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 malonyl-CoA: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 malonyl-CoA/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.
Highlights
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 [1,2,3]
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 function as an acceptor for the mitochondrial transferase
The coding sequence for the putative mitochondrial MT is located on human chromosome 22 (22q13.31), and that for the mitochondrial acyl carrier proteins (ACPs) is located on chromosomes 16 (16p12.3) in the human genome sequence data base
Summary
T/B in primer names indicates sense/antisense primer, respectively, while the numbers 1, 22, 60, and 67 correspond to the residue at which the encoded protein begins. Uppercase letters indicate the primer sequence matches cDNA sequence, while bases in lowercase are not present in cDNA; these were incorporated into the primers to engineer modifications in the amplified fragments. Engineered start/stop codons are in bold letters. Restriction sites or restriction site-specific overhangs are underlined. Restriction sites or restriction site-specific overhangs are underlined. †, the base pair numbers for MT-specific primers are according to expressed sequence tag cDNA IMAGE clone 6052380, while for ACP the numbers are according to IMAGE clone 5287610
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