The N-terminal Domain of Rat Liver Carnitine Palmitoyltransferase 1 Mediates Import into the Outer Mitochondrial Membrane and Is Essential for Activity and Malonyl-CoA Sensitivity

Abstract

The rat liver carnitine palmitoyltransferase 1 (L-CPT1), an integral outer mitochondrial membrane (OMM) protein, is the key regulatory enzyme of fatty acid oxidation and is inhibited by malonyl-CoA. In vitro import of L-CPT1 into the OMM requires the presence of mitochondrial receptors and is stimulated by ATP but is membrane potential-independent. Its N-terminal domain (residues 1-150), which contains two transmembrane segments, possesses all of the information for mitochondrial targeting and OMM insertion. Deletion of this domain abrogates protein targeting, whereas its fusion to non-OMM-related proteins results in their mitochondrial targeting and OMM insertion in a manner similar to L-CPT1. Functional analysis of chimeric CPTs expressed in Saccharomyces cerevisiae shows that this domain also mediates in vivo protein insertion into the OMM. When the malonyl-CoA-insensitive CPT2 was anchored at the OMM either by a specific OMM signal anchor sequence (pOM29) or by the N-terminal domain of L-CPT1, its activity remains insensitive to malonylCoA inhibition. This indicates that malonyl-CoA sensitivity is an intrinsic property of L-CPT1 and that its N-terminal domain cannot confer malonyl-CoA sensitivity to CPT2. Replacement of the N-terminal domain by pOM29 results in a less folded and less active protein, which is also malonyl-CoA-insensitive. Thus, in addition to its role in mitochondrial targeting and OMM insertion, the N-terminal domain of L-CPT1 is essential to maintain an optimal conformation for both catalytic function and malonyl-CoA sensitivity.

Molecular characterization of a gilthead sea bream (Sparus aurata) muscle tissue cDNA for carnitine palmitoyltransferase 1B (CPT1B)

“…Thus, the residues of the amino-terminal region established to be important for malonyl-CoA sensitivity in the liver (CPT1A) and muscle (CPT1B) isoforms of mammalian CPT1 (Shi et al, 1999, Shi et al, 2000 are conserved in the sea bream protein. Furthermore, the N-terminal domain (residues 1-150) of mammalian CPT1 has been implicated in the insertion of the protein in the outer mitochondrial membrane (Cohen et al, 1998). The high identity between the sea bream and mammalian sequences in this region suggests that the elements responsible for membrane insertion are also conserved in the sea bream CPT1.…”

Carnitine palmitoyltransferase 1C: From cognition to cancer

“…In CPT1A, it provides an intricate molecular mechanism for the modulation of malonyl-CoA sensitivity. Key residues within the N-terminal regulatory domain act as positive or negative determinants of the sensitivity of CPT1A to this metabolite [8][9][10][11][12][13][14][15].…”

Inhibition of hepatic carnitine palmitoyl-transferase I (CPT IA) by valproyl-CoA as a possible mechanism of valproate-induced steatosis

“…The kinetic characteristics of CPT IA and CPT IB differ in several 4 important aspects. CPT IB is more sensitive to malonyl-CoA [2,[15][16][17] whereas CPT IA has higher affinity for L-carnitine, one of the substrates [16,18,19]. The requirement for carnitine and the sensitivity to malonyl-CoA appear to be inversely related [20].…”

“…The polytopic membrane topology of the protein results in both a short regulatory N-terminal segment and a large catalytic C-terminal segment, both exposed to the cytosolic side of the outer mitochondrial membrane [1,2,15,16,18]. The Nterminal domain, which contains both TM1 and TM2 domains, was shown to be responsible for mitochondrial import and for maintenance of a folded enzymatically active and malonyl-CoAsensitive conformation [19]. Moreover, the nature of the cytosolic N-C (N-and C-terminal domain) interactions determine the degree of malonyl-CoA sensitivity of the liver isoform [2,21].…”