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J. Biol. Chem., Vol. 283, Issue 11, 6878-6885, March 14, 2008

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CPT1c Is Localized in Endoplasmic Reticulum of Neurons and Has Carnitine Palmitoyltransferase Activity^*

Adriana Y. Sierra¹², Esther Gratacós¹, Patricia Carrasco², Josep Clotet, Jesús Ureña, Dolors Serra^¶||, Guillermina Asins^¶||, Fausto G. Hegardt^¶||, and Núria Casals^||3

From the Molecular and Cellular Unit, School of Health Sciences, Universitat Internacional de Catalunya, Josep Trueta s/n, Sant Cugat del Vallés, Barcelona 08195, the Department of Cell Biology, Faculty of Biology, University of Barcelona, Diagonal 645, and Institute de Recerca Biomédica de Barcelona, Josep Samitier 1–5, 08028 Barcelona, the ^¶Department of Biochemistry and Molecular Biology, University of Barcelona, School of Pharmacy, 08028 Barcelona, and the ^||Centro de Investigación Biomédica en Red (CIBER) Institute of Fisiopatología de la Obesidad y Nutrición (CB06/03), Instituto de Salud Carlos III, 28029 Madrid, Spain

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 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.

Received for publication, September 24, 2007 , and in revised form, December 13, 2007.

^* This work was supported in part by the Ministerio de Educación y Ciencia, Spain (Grants SAF2004-06843-C03 and SAF2007-61926), by Ajut de Suport als Grups de Recerca de Catalunya (Grant 2005SGR-00733), and by the Fundació La Marató de TV3 (2007), Catalunya. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Both authors contributed equally to this work.

² Recipients of fellowships from the Universitat Internacional de Catalunya.

³ To whom correspondence should be addressed. Tel.: 93-50-42005; Fax: 93-50-42001; E-mail: ncasals{at}csc.uic.es.

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