An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

Higgins, Malcolm J. P.; Kekwick, R. G. O.

doi:10.1042/bj1340295

Cited by 21 publications

(11 citation statements)

References 49 publications

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“…1). It may also be a substrate for acetoacetyl-CoA synthesis and thus may participate in sterol biosynthesis (33,43). We therefore asked whether inhibition of de novo fatty acid or sterol synthesis results in an ultrastructural phenotype similar to that of acc1-7-1 or accumulation of poly(A) ϩ RNA.…”

Section: Resultsmentioning

confidence: 99%

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A Yeast Acetyl Coenzyme A Carboxylase Mutant Links Very-Long-Chain Fatty Acid Synthesis to the Structure and Function of the Nuclear Membrane-Pore Complex

Schneiter

Hitomi

Ivessa

et al. 1996

Molecular and Cellular Biology

183

185

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The conditional mRNA transport mutant of Saccharomyces cerevisiae, acc1-7-1 (mtr7-1), displays a unique alteration of the nuclear envelope. Unlike nucleoporin mutants and other RNA transport mutants, the intermembrane space expands, protuberances extend from the inner membrane into the intermembrane space, and vesicles accumulate in the intermembrane space. MTR7 is the same gene as ACC1, encoding acetyl coenzyme A (CoA) carboxylase (Acc1p), the rate-limiting enzyme of de novo fatty acid synthesis. Genetic and biochemical analyses of fatty acid synthesis mutants and acc1-7-1 indicate that the continued synthesis of malonyl-CoA, the enzymatic product of acetyl-CoA carboxylase, is required for an essential pathway which is independent from de novo synthesis of fatty acids. We provide evidence that synthesis of very-long-chain fatty acids (C 26 atoms) is inhibited in acc1-7-1, suggesting that very-long-chain fatty acid synthesis is required to maintain a functional nuclear envelope.The regulation of synthesis and transport of phospholipids has been characterized at length, but the signals which determine the phospholipid and fatty acid composition of particular membranes and membrane-specific lipid requirements are not well understood (7,62,81,83). The present investigation, which is the outgrowth of analysis of a yeast (Saccharomyces cerevisiae) mutant which accumulates poly(A) ϩ RNA in the nucleus at the restrictive temperature (41), documents a critical relationship between fatty acid chain length and the integrity of the nuclear membrane and nuclear pore complex (NPC).The biosynthesis of very-long-chain fatty acids requires four enzyme systems: acetyl coenzyme A (CoA) carboxylase, fatty acid synthase, fatty acid desaturase, and the fatty acyl chain elongation system (Fig. 1). The rate-limiting step of the de novo synthesis of fatty acids is under control of the first of these, acetyl-CoA carboxylase (Acc1p; EC 6.4.1.2). This biotinylated enzyme catalyzes the ATP-dependent carboxylation of acetyl-CoA to yield malonyl-CoA, which serves as the twocarbon-unit donor for the subsequent synthesis of long-chain fatty acids by the fatty acid synthase complex. The chain length of newly synthesized fatty acids appears to depend on the concentration of malonyl-CoA rather than on the activity of the fatty acid synthase complex (35, 43a, 82). Acc1p thus regulates both the overall rate of de novo synthesis and chainlength distribution of long-chain fatty acids. It is perhaps for this reason that its activity is subject to complex regulation (48). Yeast Acc1p/Fas3p has a subunit molecular mass of 250 kDa, is active as a tetramer, and is subject to short-term regulation by phosphorylation (2, 60, 90). Its transcription is positively regulated by Ino2p and Ino4p and negatively regulated by Opi1p; i.e., it is under the general control of phospholipid synthesis (14, 31).Many yeast long-chain fatty acid auxotrophs have been isolated (72). Sixty-one are alleles of acc1 (55, 67), while others bear mutations in fatty acid synthase (19,...

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Section: Resultsmentioning

confidence: 99%

“…It may be a substrate for polyketide synthesis (12,76) or for sterol biosynthesis (33,43), or it may serve as a two-carbon unit in acyl-chain elongation (26). We investigated a possible requirement for malonyl-CoA in these pathways by determining whether their inhibition results in an acc1-7-1 phenotype.…”

Section: Discussionmentioning

confidence: 99%

A Yeast Acetyl Coenzyme A Carboxylase Mutant Links Very-Long-Chain Fatty Acid Synthesis to the Structure and Function of the Nuclear Membrane-Pore Complex

Schneiter

Hitomi

Ivessa

et al. 1996

Molecular and Cellular Biology

183

185

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“…The excess CoA was removed by reaction with DTNB (10). Separation of the resulting mixture of (2-14C) -malonyl-CoA and the mixed disulfide of DTNBCoA was accomplished by column chromatography using DEAE cellulose (22). The malonylCoA fractions were pooled and lyophilized before desalting on a Sephadex G-i 0 column (2.5 x 90 cm) equilibrated in 3 mM-HC1.…”

Section: Synthesis and Purification Of Substratesmentioning

confidence: 99%

“…(I -t 4C)-acetylCoA was then prepared from the purified CoA and (1-14C)-acetic anhydride (46), and chromatographed on a DEAE cellulose column (22). (2-14C)-malonyl-CoA was synthesized (43), the reaction being followed by testing aliquots on cellulose TLC plates developed in l-butanol: acetic acid:water 5:2:3 (v/v/v).…”

Section: Synthesis and Purification Of Substratesmentioning

confidence: 99%

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

Høj

Mikkelsen

1982

Carlsberg Res. Commun.

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An acyl carrier protein (ACP) dependent fatty acid synthetase (fas) from barley chloroplast stroma was purified five-fold by ammonium sulphate precipitation and gel filtration on Sephacryl S-300. The {~-ketoacyl-ACP reductase, ~-ketoacyl-ACP synthetase, acetyl-CoA:ACP transacylase and malonyl-CoA:ACP transacylase activities were resolved on the Sephacryl S-300 column with apparent molecular weights of respectively 125, 92, 82 and 41 kilodalton. The fas activity exhibited an apparent molecular weight of 87 kilodalton resulting from the overlapping portions of the component activities. A fifth component of the active fas, ACP, was separated completely from the other four individual enzyme activities by the ammonium sulphate precipitation. When the fas purified by gel filtration was applied to a Ma-trex Gel Blue B column, the component activities were separated into two groups. A bound fraction contained all the malonyl-CoA:ACP transacylase whereas the [~-ketoacyl synthetase activity was exclusively present in the non-bound fraction. Neither the bound nor the non-bound fraction showed any fas activity alone, but complete reconstitution of fas activity was obtained when both protein fractio~ were combined. The barley chloroplast fas is therefore not a multifunctional protein but consists of at least five separable components. Characterization with respect to cofactor requirements was also performed. Variation of certain cofactor concentrations markedly altered the pattern of fatty acid synthesis.Abbreviations: ACP = acyl carrier protein; DTNB = 5",5-dithiobis(2-nitro benzoic acid); DT1 ~ = 1,4-dithiotreitol; Hepes = N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; HPLC = high pressure liquid chromatography; kD = kilodalton; Tricine = N-(Tris(hydroxy-methyl)-methyl)-glycin; SDS = sodium dodecyl sulphate.

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“…Malonyl-CoA was prepared according to Eggerer and Lynen [7]. [3-'4C]Malonyl-CoA was prepared enzymatically with yeast acetylCoA-carboxylase and purified according to Higgins and Kekwick [8].…”

Section: Substratesmentioning

confidence: 99%

Localization of three non‐thiol binding sites on polypeptide chain β yeast fatty acid synthetase

1977

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An investigation into the role of malonyl-coenzyme A in isoprenoid biosynthesis

Cited by 21 publications

References 49 publications

A Yeast Acetyl Coenzyme A Carboxylase Mutant Links Very-Long-Chain Fatty Acid Synthesis to the Structure and Function of the Nuclear Membrane-Pore Complex

A Yeast Acetyl Coenzyme A Carboxylase Mutant Links Very-Long-Chain Fatty Acid Synthesis to the Structure and Function of the Nuclear Membrane-Pore Complex

Partial separation of individual enzyme activities of an ACP-dependent fatty acid synthetase from barley chloroplasts

Localization of three non‐thiol binding sites on polypeptide chain β yeast fatty acid synthetase

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