ACBD6 binds long-chain acyl-CoAs with a strong preference for unsaturated, C18:1-CoA and C20:4-CoA, over saturated, C16:0-CoA, acyl species. Deletion of the C terminus, which is not conserved among the members of this family, did not affect the binding capacity or the substrate specificity of the protein. ACBD6 is not a ubiquitous protein, and its expression is restricted to tissues and progenitor cells with functions in blood and vessel development. ACBD6 was detected in bone marrow, spleen, placenta, cord blood, circulating CD34+ progenitors, and embryonic-like stem cells derived from placenta. In placenta, the protein was only detected in CD34+ progenitor cells present in blood and in CD31+ endothelial cells surrounding the blood vessels. These cells were also positive for the marker CD133, and they probably constitute hemangiogenic stem cells, precursors of both blood and vessels. We propose that human ACBD6 represents a cellular marker for primitive progenitor cells with functions in hematopoiesis and vascular endothelium development.     

An N-terminal fragment of mouse DGAT1 binds different acyl-CoAs with varying affinity

A histidine-tagged recombinant N-terminal fragment of type-1 mouse liver diacylglycerol acyltransferase (DGAT; EC 2.3.1.20), MmDGAT1_(1-95)His6, was expressed in Escherichia coli, and used to investigate possible acyl-CoA-binding properties. Analysis of the purified fragment by MALDI-TOF mass spectrometry revealed a polypeptide with molecular mass of about 11 kDa which was consistent with the calculated molecular mass based on the deduced amino acid sequence. Lipidex-1000 binding assays indicated that MmDGAT1_(1-95)His₆ interacted with long chain fatty acyl-CoAs similar to observations on DGAT1 from oilseed rape (Brassica napus). Binding, as a function of acyl-CoA concentration, differed for palmitoyl (16:0), stearoyl (18:0), and erucoyl (cisΔ¹³22:1)-CoA. Binding of stearoyl- or erucoyl-CoA to MmDGAT1_(1-95)His₆ as a function of acyl-CoA concentration, however, was sigmoid and displayed positive cooperativity suggesting that MmDGAT1 may be subject to allosteric modulation by acyl-CoAs. An intra-polypeptide segment within the N-terminal region of MmDGAT1 contained remnants of an acyl-CoA-binding signature initially identified in plant DGAT1. The acyl-CoA-binding site in mammalian DGAT1 could represent a potential target for therapeutic interventions for disorders such as type-2 diabetes and obesity.     

Corresponding increase in long-chain acyl-CoA and acylcarnitine after exercise in muscle from VLCAD mice

Long-chain acylcarnitines accumulate in long-chain fatty acid oxidation defects, especially during periods of increased energy demand from fat. To test whether this increase in long-chain acylcarnitines in very long-chain acyl-CoA dehydrogenase (VLCAD(-/-)) knock-out mice correlates with acyl-CoA content, we subjected wild-type (WT) and VLCAD(-/-) mice to forced treadmill running and analyzed muscle long-chain acyl-CoA and acylcarnitine with tandem mass spectrometry (MS/MS) in the same tissues. After exercise, long-chain acyl-CoA displayed a significant increase in muscle from VLCAD(-/-) mice [C16:0-CoA, C18:2-CoA and C18:1-CoA in sedentary VLCAD(-/-): 5.95 +/- 0.33, 4.48 +/- 0.51, and 7.70 +/- 0.30 nmol x g(-1) wet weight, respectively; in exercised VLCAD(-/-): 8.71 +/- 0.42, 9.03 +/- 0.93, and 14.82 +/- 1.20 nmol x g(-1) wet weight, respectively (P < 0.05)]. Increase in acyl-CoA in VLCAD-deficient muscle was paralleled by a significant increase in the corresponding chain length acylcarnitine. Exercise resulted in significant lowering of the free carnitine pool in VLCAD(-/-) muscle. This is the first study demonstrating that acylcarnitines and acyl-CoA directly correlate and concomitantly increase after exercise in VLCAD-deficient muscle.     

Microsomal fatty acyl-CoA transacylation and hydrolysis: fatty acyl-CoA species dependent modulation by liver fatty acyl-CoA binding proteins1

Liver and intestinal cytosol contain abundant levels of long chain fatty acyl-CoA binding proteins such as liver fatty acid binding protein (L-FABP) and acyl-CoA binding protein (ACBP). However, the relative function and specificity of these proteins in microsomal utilization of long chain fatty acyl-CoAs (LCFA-CoAs) for sequential transacylation of glycerol-3-phosphate to form phosphatidic acid is not known. The results showed for the first time that L-FABP and ACBP both stimulated microsomal incorporation of the monounsaturated oleoyl-CoA and polyunsaturated arachidonoyl-CoA 8–10-fold and 2–3-fold, respectively. In contrast, these proteins inhibited microsomal utilization of the saturated palmitoyl-CoA by 69% and 62%, respectively. These similar effects of L-FABP and ACBP on microsomal phosphatidic acid biosynthesis were mediated primarily through the activity of glycerol-3-phosphate acyltransferase (GPAT), the rate limiting step, rather than by protecting the long chain acyl-CoAs from microsomal hydrolase activity. In fact, ACBP but not L-FABP protected long chain fatty acyl-CoAs from microsomal acyl-CoA hydrolase activity in the order: palmitoyl-CoA>oleoyl-CoA>arachidonoyl-CoA. In summary, the data established for the first time a role for both L-FABP and ACBP in microsomal phosphatidic acid biosynthesis. By preferentially stimulating microsomal transacylation of unsaturated long chain fatty acyl-CoAs while concomitantly exerting their differential protection from microsomal acyl-CoA hydrolase, L-FABP and ACBP can uniquely function in modulating the pattern of fatty acids esterified to phosphatidic acid, the de novo precursor of phospholipids and triacylglycerols. This may explain in part the simultaneous presence of these proteins in cell types involved in fatty acid absorption and lipoprotein secretion.     

Carnitine palmitoyltransferase activities: Effects of serum albumin,acyl-CoA binding protein and fatty acid binding protein

The carnitine palmitoyltransferase activity of various subcellular preparations measured with octanoyl-CoA as substrate was markedly increased by bovine serum albumin at low M concentrations of octanoyl-CoA. However, even a large excess (500 M) of this acyl-CoA did not inhibit the activity of the mitochondrial outer carnitine palmitoyltransferase, a carnitine palmitoyltransferase isoform that is particularly sensitive to inhibition by low M concentrations of palmitoyl-CoA. This bovine serum albumin stimulation was independent of the salt activation of the carnitine palmitoyltransferase activity. The effects of acyl-CoA binding protein (ACBP) and the fatty acid binding protein were also examined with palmitoyl-CoA as substrate. The results were in line with the findings of stronger binding of acyl-CoA to ACBP but showed that fatty acid binding protein also binds acyl-CoA esters. Although the effects of these proteins on the outer mitochondrial carnitine palmitoyltransferase activity and its malonyl-CoA inhibition varied with the experimental conditions, they showed that the various carnitine palmitoyltransferase preparations are effectively able to use palmitoyl-CoA bound to ACBP in a near physiological molar ratio of 1:1 as well as that bound to the fatty acid binding protein. It is suggested that the three proteins mentioned above effect the carnitine palmitoyltransferase activities not only by binding of acyl-CoAs, preventing acyl-CoA inhibition, but also by facilitating the removal of the acylcarnitine product from carnitine palmitoyltransferase. These results support the possibility that the acyl-CoA binding ability of acyl-CoA binding protein and of fatty acid binding protein have a role in acyl-CoA metabolismin vivo.Abbreviations ACBP acyl-CoA binding protein - BSA bovine serum albumin - CPT carnitine palmitoyltransferase - CPT₀ malonyl-CoA sensitive CPT of the outer mitochondrial membrane - CPT malonyl-CoA insensitive CPT of the inner mitochondrial membrane - OG octylglucoside - OMV outer membrane vesicles - IMV inner membrane vesicles Affiliated to the Department of Experimental Medicine, University of Montreal     

Chain elongation of fatty acid in brain: a comparison of mitochondrial and microsomal enzyme activities

The activities of mitochondrial and microsomal fatty acid-elongating enzymes have been measured in rat brain during postnatal development and in brains of jimpy, msd, and quaking mice. The microsomal enzyme activity rose from a low in the immature brain to a maximum at 21 days of age and then declined to low levels in the mature brain. The developmental patterns were similar for all acyl-CoAs tested. The maximum activity fell sharply from C₁₆ to C₁₈ and then fell gradually with increase in fatty acid chain length up to C₂₄. The activities for monounsaturated acyl-CoAs were slightly higher than for corresponding saturated esters. The mitochondrial enzyme activity was high in the immature brain and remained virtually unchanged during further brain development. This activity steadily decreased with increasing chain length from C₁₆ to C₂₄. The microsomal enzyme activity was reduced in myelin-deficient mutants compared to their controls. The extent of reduction was most severe for C₂₀- to C₂₄-CoAs followed by C₁₈-CoA and then C₁₆-CoA, for which the activity was reduced only in the jimpy mouse. The activities for C₂₀- to C₂₄-CoAs in jimpy, msd, and quaking mice were 12, 38, and 52% of the control, respectively. The mitochondrial enzyme activity was not affected by these mutations. Fatty acid synthetase activity was similar in the mutant and control mice. These results suggest that the deficiency of long-chain fatty acids in the central nervous system of myelin-deficient mouse mutants is due to reduced synthesis by the microsomal enzyme, which is directly related to myelination. The brain mitochondrial enzyme appears to be unrelated to myelination.     

Changes of free fatty acids and acyl-CoAs in rat brain hippocampal slice with tetraethylammonium-induced long-term potentiation

We investigated the role of acyl-CoAs during induction and maintenance of long-term potentiation in rat brain hippocampus. Changes of acyl-CoA and free fatty acids (FFA) in hippocampus were measured during tetraethylammonium (TEA)-induced LTP. Results indicated that concentrations of acyl-CoAs and FFAs in slices were changed during TEA-induced LTP and 16:0-CoA and 18:0-CoA were increased in the early phase of stimulation, whereas free fatty acids in this phase were rather decreased. The increase of 20:4-CoA was delayed more than saturated acyl-CoAs. To examine the role of acyl-CoA in LTP of evoked transmitter release, we measured the glutamate release from hippocampal slice with the addition of acyl-CoA using glutamate electrode. Acyl-CoA (16:0-, 18:1-, and 20:4-CoA) could enhance glutamate release in hippocampal slice. It is suggested that saturated acyl-CoAs may play a functional role in the early phase of LTP.     

Human Fatty Acid Transport Protein 2a/Very Long Chain Acyl-CoA Synthetase 1 (FATP2a/Acsvl1) Has a Preference in Mediating the Channeling of Exogenous n-3 Fatty Acids into Phosphatidylinositol

The trafficking of fatty acids across the membrane and into downstream metabolic pathways requires their activation to CoA thioesters. Members of the fatty acid transport protein/very long chain acyl-CoA synthetase (FATP/Acsvl) family are emerging as key players in the trafficking of exogenous fatty acids into the cell and in intracellular fatty acid homeostasis. We have expressed two naturally occurring splice variants of human FATP2 (Acsvl1) in yeast and 293T-REx cells and addressed their roles in fatty acid transport, activation, and intracellular trafficking. Although both forms (FATP2a (M_r 70,000) and FATP2b (M_r 65,000 and lacking exon3, which encodes part of the ATP binding site)) were functional in fatty acid import, only FATP2a had acyl-CoA synthetase activity, with an apparent preference toward very long chain fatty acids. To further address the roles of FATP2a or FATP2b in fatty acid uptake and activation, LC-MS/MS was used to separate and quantify different acyl-CoA species (C14–C24) and to monitor the trafficking of different classes of exogenous fatty acids into intracellular acyl-CoA pools in 293T-REx cells expressing either isoform. The use of stable isotopically labeled fatty acids demonstrated FATP2a is involved in the uptake and activation of exogenous fatty acids, with a preference toward n-3 fatty acids (C18:3 and C22:6). Using the same cells expressing FATP2a or FATP2b, electrospray ionization/MS was used to follow the trafficking of stable isotopically labeled n-3 fatty acids into phosphatidylcholine and phosphatidylinositol. The expression of FATP2a resulted in the trafficking of C18:3-CoA and C22:6-CoA into both phosphatidylcholine and phosphatidylinositol but with a distinct preference for phosphatidylinositol. Collectively these data demonstrate FATP2a functions in fatty acid transport and activation and provides specificity toward n-3 fatty acids in which the corresponding n-3 acyl-CoAs are preferentially trafficked into acyl-CoA pools destined for phosphatidylinositol incorporation.     

Plant Acyl-CoA:Lysophosphatidylcholine Acyltransferases (LPCATs) Have Different Specificities in Their Forward and Reverse Reactions

Acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) enzymes have central roles in acyl editing of phosphatidylcholine (PC). Plant LPCAT genes were expressed in yeast and characterized biochemically in microsomal preparations of the cells. Specificities for different acyl-CoAs were similar for seven LPCATs from five different species, including species accumulating hydroxylated acyl groups in their seed oil, with a preference for C₁₈-unsaturated acyl-CoA and low activity with palmitoyl-CoA and ricinoleoyl (12-hydroxyoctadec-9-enoyl)-CoA. We showed that Arabidopsis LPCAT1 and LPCAT2 enzymes catalyzed the acylation and de-acylation of both sn positions of PC, with a preference for the sn-2 position. When acyl specificities of the Arabidopsis LPCATs were measured in the reverse reaction, sn-2-bound oleoyl, linoleoyl, and linolenoyl groups from PC were transferred to acyl-CoA to a similar extent. However, a ricinoleoyl group at the sn-2-position of PC was removed 4–6-fold faster than an oleoyl group in the reverse reaction, despite poor utilization in the forward reaction. The data presented, taken together with earlier published reports on in vivo lipid metabolism, support the hypothesis that plant LPCAT enzymes play an important role in regulating the acyl-CoA composition in plant cells by transferring polyunsaturated and hydroxy fatty acids produced on PC directly to the acyl-CoA pool for further metabolism or catabolism.     

Regulation of sex pheromone biosynthesis in the housefly, Musca domestica: relative contribution of the elongation and reductive steps.

The regulation of production of the sex pheromone (Z)-9-tricosene (Z9-23:Hy) in the housefly, Musca domestica, was studied by examining the chain length specificity of the fatty acyl-CoA elongation reactions and the reductive conversion of fatty acyl-CoAs to alkenes in 1- and 4-day-old male and female houseflies. Microsomal preparations from 4-day-old female insects produced as the predominant alkene Z9-23:Hy when incubated with malonyl-CoA, NADPH, and [9,10-3H2]oleoyl-CoA (18:1-CoA), whereas microsomal preparations from 4-day-old male insects produced predominantly (Z)-9-heptacosene (Z9-27:Hy). These are the major alkenes produced in vivo by Day 4 females and males, respectively. Microsomes prepared from both Day 1 males and Day 1 females produced Z9-27:Hy as the major alkene from labeled 18:1-CoA. This is the major alkene produced in vivo by both sexes at Day 1. An examination of the chain length specificity of the elongation reactions showed that microsomes prepared from Day 4 male insects readily elongated both 18:1-CoA and 15-[15,16-3H2]tetracosenoyl-CoA (24:1-CoA) to 28-carbon moieties, whereas microsomes from Day 4 female insects did not efficiently elongate either substrate beyond 24 carbons. With high substrate concentrations, microsomes prepared from male insects converted 24:1-CoA to Z9-23:Hy more efficiently than did those from females, whereas under lower and presumably more physiological substrate concentrations, microsomes from females had slightly higher activity than did those from males. Taken together, these data show that the regulation of the chain length of the alkenes, and thus sex pheromone production, in the housefly resides predominantly in the elongation reactions and not in the step which converts the fatty acyl-CoA to hydrocarbon.     

Atg37 regulates the assembly of the pexophagic receptor protein complex

Like other selective autophagy pathways, the selective autophagy of peroxisomes, pexophagy, is controlled by receptor protein complexes (RPCs). The pexophagic RPC in Pichia pastoris consists of several proteins: Pex3 and Pex14 ligands in the peroxisomal membrane, Atg30 receptor, Atg11, and Atg17 scaffolds, and the phagophore protein Atg8. Recently, we identified a new component of the pexophagic RPC, Atg37, which is involved in the assembly of this complex. Atg37 is an integral peroxisomal membrane protein (PMP) that binds Pex3 and Atg30, but not Pex14 or Atg8. In the absence of Atg37, the recognition of Pex3 and recruitment of Atg17 by Atg30 are normal. However, the recruitment of Atg11 is severely affected suggesting that the role of Atg37 is to facilitate the Atg30-Atg11 interaction. Palmitoyl-CoA competes with Atg30 for the acyl-CoA binding domain of Atg37 in vitro and might regulate the dynamics of the pexophagic RPC in vivo. The human counterpart of Atg37, ACBD5, also localizes to peroxisomes and is specifically required for pexophagy. Therefore, it is tempting to speculate that ACBD5/ATG37 regulates the assembly of the pexophagic RPC in mammalian cells.     

Substrate inhibition and affinities of the glyoxysomal β-oxidation of sunflower cotyledons

During the glyoxysomal β-oxidation of long-chain acyl-CoAs, short-chain intermediates accumulate transiently (Kleiter and Gerhardt 1998, Planta 206: 125–130). The studies reported here address the underlying factors. The studies concentrated upon the aspects of (i) chain length specificity and (ii) metabolic regulation of the glyoxysomal β-oxidation of sunflower (Helianthus annuus L.) cotyledons. (i) Concentration-rate curves of the β-oxidation of acyl-CoAs of various chain lengths showed that the β-oxidation activity towards long-chain acyl-CoAs was higher than that towards short-chain acyl-CoAs at substrate concentrations <20 μM. At substrate concentrations >20 μM, long-chain acyl-CoAs were β-oxidized more slowly than short-chain acyl-CoAs because the β-oxidation of long-chain acyl-CoAs is subject to substrate inhibition which had already started at 5–10 μM substrate concentration and results from an inhibition of the multifunctional protein (MFP) of the β-oxidation reaction sequence. However, low concentrations of free long-chain acyl-CoAs are rather likely to exist within the glyoxysomes due to the acyl-CoA-binding capacity of proteins. Consequently, the β-oxidation rate towards a parent long-chain acyl-CoA will prevail over that towards the short-chain intermediates. (ii) Low concentrations (≤5 μM) of a long-chain acyl-CoA exerted an inhibitory effect on the β-oxidation rate of butyryl-CoA. Reversibility of the inhibition was observed as well as metabolization of the inhibiting long-chain acyl-CoA. Regarding the activities of the individual β-oxidation enzymes towards their C₄ substrates in the presence of a long-chain acyl-CoA, the MFP activity exhibited strong inhibition. This inhibition appears not to be due to the detergent-like physical properties of long-chain acyl-CoAs. The results of the studies, which are consistent with the observation that short-chain intermediates accumulate transiently during complete degradation of a long-chain acyl-CoA, suggest that the substrate concentration-dependent chain-length specificity of the β-oxidation and a metabolic regulation at the level of MFP are factors determining this transient accumulation. Received: 2 February 1999 / Accepted: 14 April 1999     

Acyl-CoA binding domain containing 3 (ACBD3) recruits the protein phosphatase PPM1L to ER-Golgi membrane contact sites

The metal-dependent protein phosphatase family (PPM) governs a number of signaling pathways. PPM1L, originally identified as a negative regulator of stress-activated protein kinase signaling, was recently shown to be involved in the regulation of ceramide trafficking at ER-Golgi membrane contact sites. Here, we identified acyl-CoA binding domain containing 3 (ACBD3) as an interacting partner of PPM1L. We showed that this association, which recruits PPM1L to ER-Golgi membrane contact sites, is mediated by a GOLD (Golgi dynamics) domain in ACBD3. These results suggested that ACBD3 plays a pivotal role in ceramide transport regulation at the ER-Golgi interface.

Structured summary of protein interactions

ACBD3 and PPM1Lcolocalize by fluorescence microscopy (View interaction)FYCO1physically interacts with PPM1L by pull down (View interaction)SEC14L2physically interacts with PPM1L by pull down (View interaction)ACBD3physically interacts with PPM1L by pull down (View interaction)SEC14L1physically interacts with PPM1L by pull down (View interaction)PPM1Lphysically interacts with ACBD3 by two hybrid (View interaction)     

Long-chain acyl-CoA oxidases of Arabidopsis

Full-length cDNAs coding for two distinct acyl-CoA oxidases were isolated by screening an Arabidopsis cDNA library. The genes for the two acyl-CoA oxidases have been termed AtACX1 and AtACX2. AtACX1 encodes a peptide of 664 amino acids possessing a molecular mass of 74.3 kDa. AtACX2 encodes a peptide of 691 amino acids in length with a molecular mass of 77.5 kDa. Peroxisomal targeting signals were identified in the primary sequences. AtACX1 has a putative PTS1, whereas AtACX2 has a characteristic PTS2. Expression of AtACX1 and AtACX2 in Escherichia coli gave active enzymes for enzymatic and biochemical analysis. AtACX1 was active with both medium-and long-chain saturated fatty acyl-CoAs and showed maximal activity with C14-CoA. Activity with mono-unsaturated acyl-CoAs was slightly higher than with the corresponding saturated acyl-CoA. AtACX2 was active with long-chain acyl-CoAs and showed maximal activity with C18-CoA. AtACX2 activity with mono-unsaturated acyl-CoAs was approximately twice as high as with the corresponding saturated acyl-CoA. Both enzymes have an apparent Km of approximately 5 microM with the preferred substrate. Northern analysis was conducted to determine the expression patterns of AtACX1 and AtACX2 during germination and in various tissues of a mature plant. The two genes showed generally similar expression profiles and steady-state mRNA levels in seedlings and mature tissues, but subtle differences were observed. Enzymatic analyses of plant extracts revealed that AtACX1 and AtACX2 are members of a family that includes acyl-CoA oxidases specific for shorter-chain acyl-CoAs. Through expression of antisense constructs of the individual genes, we were able to decrease long-chain oxidase activity only in antisense AtACX1 plants. Seedlings with long-chain oxidase activity reduced down to 30% of wild-type levels germinated and established normally; however, reduced root growth appeared to be a general feature of antisense AtACX1 plants.     

Influence of phospholipid chain length on verotoxin/globotriaosyl ceramide binding in model membranes: comparison of a supported bilayer film and liposomes

The importance of the surrounding lipid environment on the availability of glycolipid carbohydrate for ligand binding was demonstrated by studying the influence of phosphatidylcholine fatty acid chain length on binding of verotoxins (VT1 and VT2c) to their specific cell surface receptor, globotriaosylceramide (Gb₃) in the presence of auxiliary lipids both in a microtitre plate surface bilayer film and in a liposome membrane model system. In the microtitre assay, both VT1 and VT2c binding to Gb₃ was increased as a function of decreasing PC acyl chain length likely resulting in increased Gb₃ exposure. In the liposome assay VT1 binding was similarly modulated, however the effect on VT2c binding was more complex and did not follow a simple function of increased carbohydrate exposure. Earlier work established that C22:1 and C18:1Gb₃ fatty acid homologues were the preferred Gb₃ receptor isoforms in the microtitre assay for VT1 and VT2c respectively. This selectivity was maintained in C16PC containing liposomes, but in C14PC liposomes, binding to C22:1Gb₃ (but not C18:1Gb₃) was elevated such that this Gb₃ species now became the preferred receptor for both toxins. This change in verotoxin/Gb₃ homologue binding selectivity in the presence of C14PC did not occur in the microtitre bilayer format. These results are consistent with our proposal that these toxins recognize different epitopes on the Gb₃ oligosaccharide. We infer that relative availability of these epitopes for toxin binding in an artificial bilayer is influenced not only by the exposure due to the discrepancy between the fatty acyl chain lengths of Gb₃ and PC, but by the physical mode of presentation of the bilayer structure. Such acyl chain length differences have a more marked effect in a supported bilayer film whereas only the largest discrepancies affect Gb₃ receptor function in liposomes. The basis of phospholipid modulation of glycolipid carbohydrate accessibility for receptor function is likely complex and will involve phase separation, gel/liquid crystalline transition, packing and lateral mobility within the bilayer, suggesting that such parameters should be considered in the assessment of glycolipid receptor function in cells.     

Fatty acyl-CoA elongation in Blatella germanica integumental microsomes

Insect cuticular hydrocarbons are synthesized de novo in integumental tissue through the concerted action of fatty acid synthases (FASs), fatty acyl-CoA elongases, a reductase, and a decarboxylase to produce hydrocarbons and CO2. Elongation of fatty acyl-CoAs to very long chain fatty acids was studied in the integumental microsomes of the German cockroach, Blatella germanica. Incubation of [1-14C]palmitoyl-CoA, malonyl-CoA, and NADPH resulted in the production of 18-CoA with minor amounts of C20, C22, C24, C30, and C32 labeled acyl-CoA moieties. Similar experiments with [1-14C]stearoyl-CoA rendered C20-CoA as the major product, and lesser amounts of C22 and C24-CoAs were also detected. After solubilization of the microsomal FAS, kinetic parameters were determined radiochemically or by measuring NADPH consumption. The reaction velocity was linear for up to 3 min incubation time, and with a protein concentration up to 0.025 microg/microl. The effect of the chain length on the reaction velocity was compared for palmitoyl-CoA, stearoyl-CoA, and eicosanoyl-CoA. The optimal substrate concentration was 10 microM for C16-CoA, between 8 and 12 microM for C18-CoA, and close to 3 microM for C20-CoA. In vivo hydrocarbon biosynthesis was inhibited from 55.5 to 72.5% in the presence of 1 mM trichloroacetic acid, a known inhibitor of elongation reactions.     

Characterization of the Bubblegum acyl-CoA synthetase of Microchloropsis gaditana

The metabolic pathways of glycerolipids are well described in cells containing chloroplasts limited by a two-membrane envelope but not in cells containing plastids limited by four membranes, including heterokonts. Fatty acids (FAs) produced in the plastid, palmitic and palmitoleic acids (16:0 and 16:1), are used in the cytosol for the synthesis of glycerolipids via various routes, requiring multiple acyl-Coenzyme A (CoA) synthetases (ACS). Here, we characterized an ACS of the Bubblegum subfamily in the photosynthetic eukaryote Microchloropsis gaditana, an oleaginous heterokont used for the production of lipids for multiple applications. Genome engineering with TALE-N allowed the generation of MgACSBG point mutations, but no knockout was obtained. Point mutations triggered an overall decrease of 16:1 in lipids, a specific increase of unsaturated 18-carbon acyls in phosphatidylcholine and decrease of 20-carbon acyls in the betaine lipid diacylglyceryl–trimethyl–homoserine. The profile of acyl-CoAs highlighted a decrease in 16:1-CoA and 18:3-CoA. Structural modeling supported that mutations affect accessibility of FA to the MgACSBG reaction site. Expression in yeast defective in acyl-CoA biosynthesis further confirmed that point mutations affect ACSBG activity. Altogether, this study supports a critical role of heterokont MgACSBG in the production of 16:1-CoA and 18:3-CoA. In M. gaditana mutants, the excess saturated and monounsaturated FAs were diverted to triacylglycerol, thus suggesting strategies to improve the oil content in this microalga.

A heterokont Bubblegum acyl-CoA synthetase (ACSBG), or lipidosin, is essential in Microchloropsis (Nannochloropsis) gaditana and thio-esterifies 16:1 and 18:3 fatty acids to Coenzyme A in vivo.     

Eukaryotic protein recruitment into the Chlamydia inclusion: implications for survival and growth

Chlamydia trachomatis (Ct) is an obligate intracellular human pathogen that multiplies within a parasitophorous vacuole called an inclusion. We report that the location of several host-cell proteins present in the cytosol, the nucleus, and membranes was altered during Ct development. The acyl-CoA synthetase enzyme ACSL3 and the soluble acyl-CoA binding protein ACBD6 were mobilized from organelle membranes and the nucleus, respectively, into the lumen of the inclusion. The nuclear protein ZNF23, a pro-apoptosis factor, was also translocated into the inclusion lumen. ZNF23, among other proteins, might be targeted by Ct to inhibit host cell apoptosis, thereby enabling bacterial survival. In contrast, the acyl-CoA:lysophosphatidylcholine acyltransferase LPCAT1, an endoplasmic reticulum membrane protein, was recruited to the inclusion membrane. The coordinated action of ACBD6, ACSL3 and LPCAT1 likely supports remodeling and scavenging of host lipids into bacterial-specific moieties essential to Ct growth. To our knowledge, these are the first identified host proteins known to be intercepted and translocated into the inclusion.     

Engineering and mechanistic studies of the Arabidopsis FAE1 beta-ketoacyl-CoA synthase, FAE1 KCS.

The Arabidopsis FAE1 beta-ketoacyl-CoA synthase (FAE1 KCS) catalyzes the condensation of malonyl-CoA with long-chain acyl-CoAs. Sequence analysis of FAE1 KCS predicted that this condensing enzyme is anchored to a membrane by two adjacent N-terminal membrane-spanning domains. In order to characterize the FAE1 KCS and analyze its mechanism, FAE1 KCS and its mutants were engineered with a His6-tag at their N-terminus, and expressed in Saccharomyces cerevisiae. The membrane-bound enzyme was then solubilized and purified to near homogeneity on a metal affinity column. Wild-type recombinant FAE1 KCS was active with several acyl-CoA substrates, with highest activity towards saturated and monounsaturated C16 and C18. In the absence of an acyl-CoA substrate, FAE1 KCS was unable to carry out decarboxylation of [3-(14)C]malonyl-CoA, indicating that it requires binding of the acyl-CoA for decarboxylation activity. Site-directed mutagenesis was carried out on the FAE1 KCS to assess if this condensing enzyme was mechanistically related to the well characterized soluble condensing enzymes of fatty acid and flavonoid syntheses. A C223A mutant enzyme lacking the acylation site was unable to carry out decarboxylation of malonyl-CoA even when 18:1-CoA was present. Mutational analyses of the conserved Asn424 and His391 residues indicated the importance of these residues for FAE1-KCS activity. The results presented here provide the initial analysis of the reaction mechanism for a membrane-bound condensing enzyme from any source and provide evidence for a mechanism similar to the soluble condensing enzymes.