Immunolocalization of long-chain acyl-CoAs in plant cells

Long chain acyl-Coenzyme A esters (acyl-CoAs) are key substrates in many enzymic reactions of lipid metabolism. Due to their amphiphilic nature, the membrane localization of these molecules cannot be established by subcellular membrane fractionation and usual biochemical studies. We have developed another approach based on ultrastructural immunogold cytochemistry. To preserve the acyl-CoA membrane content, the plant material was freeze substituted and cryoembedded after short aldehyde fixation followed by quick freezing. Using Arabidopsis thaliana root cells and specific antibodies raised against acyl-CoAs, we show that acyl-CoAs are mainly localized in endoplasmic reticulum membranes.Our results demonstrate the value of cryo-methods for the accurate localization of labile metabolites in plant cells.     

Quantitative analysis of short-chain acyl-coenzymeAs in plant tissues by LC–MS–MS electrospray ionization method

Because acyl-CoAs play major roles in numerous anabolic and catabolic pathways, the quantitative determination of these metabolites in biological tissues is paramount to understanding the regulation of these metabolic processes. Here, we report a method for the analysis of a collection of short-chain acyl-CoAs (<6 carbon chain length) from plant extracts. Identification of each individual acyl-CoA was conducted by monitoring specific mass-fragmentation ions that are derived from common chemical moieties of all Coenzyme A (CoA) derivatives, namely the adenosine triphosphate nucleotide, pantothenate and acylated cysteamine. This method is robust and quick, enabling the quantitative analysis of up to 12 different acyl-CoAs in plant metabolite extracts with minimal post-extraction processing, using a 30 min chromatographic run-time.     

Reversible high affinity inhibition of phosphofructokinase-1 by acyl-CoA: a mechanism integrating glycolytic flux with lipid metabolism

The enzyme phosphofructokinase-1 (PFK-1) catalyzes the first committed step of glycolysis and is regulated by a complex array of allosteric effectors that integrate glycolytic flux with cellular bioenergetics. Here, we demonstrate the direct, potent, and reversible inhibition of purified rabbit muscle PFK-1 by low micromolar concentrations of long chain fatty acyl-CoAs (apparent Ki～1 μM). In sharp contrast, short chain acyl-CoAs, palmitoylcarnitine, and palmitic acid in the presence of CoASH were without effect. Remarkably, MgAMP and MgADP but not MgATP protected PFK-1 against inhibition by palmitoyl-CoA indicating that acyl-CoAs regulate PFK-1 activity in concert with cellular high energy phosphate status. Furthermore, incubation of PFK-1 with [1-(14)C]palmitoyl-CoA resulted in robust acylation of the enzyme that was reversible by incubation with acyl-protein thioesterase-1 (APT1). Importantly, APT1 reversed palmitoyl-CoA-mediated inhibition of PFK-1 activity. Mass spectrometric analyses of palmitoylated PFK-1 revealed four sites of acylation, including Cys-114, Cys-170, Cys-351, and Cys-577. PFK-1 in both skeletal muscle extracts and in purified form was inhibited by S-hexadecyl-CoA, a nonhydrolyzable palmitoyl-CoA analog, demonstrating that covalent acylation of PFK-1 was not required for inhibition. Tryptic footprinting suggested that S-hexadecyl-CoA induced a conformational change in PFK-1. Both palmitoyl-CoA and S-hexadecyl-CoA increased the association of PFK-1 with Ca2+/calmodulin, which attenuated the binding of palmitoylated PFK-1 to membrane vesicles. Collectively, these results demonstrate that fatty acyl-CoA modulates phosphofructokinase activity through both covalent and noncovalent interactions to regulate glycolytic flux and enzyme membrane localization via the branch point metabolic node that mediates lipid flux through anabolic and catabolic pathways.     

Interaction of amphiphilic substrates (acyl-CoAs) and their metabolites (free fatty acids) with microsomes from mouse sciatic nerves

We have measured the partition of stearoyl-CoA and oleoyl-CoA between an aqueous phase and the microsomes from mouse sciatic nerves. A method of microultracentrifugation was used which allowed us to study separately the aqueous phase and the biological membranes. We observed that the partition is dependent upon the amount of acyl-CoAs and membrane proteins but seems to be independent of time. A theoretical analysis of these data allowed interpretation of the binding and release in terms of acyl-CoA surface density in the vesicles. We have also analyzed the fate of the membrane-bound acyl-CoAs. We show that, whereas the apparent partition does not seem to vary, the hydrolysis of the membrane-bound acyl-CoAs followed by the release of free fatty acids from the membrane leads to a modification of the partition of acyl-CoAs between the membrane and the aqueous phase. We propose that there is a constant partition of the aliphatic chains (acyl-CoAs + free fatty acids).     

Carboxyl-methylation of prenylated calmodulin CaM53 is required for efficient plasma membrane targeting of the protein

Prenylation is necessary for association of the petunia calmodulin CaM53 with the plasma membrane. To determine whether post-prenylation processing of the protein was also required for plasma membrane targeting, we studied the subcellular localization of a GFP-labelled CaM53 reporter in yeast and plant cells. Blocking of carboxyl-methylation of prenylated proteins either by a specific inhibitor or in mutant yeast cells resulted in localization of green fluorescence to what appears to be the endomembrane system, in contrast with the plasma membrane localization observed in control cells. We show that a prenyl-cysteine methyltransferase (PCM) activity that carboxyl-methylates prenylated CaM53 also exists in plant cells, and that it is required for efficient plasma membrane targeting. We also report an Arabidopsis gene with homology to PCM and demonstrate that it encodes a protein with PCM activity that localizes to the endomembrane system of plant cells, similar to prenylated but unmethylated CaM53. Together, our data suggest that, following prenylation, CaM53 is probably associated with the endomembrane system, where a PCM activity methylates the prenylated protein prior to targeting it to its final destination in the plasma membrane.     

Secretory carrier membrane proteins

Secretory carrier membrane proteins (SCAMPs) are a family of integral membrane proteins that play roles in mediating exocytosis in animal cells. However, relatively little is known about the subcellular localization, trafficking, and function of SCAMPs in plants. Several recent studies in plant cells indicate that plant SCAMPs share many similarities with their mammalian homologs although there are differences. In this review, we will first summarize and compare animal and plant SCAMPs in terms of their subcellular localization, trafficking, and possible functions. We will then present a phylogenetic analysis of plant and animal SCAMPs. Finally, we will present expression analysis on selective Arabidopsis SCAMPs in the hope of pointing to directions for functional characterization of plant SCAMPs in the future.     

Quantitation of fatty acyl-coenzyme As in mammalian cells by liquid chromatography-electrospray ionization tandem mass spectrometry

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.     

Regulation of mitochondrial carbamoyl-phosphate synthetase 1 activity by active site fatty acylation

In addition to its role in reversible membrane localization of signal-transducing proteins, protein fatty acylation could play a role in the regulation of mitochondrial metabolism. Previous studies have shown that several acylated proteins exist in mitochondria isolated from COS-7 cells and rat liver. Here, a prominent fatty-acylated 165-kDa protein from rat liver mitochondria was identified as carbamoyl-phosphate synthetase 1 (CPS 1). Covalently attached palmitate was linked to CPS 1 via a thioester bond resulting in an inhibition of CPS 1 activity at physiological concentrations of palmitoyl-CoA. This inhibition corresponds to irreversible inactivation of CPS 1 and occurred in a time- and concentration-dependent manner. Fatty acylation of CPS 1 was prevented by preincubation with N-ethylmaleimide and 5'-p-fluorosulfonylbenzoyladenosine, an ATP analog that reacts with CPS 1 active site cysteine residues. Our results suggest that fatty acylation of CPS 1 is specific for long-chain fatty acyl-CoA and very likely occurs on at least one of the essential cysteine residues inhibiting the catalytic activity of CPS 1. Inhibition of CPS 1 by long-chain fatty acyl-CoAs could reduce amino acid degradation and urea secretion, thereby contributing to nitrogen sparing during starvation.     

The yeast osmosensitive mutant fps1Delta transformed by the cauliflower BobTIP1;1 aquaporin withstand a hypo-osmotic shock

Osmoregulation plays an important role in cellular responses to osmotic stress in plants and in yeast. Aquaporins contribute to osmotic adjustment by facilitating transport of water or solutes across membranes. The tonoplastic water channel BobTIP1;1 (original name BobTIP26-1) genes are upregulated during dessication stress in cauliflower meristematic tissue. To investigate the physiological importance of BobTIP1;1, we expressed it in a Saccharomyces cerevisiae osmosensitive mutant fps1Delta. We showed that the defect in the yeast glycerol plasma membrane transporter is complemented by a plant cDNA encoding the aquaporin BobTIP1;1 which is localized in the vacuolar membrane of the complemented yeast cells. To our knowledge, this is the first example of a plant aquaporin for which localization in the vacuolar membrane of yeast cells is related to an osmoresistant phenotype under hypo-osmotic shock.     

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.     

Profiling hydroxycinnamoyl-coenzyme A thioesters: unlocking the back door of phenylpropanoid metabolism

In plants, 20 to 30% of photosynthetically fixed carbon is directed toward lignin and other phenylpropanoid compounds for which hydroxycinnamoyl-coenzyme A (CoA) esters are key intermediates. CoA thioesters, ubiquitous metabolites found in all living cells (often at trace levels), have traditionally been challenging to measure. Here we report a hydrophilic interaction liquid chromatography (HILIC) method, coupled with tandem mass spectrometry (MS/MS), that allows simultaneous sensitive quantification of previously undetectable hydroxycinnamoyl-CoA esters and an extended range of acyl-CoAs from plant tissues. This method provides rapid liquid chromatography (LC) analysis (10 min/sample) and the ability for qualitative assessment of acyl-CoAs by MS/MS precursor ion scanning.     

Modulation by nutrients and drugs of liver acyl-CoAs analyzed by mass spectrometry

The profile of liver acyl-CoAs induced by dietary fats of variable compositions or by xenobiotic hypolipidemic amphipathic carboxylates was evaluated in vivo using a novel electrospray ionization tandem mass spectrometry methodology of high resolution, sensitivity, and reliability. The composition of liver fatty acyl-CoAs was found to reflect the composition of dietary fat. Treatment with hypolipidemic carboxylates resulted in liver dominant abundance of their respective acyl-CoAs accompanied by an increase in liver fatty acyl-CoAs. Cellular effects exerted by dietary fatty acids and/or xenobiotic carboxylic drugs may be transduced in vivo by their respective acyl-CoAs.     

Subcellular localization of the Hpa RxLR effector repertoire identifies a tonoplast-associated protein HaRxL17 that confers enhanced plant susceptibility

Filamentous phytopathogens form sophisticated intracellular feeding structures called haustoria in plant cells. Pathogen effectors are likely to play a role in the establishment and maintenance of haustoria in addition to their better-characterized role in suppressing plant defence. However, the specific mechanisms by which these effectors promote virulence remain unclear. To address this question, we examined changes in subcellular architecture using live-cell imaging during the compatible interaction between the oomycete Hyaloperonospora arabidopsidis (Hpa) and its host Arabidopsis. We monitored host-cell restructuring of subcellular compartments within plant mesophyll cells during haustoria ontogenesis. Live-cell imaging highlighted rearrangements in plant cell membranes upon infection, in particular to the tonoplast, which was located close to the extra-haustorial membrane surrounding the haustorium. We also investigated the subcellular localization patterns of Hpa RxLR effector candidates (HaRxLs) in planta. We identified two major classes of HaRxL effector based on localization: nuclear-localized effectors and membrane-localized effectors. Further, we identified a single effector, HaRxL17, that associated with the tonoplast in uninfected cells and with membranes around haustoria, probably the extra-haustorial membrane, in infected cells. Functional analysis of selected effector candidates in planta revealed that HaRxL17 enhances plant susceptibility. The roles of subcellular changes and effector localization, with specific reference to the potential role of HaRxL17 in plant cell membrane trafficking, are discussed with respect to Hpa virulence.     

Human brain acyl-CoA hydrolase isoforms encoded by a single gene

Acyl-CoA hydrolases are a group of enzymes that catalyze the hydrolysis of acyl-CoA thioesters to free fatty acids and CoA-SH. The human brain acyl-CoA hydrolase (BACH) gene comprises 13 exons, generating several isoforms through the alternative use of exons. Four first exons (1a-1d) can be used, and three patterns of splicing occur at exon X located between exons 7 and 8 that contains an internal 3(')-splice acceptor site and creates premature stop codons. When examined with green fluorescent protein-fusion constructs expressed in Neuro-2a cells, the nuclear localization signal encoded by exon 9 was functional by itself, whereas the whole structure was cytosolic, suggesting nuclear translocation of the enzyme. This was consistent with dual staining of the cytosol and nucleus in certain neurons by immunohistochemistry using anti-BACH antibody. The mitochondrial targeting signals encoded by exons 1b and 1c were also functional and directed mitochondrial localization of BACH isoforms with the signals. Although BACH mRNA containing the sequence derived from exon 1a, but not exon X, was exclusively expressed in human brain, these results suggest that the human BACH gene can express long-chain acyl-CoA hydrolase activity in multiple intracellular compartments by generating BACH isoforms with differential localization signals to affect various cellular functions that involve acyl-CoAs.     

Sorting signals in the cytosolic tail of membrane proteins involved in the interaction with plant ARF1 and coatomer

In mammals and yeast, a cytosolic dilysine motif is critical for endoplasmic reticulum (ER) localization of type I membrane proteins. Retrograde transport of type I membrane proteins containing dilysine motifs at their cytoplasmic carboxy (C)-terminal tail involves the interaction of these motifs with the COPI coat. The C-terminal dilysine motif has also been shown to confer ER localization to type I membrane proteins in plant cells. Using in vitro binding assays, we have analyzed sorting motifs in the cytosolic tail of membrane proteins, which may be involved in the interaction with components of the COPI coat in plant cells. We show that a dilysine motif in the -3,-4 position (relative to the cytosolic C-terminus) recruits in a very specific manner all the subunits of the plant coatomer complex. Lysines cannot be replaced by arginines or histidines to bind plant coatomer. A diphenylalanine motif in the -7,-8 position, which by itself has a low ability to bind plant coatomer, shows a clear cooperativity with the dilysine motif. Both dilysine and diphenylalanine motifs are present in the cytosolic tail of several proteins of the p24 family of putative cargo receptors, which has several members in plant cells. The cytosolic tail of a plant p24 protein is shown to recruit not only coatomer but also ADP ribosylation factor 1 (ARF1), a process which depends on both dilysine and diphenylalanine motifs. ARF1 binding increases twofold upon treatment with brefeldin A (BFA) and is completely abolished upon treatment with GTPgammaS, suggesting that ARF1 can only interact with the cytosolic tail of p24 proteins in its GDP-bound form.     

Identification of a calmodulin-regulated soybean Ca(2+)-ATPase (SCA1) that is located in the plasma membrane

Ca(2)+-ATPases are key regulators of Ca(2+) ion efflux in all eukaryotes. Animal cells have two distinct families of Ca(2+) pumps, with calmodulin-stimulated pumps (type IIB pumps) found exclusively at the plasma membrane. In plants, no equivalent type IIB pump located at the plasma membrane has been identified at the molecular level, although related isoforms have been identified in non-plasma membrane locations. Here, we identify a plant cDNA, designated SCA1 (for soybean Ca(2+)-ATPase 1), that encodes Ca(2+)-ATPase and is located at the plasma membrane. The plasma membrane localization was determined by sucrose gradient and aqueous two-phase membrane fractionations and was confirmed by the localization of SCA1p tagged with a green fluorescent protein. The Ca(2+)-ATPase activity of the SCA1p was increased approximately sixfold by calmodulin (K(1/2) approximately 10 nM). Two calmodulin binding sequences were identified in the N-terminal domain. An N-terminal truncation mutant that deletes sequence through the two calmodulin binding sites was able to complement a yeast mutant (K616) that was deficient in two endogenous Ca(2+) pumps. Our results indicate that SCA1p is structurally distinct from the plasma membrane-localized Ca(2+) pump in animal cells, belonging instead to a novel family of plant type IIB pumps found in multiple subcellular locations. In plant cells from soybean, expression of this plasma membrane pump was highly and rapidly induced by salt (NaCl) stress and a fungal elicitor but not by osmotic stress.     

Localization of the Sodium-Taurocholate cotransporting polypeptide in membrane rafts and modulation of its activity by cholesterol in vitro

BACKGROUND: The relevance of discrete localization of hepatobiliary transporters in specific membrane microdomains is not well known. AIM: To determine whether the Na+/taurocholate cotransporting polypeptide (Ntcp), the main hepatic sinusoidal bile salt transporter, is localized in specific membrane microdomains. METHODS: Presence of Ntcp in membrane rafts obtained from mouse liver was studied by immunoblotting and immunofluorescence. HEK-293 cells stably transfected with rat Ntcp were used for in vitro studies. Expression, localization and function of Ntcp in these cells were assessed by immunoblotting, immunofluorescence and biotinylation studies and Na+ -dependent taurocholate uptake assays, respectively. The effect of cholesterol depletion/repletion assays on Ntcp function was also investigated. RESULTS: Ntcp localized primarily to membrane rafts in in vivo studies and localized partially in membrane rafts in transfected HEK-293 cells. In these cells, membrane cholesterol depletion resulted in a shift of Ntcp localization into non-membrane rafts, which correlated with a 2.5-fold increase in taurocholate transport. Cholesterol repletion shifted back part of Ntcp into membrane rafts, and normalized taurocholate transport to values similar to control cells. CONCLUSION: Ntcp localizes in membrane rafts and its localization and function are regulated by membrane cholesterol content. This may serve as a novel regulatory mechanism of bile salt transport in liver.     

Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1

Sucrose (Suc) synthase (SUS) cleaves Suc to form UDP glucose and fructose, and exists in soluble and membrane-associated forms, with the latter proposed to channel UDP glucose to the cellulose-synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However, the structural features responsible for membrane localization and the mechanisms regulating its dual intracellular localization are unknown. The maize (Zea mays) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (1) partial membrane localization when expressed in Escherichia coli, and (2) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and alanine substitutions) and in vitro microsome-binding assays with the SUS1 protein to define intrinsic membrane-binding regions and potential regulatory factors that could be provided by cellular microenvironment. The results suggest that two regions of SUS1 contribute to membrane affinity: (1) the amino-terminal noncatalytic domain, and (2) a region with sequence similarity to the C-terminal pleckstrin homology domain of human pleckstrin. Alanine substitutions within the pleckstrin homology-like domain of SUS1 reduced membrane association in E. coli and with plant microsomes in vitro without reducing enzymatic activity. Microsomal association of wild-type SUS1 displayed cooperativity with SUS1 protein concentration and was stimulated by both lowering the pH and adding Suc. These studies offer insight into the molecular level regulation of SUS1 localization and its participation in carbon partitioning in plants. Moreover, transgenics with active SUS mutants altered in membrane affinity may be of technological utility.     

Fatty acyl-CoA as an endogenous activator of UDP-glucuronosyltransferases

The acyl-CoA-dependent modulation of hepatic microsomal UDP-glucuronosyltransferase (UGT) function in rats was studied. Oleoyl- and palmitoyl-CoAs inhibited UGT activity toward 4-methylumbelliferone in the presence of Brij 58. However, acyl-CoAs enhanced UGT activity in untreated microsomes. A maximum activation of about 8-fold over the control was observed at 15 microM oleoyl-CoA, whereas 50 microM or more oleoyl-CoA had an inhibitory effect on UGT function. Medium- and long-chain acyl-CoAs also exhibited similar effects. On the basis of resistance to tryptic digestion of UGTs, oleoyl-CoA at 15 microM has no ability to change the permeability of the endoplasmic reticulum (ER) membrane, although perturbation of the membrane occurred with 50 microM oleoyl-CoA. N-Ethylmaleimide and 5,5'-dithiobis(2-nitrobenzoic acid) abolished the oleoyl-CoA (15 microM)-dependent activation of microsomal UGT. These results suggest that: (1) acyl-CoAs play a role as an endogenous activator of UGTs, and (2) a sulfhydryl group is required for the activation of UGT by physiological concentrations of acyl-CoAs.