CPT1c is localized in endoplasmic reticulum of neurons and has carnitine palmitoyltransferase activity.
CPT1c is a carnitine palmitoyltransferase 1 (CPT1) isoform that is expressed only in the brain. The enzyme has recently been localized in neuron mitochondria. Although it has high sequence identity with the other two CPT1 isoenzymes (a and b), no CPT activity has been detected to date. Our results indicate that CPT1c is expressed in neurons but not in astrocytes of mouse brain sections. Overexpression of CPT1c fused to the green fluorescent protein in cultured cells demonstrates that CPT1c is localized in the endoplasmic reticulum rather than mitochondria and that the N-terminal region of CPT1c is responsible for endoplasmic reticulum protein localization. Western blot experiments with cell fractions from adult mouse brain corroborate these results. In addition, overexpression studies demonstrate that CPT1c does not participate in mitochondrial fatty acid oxidation, as would be expected from its subcellular localization. To identify the substrate of CPT1c enzyme, rat cDNA was overexpressed in neuronal PC-12 cells, and the levels of acylcarnitines were measured by high-performance liquid chromatography-mass spectrometry. Palmitoylcarnitine was the only acylcarnitine to increase in transfected cells, which indicates that palmitoyl-CoA is the enzyme substrate and that CPT1c has CPT1 activity. Microsomal fractions of PC-12 and HEK293T cells overexpressing CPT1c protein showed a significant increase in CPT1 activity of 0.57 and 0.13 nmol.mg(-1).min(-1), respectively, which is approximately 50% higher than endogenous CPT1 activity. Kinetic studies demonstrate that CPT1c has similar affinity to CPT1a for both substrates but 20-300 times lower catalytic efficiency.
Sierra
Adriana Y
AY
Molecular and Cellular Unit, School of Health Sciences, Universitat Internacional de Catalunya, Josep Trueta s/n, Sant Cugat del Vallés, Barcelona 08195, Spain.
Gratacós
Esther
E
Carrasco
Patricia
P
Clotet
Josep
J
Ureña
Jesús
J
Serra
Dolors
D
Asins
Guillermina
G
Hegardt
Fausto G
FG
Casals
Núria
N
D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000737:chemistry* / Q000378:metabolism*; D002384:Catalysis; D002460:Cell Line; D005227:Fatty Acids / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D008928:Mitochondria / Q000378:metabolism; D008954:Models, Biological; D009474:Neurons / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism; D016716:PC12 Cells; D020033:Protein Isoforms; D051381:Rats
D005227:Fatty Acids; D020033:Protein Isoforms; D002334:Carnitine O-Palmitoyltransferase; D010100:Oxygen