The liver isoform of carnitine palmitoyltransferase 1 is not targeted to the endoplasmic reticulum

Liver microsomal fractions contain a malonyl-CoA-inhibitable carnitine acyltransferase (CAT) activity. It has been proposed [Fraser, Corstorphine, Price and Zammit (1999) FEBS Lett. 446, 69-74] that this microsomal CAT activity is due to the liver form of carnitine palmitoyltransferase 1 (L-CPT(1)) being targeted to the endoplasmic reticulum (ER) membrane as well as to mitochondria, possibly by an N-terminal signal sequence [Cohen, Guillerault, Girard and Prip-Buus (2001) J. Biol. Chem. 276, 5403-5411]. COS- I cells were transiently transfected to express a fusion protein in which enhanced green fluorescent protein was fused to the C-terminus of L-CPT(1). Confocal microscopy showed that this fusion protein was localized to mitochondria, and possibly to peroxisomes, but not to the ER. cDNAs corresponding to truncated (amino acids 1-328) or full-length L-CPT(1) were transcribed and translated in the presence of canine pancreatic microsomes. However, there was no evidence of authentic insertion of CPT(1) into the ER membrane. Rat liver microsomal fractions purified by sucrose-density-gradient centrifugation contained all 88 kDa protein (p88) which was recognized by an anti-L-CPT(1) antibody and by 2,4-dinitrophenol-etomoxiryl-CoA, a covalent inhibitor of L-CPT(1). Abundance of p88 and malonyl-CoA-inhibitable CAT activity were increased approx. 3-fold by starvation for 24 h. Deoxycholate solubilized p88 and malonyl-CoA-inhibitable CAT activity from microsomes to approximately the same extent. The microsomal fraction contained porin, which, relative to total protein, was Lis abundant as in crude mitochondrial outer membranes fractions. It is concluded that L-CPT(1) is not targeted to the ER membrane and that malonyl-CoA CAT in microsomal fractions is L-CPT(1) that is derived from mitochoildria, possibly from membrane contact sites.