Carnitine short-chain acyltransferase in pea mitochondria

Wood, C.; Jalil, Muhammad; Ariffin, Azis; Yong, Bob C. S.; Thomas, David

doi:10.1007/bf00397711

Cited by 21 publications

(9 citation statements)

References 14 publications

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“…Overall, our observations imply that ACL is near-limiting in generating cytosolic acetyl-CoA and that other mechanisms (Wood et al, 1983;Burgess and Thomas, 1986;Masterson et al, 1990) for generating cytosolic acetyl-CoA cannot substitute for ACL-derived acetyl-CoA.…”

Section: Acl-derived Acetyl-coa Is Nonredundant In Cytosolic Acetyl-cmentioning

confidence: 65%

Reverse Genetic Characterization of Cytosolic Acetyl-CoA Generation by ATP-Citrate Lyase in Arabidopsis

2005

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Acetyl-CoA provides organisms with the chemical flexibility to biosynthesize a plethora of natural products that constitute much of the structural and functional diversity in nature. Recent studies have characterized a novel ATP-citrate lyase (ACL) in the cytosol of Arabidopsis thaliana. In this study, we report the use of antisense RNA technology to generate a series of Arabidopsis lines with a range of ACL activity. Plants with even moderately reduced ACL activity have a complex, bonsai phenotype, with miniaturized organs, smaller cells, aberrant plastid morphology, reduced cuticular wax deposition, and hyperaccumulation of starch, anthocyanin, and stress-related mRNAs in vegetative tissue. The degree of this phenotype correlates with the level of reduction in ACL activity. These data indicate that ACL is required for normal growth and development and that no other source of acetyl-CoA can compensate for ACL-derived acetyl-CoA. Exogenous malonate, which feeds into the carboxylation pathway of acetyl-CoA metabolism, chemically complements the morphological and chemical alterations associated with reduced ACL expression, indicating that the observed metabolic alterations are related to the carboxylation pathway of cytosolic acetyl-CoA metabolism. The observations that limiting the expression of the cytosolic enzyme ACL reduces the accumulation of cytosolic acetyl-CoA-derived metabolites and that these deficiencies can be alleviated by exogenous malonate indicate that ACL is a nonredundant source of cytosolic acetyl-CoA.

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Section: Acl-derived Acetyl-coa Is Nonredundant In Cytosolic Acetyl-cmentioning

confidence: 65%

Reverse Genetic Characterization of Cytosolic Acetyl-CoA Generation by ATP-Citrate Lyase in Arabidopsis

2005

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“…other acyl-CoAs across these organellar membranes (Wood et al, 1983;Burgess and Thomas, 1986;Masterson et al, 1990;Schwabedissen-Gerbling and Gerhardt, 1995;Masterson and Wood, 2000). Most of these studies were undertaken with the goal of determining whether the mitochondrial acetyl-CoA pool can be translocated to the chloroplast as acetylcarnitine, rather than to establish the export of acetylCoA from mitochondria into the cytosol.…”

Section: Discussionmentioning

confidence: 99%

Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis,

Fatland¹,

Ke²,

Anderson³

et al. 2002

Plant Physiology

196

194

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Acetyl-coenzyme A (CoA) is used in the cytosol of plant cells for the synthesis of a diverse set of phytochemicals including waxes, isoprenoids, stilbenes, and flavonoids. The source of cytosolic acetyl-CoA is unclear. We identified two Arabidopsis cDNAs that encode proteins similar to the amino and carboxy portions of human ATP-citrate lyase (ACL). Coexpression of these cDNAs in yeast (Saccharomyces cerevisiae) confers ACL activity, indicating that both the Arabidopsis genes are required for ACL activity. Arabidopsis ACL is a heteromeric enzyme composed of two distinct subunits, ACLA (45 kD) and ACLB (65 kD). The holoprotein has a molecular mass of 500 kD, which corresponds to a heterooctomer with an A 4 B 4 configuration. ACL activity and the ACLA and ACLB polypeptides are located in the cytosol, consistent with the lack of targeting peptides in the ACLA and ACLB sequences. In the Arabidopsis genome, three genes encode for the ACLA subunit (ACLA-1, At1g10670; ACLA-2, At1g60810; and ACLA-3, At1g09430), and two genes encode the ACLB subunit (ACLB-1, At3g06650 and ACLB-2, At5g49460). The ACLA and ACLB mRNAs accumulate in coordinated spatial and temporal patterns during plant development. This complex accumulation pattern is consistent with the predicted physiological needs for cytosolic acetyl-CoA, and is closely coordinated with the accumulation pattern of cytosolic acetyl-CoA carboxylase, an enzyme using cytosolic acetyl-CoA as a substrate. Taken together, these results indicate that ACL, encoded by the ACLA and ACLB genes of Arabidopsis, generates cytosolic acetyl-CoA. The heteromeric organization of this enzyme is common to green plants (including Chlorophyceae, Marchantimorpha, Bryopsida, Pinaceae, monocotyledons, and eudicots), species of fungi, Glaucophytes, Chlamydomonas, and prokaryotes. In contrast, all known animal ACL enzymes have a homomeric structure, indicating that a evolutionary fusion of the ACLA and ACLB genes probably occurred early in the evolutionary history of this kingdom.Acetyl-coenzyme A (CoA) is an intermediate metabolite that is juxtaposed between catabolic and anabolic processes. As the entry point for the tricarboxylic acid (TCA) cycle, acetyl-CoA can be considered the gateway in the oxidation of carbon derived from the catabolism of fatty acids, certain amino acids (e.g. Leu, Ile, Lys, and Trp), and carbohydrates. Furthermore, acetyl-CoA is the intermediate precursor for the biosynthesis of a wide variety of phytochemicals. Because membranes are impermeable to CoA derivatives, it can be inferred that acetyl-CoA is generated in at least four distinct metabolic pools representing the four subcellular compartments where acetyl-CoA metabolism occurs: plastids, mitochondria, peroxisomes, and the cytosol (Fig. 1). Therefore, plants should have distinct acetyl-CoA-generating systems in mitochondria (for the TCA cycle), in plastids (for de novo fatty acid biosynthesis), in peroxisomes (the product of ␤-oxidation of fatty acids), and in the cytosol (for the biosynthesis of isoprenoids, flavo...

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“…It is suprising that Roughan et al (1993) found no CAT activity in homogenates of pea shoots and spinach and amaranthus leaves as it is fairly well established that mitochondria at least possess CAT activity Wood et al 1983;Burgess & Thomas 1986;Gerbling & Gerhardt 1988;Budde et al 1991) and this mitochondrial CAT should have been detected.…”

Section: Discussionmentioning

confidence: 99%

“…CAT has been detected in pea (Pisum sativum L.) mitochondria (Thomas & McNeil 1976;Wood et al 1983;Burgess & Thomas 1986;Gerbling & Gerhardt 1988;Budde et al 1991). Burgess & Thomas (1986) demonstrated that this CAT was located on the matrix side of the mitochondrial inner membrane, where it was able to alleviate acetyl CoA pressure and pyruvate dehydrogenase inhibition during inhibition of the Krebs cycle.…”

Section: Introductionmentioning

confidence: 99%

Pea chloroplast carnitine acetyltransferase

Masterson

Wood

2000

Proc. R. Soc. Lond. B

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The purpose of this study was to resolve the controversy as to whether or not chloroplasts possess the enzyme carnitine acetyltransferase (CAT) and whether the activity of this enzyme is su¤cient to support previously reported rates of fatty acid synthesis from acetylcarnitine. CAT catalyses the freely reversible reaction: carnitine + short-chain acylCoA 6 short-chain acylcarnitine + CoASH. CAT activity was detected in the chloroplasts of Pisum sativum L. With membrane-impermeable acetyl CoA as a substrate, activity was only detected in ruptured chloroplasts and not with intact chloroplasts, indicating that the enzyme was located on the stromal side of the envelope. In crude preparations, CAT could only be detected using a sensitive radioenzymatic assay due to competing reactions from other enzymes using acetyl CoA and large amounts of ultraviolet-absorbing materials. After partial puri¢cation of the enzyme, CAT was detected in both the forward and reverse directions using spectrophotometric assays. Rates of 100 nmol of product formed per minute per milligram of protein were obtained, which is su¤-cient to support reported fatty acid synthesis rates from acetylcarnitine. Chloroplastic CAT showed optimal activity at pH 8.5 and had a high substrate speci¢city, handling C2^C4 acyl CoAs only. We believe that CAT has been satisfactorily demonstrated in pea chloroplasts.

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Carnitine short-chain acyltransferase in pea mitochondria

Abstract: Carnitine acetyltransferase was shown to be present in pea-cotyledon mitochondria. Acetyl-carnitine may well be exported, without excessive energy loss, from mitochondrial matrix sites to extra-mitochondrial sites.

Cited by 21 publications

References 14 publications

Reverse Genetic Characterization of Cytosolic Acetyl-CoA Generation by ATP-Citrate Lyase in Arabidopsis

Reverse Genetic Characterization of Cytosolic Acetyl-CoA Generation by ATP-Citrate Lyase in Arabidopsis

Molecular Characterization of a Heteromeric ATP-Citrate Lyase That Generates Cytosolic Acetyl-Coenzyme A in Arabidopsis,

Pea chloroplast carnitine acetyltransferase

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