Identification of the human mitochondrial linoleoyl-coenzyme A monolysocardiolipin acyltransferase (MLCL AT-1).
Here we report the identification of a previously uncharacterized human protein as the human monolysocardiolipin acyltransferase-1 (MLCL AT-1). Pig liver mitochondria were treated with n-butyl alcohol followed by Q-Sepharose chromatography, preparative gel electrophoresis, cytidine diphosphate-1,2-diacyl-sn-glycerol-Sepharose chromatography, and finally monolysocardiolipin-adriamycin-agarose affinity chromatography. Elution with either monolysocardiolipin or linoleoyl coenzyme A revealed a major band at 74 kDa with high specific activity (2,300 pmol/min/mg) for the acylation of monolysocardiolipin to cardiolipin using [1-(14)C]linoleoyl coenzyme A as substrate. Matrix-assisted laser desorption ionization time-of-flight-mass spectrometry analysis followed by search of the Mascot protein data base revealed peptide matches consistent with a 59-kDa protein identified as unknown human protein (GenBank(TM) protein accession number AAX93141; nucleotide accession number AC011742.3). The purified human recombinant MLCL AT-1 protein utilized linoleoyl coenzyme A > oleoyl coenzyme A > palmitoyl coenzyme A for the specific acylation of monolysocardiolipin to cardiolipin. Expression of MLCL AT-1 in HeLa cells increased mitochondrial monolysocardiolipin acyltransferase activity and [1-(14)C]linoleic acid incorporated into cardiolipin, whereas RNA interference knockdown of MLCL AT-1 in HeLa cells resulted in reduction in enzyme activity and [1-(14)C]linoleic acid incorporated into cardiolipin. In contrast, expression of MLCL AT-1 in HeLa cells did not alter [1-(14)C]oleic or [1-(14)C]palmitate incorporation into cardiolipin indicating in vivo specificity for the remodeling of cardiolipin with linoleate. Finally, expression of MLCL AT-1 in Barth syndrome lymphoblasts, which exhibit cardiolipin levels 20% that of normal lymphoblasts, increased mitochondrial monolysocardiolipin acyltransferase activity, [1-(14)C]linoleic acid incorporation into cardiolipin, cardiolipin mass, and succinate dehydrogenase (mitochondrial complex II) activity compared with mock-transfected Barth syndrome lymphoblasts. The results identify MLCL AT-1 as a human mitochondrial monolysocardiolipin acyltransferase involved in the remodeling of cardiolipin.
Taylor
William A
WA
Department of Pharmacology and Therapeutics, University of Manitoba, Winnipeg, Manitoba R3E 0T6, Canada.
Hatch
Grant M
GM
D000217:Acyltransferases / Q000302:isolation & purification*; D000818:Animals; D002308:Cardiolipins / Q000378:metabolism; D009202:Cardiomyopathies; D019295:Computational Biology; D006367:HeLa Cells; D006801:Humans; D019787:Linoleic Acid / Q000378:metabolism; D008214:Lymphocytes / Q000378:metabolism / Q000473:pathology; D008930:Mitochondria, Liver / Q000201:enzymology*; D024101:Mitochondrial Proteins / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D019301:Oleic Acid / Q000378:metabolism; D019308:Palmitic Acid / Q000378:metabolism; D013552:Swine
D002308:Cardiolipins; D024101:Mitochondrial Proteins; D019301:Oleic Acid; D019308:Palmitic Acid; D019787:Linoleic Acid; D000217:Acyltransferases; C401969:monolysocardiolipin acyltransferase