Cloning, Expression, Characterization, and Interaction of Two Components of a Human Mitochondrial Fatty Acid Synthase

Abstract

The possibility that human cells contain, in addition to the cytosolic type I fatty acid synthase complex, a mitochondrial type II malonyl-CoA-dependent system for the biosynthesis of fatty acids has been examined by cloning, expressing, and characterizing two putative components. Candidate coding sequences for a malonylCoA:acyl carrier protein transacylase (malonyltransferase) and its acyl carrier protein substrate, identified by BLAST searches of the human sequence data base, were located on nuclear chromosomes 22 and 16, respectively. The encoded proteins localized exclusively in mitochondria only when the putative N-terminal mitochondrial targeting sequences were present as revealed by confocal microscopy of HeLa cells infected with appropriate green fluorescent protein fusion constructs. The mature, processed forms of the mitochondrial proteins were expressed in Sf9 cells and purified, the acyl carrier protein was converted to the holoform in vitro using purified human phosphopantetheinyltransferase, and the functional interaction of the two proteins was studied. Compared with the dual specificity malonyl/ acetyltransferase component of the cytosolic type I fatty acid synthase, the type II mitochondrial counterpart exhibits a relatively narrow substrate specificity for both the acyl donor and acyl carrier protein acceptor. Thus, it forms a covalent acyl-enzyme complex only when incubated with malonyl-CoA and transfers exclusively malonyl moieties to the mitochondrial holoacyl carrier protein. The type II acyl carrier protein from Bacillus subtilis, but not the acyl carrier protein derived from the human cytosolic type I fatty acid synthase, can also function as an acceptor for the mitochondrial transferase. These data provide compelling evidence that human mitochondria contain a malonylCoA/acyl carrier protein-dependent fatty acid synthase system, distinct from the type I cytosolic fatty acid synthase, that resembles the type II system present in prokaryotes and plastids. The final products of this system, yet to be identified, may play an important role in mitochondrial function.The existence of a mitochondrial system for the biosynthesis of fatty acids in animals was first reported 40 years ago and postulated to function by a reversal of the process of fatty acid ␤-oxidation (1-3). Indeed one laboratory reported that short and medium chain-length fatty acids could be produced using the partially purified mitochondrial ␤-oxidation enzymes, ␤-ketoacyl thiolase, ␤-hydroxyacyl-CoA dehydrogenase, enoyl-CoA hydratase, and enoyl-CoA reductase (4), and another found that inhibitory antibodies to pig heart 3-ketoacyl-CoA thiolase inhibited, in a parallel fashion, the fatty acid elongation system of pig heart mitochondria (5). The picture was complicated by reports that liver and heart mitochondria have two distinct fatty acid-synthesizing systems, one acetyl-CoA-dependent, possibly involving some of the ␤-oxidation enzymes, the other malonyl-CoA-dependent and apparently resembling the cytosolic FAS 1 system. Most ...

Mitochondrial MTHFD2L Is a Dual Redox Cofactor-specific Methylenetetrahydrofolate Dehydrogenase/Methenyltetrahydrofolate Cyclohydrolase Expressed in Both Adult and Embryonic Tissues

“…In the present study, we overcame these problems by using several strategies. To increase the yield of recombinant protein, we replaced the mitochondrial targeting sequence of the rat MTHFD2L with an N-terminal His tag for localization in the yeast cytosol (31). Despite the loss of its targeting sequence, the protein remained associated with cellular membranes; so we included sodium carbonate in the extraction buffer to release MTHFD2L from membranes (32).…”

Mammalian ACSF3 Protein Is a Malonyl-CoA Synthetase That Supplies the Chain Extender Units for Mitochondrial Fatty Acid Synthesis

“…The mitochondrial systems are composed of an acyl carrier protein (ACP) 2 and malonyltransferase (which together generate the malonyl-ACP substrate used for chain extension by ␤-ketoacyl synthase) and a trio of ␤-carbon-processing enzymes (␤-ketoacyl reductase, dehydrase, and enoyl reductase) that completely saturate the acyl chain prior to the following round of chain extension. All of these enzymes have been characterized in mammalian mitochondria (1)(2)(3)(4)(5), and recent evidence indicates that one of the major functions of the pathway is to generate the octanoyl precursor required for formation of the lipoyl moieties that are essential for post-translational modification of several mitochondrial proteins (6,7). The source of malonyl-CoA as the substrate for a mitochondrial fatty acid synthase system is unknown (8).…”

“…To evaluate the effect of ACSF3 knockdown on mitochondrial function, we assessed the ability of mitochondria to utilize [2][3][4][5][6][7][8][9][10][11][12][13][14] C]malonate for fatty acid synthesis. The presence in these mitochondrial preparations of substantial thioesterase activity against malonyl-CoA precluded estimation of the amount of this metabolite formed (7).…”

“…Formation of acetyl-ACP is blocked by cerulenin (7), an inhibitor of ␤-ketoacyl synthase. There is no other known pathway for the formation of acetyl-ACP because the mitochondrial malonyltransferase shuttles exclusively malonyl moieties between CoA and ACP thiols (5,24). Thus, the total amount of radiolabeled malonyl-ACP and acetyl-ACP is representative of the malonyl-ACP moieties derived from [2-14 C]malonate that were not utilized for fatty acid synthesis.…”

Cloning, Expression, and Characterization of the Human Mitochondrial β-Ketoacyl Synthase

“…The presence of a similar mitochondrial FAS system in animals has been suspected for some time, based initially on the discovery of an ACP-like protein in animal mitochondria (8,9). However, only recently have other components of a putative mitochondrial FAS been identified, cloned, and characterized; they include the human ACP and malonyl transferase (10) and enoyl reductase (11). In continuation of the search for other components, we have now identified and characterized a single candidate for a type II human mitochondrial ␤-ketoacyl synthase, the critical enzyme required for catalysis of the chain-elongating condensation reaction.…”