Functional Studies of Yeast-Expressed Human Heart Muscle Carnitine Palmitoyltransferase I

Long-chain fatty acids are the primary source of energy production in the heart. Carnitine palmitoyltrans- ferase I (CPT-I) catalyzes the first reaction in the transport of long-chain fatty acids from the cytoplasm to the mitochondrion, a rate-limiting step in β-oxidation. In this study, we report the functional expression of the human heart/skeletal muscle isoform of CPT-I (M-CPT-I) in the yeastPichia pastoris. Screening of a human heart cDNA library with cDNA fragments encoding the rat heart M-CPT-I resulted in the isolation of a single full-length human heart M-CPT-I cDNA clone. The clone has an open reading frame of 2316 bp with a 5′ untranslated region of 38 bp and a 256-bp 3′ untranslated region with the poly(A)+addition sequence AATAAA. The predicted protein has 772 amino acids and a molecular mass of 88 kDa. Northern blot analysis of mRNAs from different human tissues using the human M-CPT-I cDNA as a probe revealed an abundant transcript of ∼3.1 kb that was only present in human heart and skeletal muscle tissue. Expression of the human M-CPT-I cDNA inP. pastoris,a yeast with no endogenous CPT activity, produced an 80-kDa protein that was located in the mitochondria. Isolated mitochondria from the M-CPT-I expression strain exhibited a malonyl-coenzyme A (CoA)-sensitive CPT activity that was detergent labile. TheI50for malonyl-CoA inhibition of the yeast-expressed M-CPT-I was 69 nm, and theKms for carnitine and palmitoyl-CoA were 666 and 42 μm, respectively. TheI50for malonyl-CoA inhibition of the heart enzyme is 30 times lower than that of the yeast-expressed liver CPT-I, and theKmfor carnitine is more than 20 times higher than that of the liver CPT-I. This is the first report of the expression of a heart CPT-I in a system devoid of endogenous CPT activity and the functional characterization of a human heart M-CPT-I in the absence of the liver isoform and CPT-II.