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.
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 malonyl-CoA 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.
Cohen
I
I
Endocrinologie, Métabolisme, et Développement, CNRS-UPR 1524, 9 Rue J. Hetzel, 92190 Meudon, France.
Kohl
C
C
McGarry
J D
JD
Girard
J
J
Prip-Buus
C
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry / Q000378:metabolism*; D017931:DNA Primers; D007425:Intracellular Membranes / Q000201:enzymology*; D008297:Male; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013696:Temperature
D017931:DNA Primers; D011993:Recombinant Fusion Proteins; D008316:Malonyl Coenzyme A; D000255:Adenosine Triphosphate; D002334:Carnitine O-Palmitoyltransferase