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Biochem. J. (2005) 387 (67–76) (Printed in Great Britain)

Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity

Audrey FAYE*, Karen BORTHWICK†, Catherine ESNOUS*, Nigel T. PRICE†, Stéphanie GOBIN*, Vicky N. JACKSON†, Victor A. ZAMMIT†, Jean GIRARD* and Carina PRIP-BUUS*1

*Département d'Endocrinologie, Institut Cochin, INSERM U567, CNRS Unité Mixte de Recherche 8104, Université René Descartes, 24 rue du Faubourg Saint-Jacques, 75014 Paris, France, and †Department of Cell Biochemistry, Hannah Research Institute, Ayr, Scotland KA6 5HL, U.K.

We have previously proposed that changes in malonyl-CoA sensitivity of rat L-CPT1 (liver carnitine palmitoyltransferase 1) might occur through modulation of interactions between its cytosolic N- and C-terminal domains. By using a cross-linking strategy based on the trypsin-resistant folded state of L-CPT1, we have now shown the existence of such N–C (N- and C-terminal domain) intramolecular interactions both in wild-type L-CPT1 expressed in Saccharomyces cerevisiae and in the native L-CPT1 in fed rat liver mitochondria. These N–C intramolecular interactions were found to be either totally (48-h starvation) or partially abolished (streptozotocin-induced diabetes) in mitochondria isolated from animals in which the enzyme displays decreased malonyl-CoA sensitivity. Moreover, increasing the outer membrane fluidity of fed rat liver mitochondria with benzyl alcohol in vitro, which induced malonyl-CoA desensitization, attenuated the N–C interactions. This indicates that the changes in malonyl-CoA sens-itivity of L-CPT1 observed in mitochondria from starved and diabetic rats, previously shown to be associated with altered membrane composition in vivo, are partly due to the disruption of N–C interactions. Finally, we show that mutations in the regulatory regions of the N-terminal domain affect the ability of the N terminus to interact physically with the C-terminal domain, irrespective of whether they increased [S24A (Ser²⁴Ala)/Q30A] or abrogated (E3A) malonyl-CoA sensitivity. Moreover, we have identified the region immediately N-terminal to transmembrane domain 1 (residues 40–47) as being involved in the chemical N–C cross-linking. These observations provide the first demonstration by a physico-chemical method that L-CPT1 adopts different conformational states that differ in their degree of proximity between the cytosolic N-terminal and the C-terminal domains, and that this determines its degree of malonyl-CoA sensitivity depending on the physiological state.

Key words: carnitine palmitoyltransferase, carnitine acyltransferase, malonyl-CoA, intramolecular interaction, cross-linking, mitochondria.

Abbreviations used: BS³, bis(sulphosuccinimidyl) suberate; (L/M-)CPT1, (liver/muscle) carnitine palmitoyltransferase 1; E3A etc., Glu³Ala etc.; EDC, 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide; f82, 82-kDa fragment; GMBS, N-(g-maleimidobutyloxy)-succinimide ester; LC-SMCC, succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate); MBS, 3-maleimidobenzoyl-N-hydroxysuccinimide ester; N–C interaction, N- and C-terminal domain interaction; OMM, mitochondrial outer membrane; sulpho-GMBS, N-(g-maleimidobutyloxy)-sulphosuccinimide ester; sulpho-KMUS, N-(k-maleimidoundecanoyloxy)-sulphosuccinimide ester; sulpho-MBS, 3-maleimidobenzoyl-N-hydroxysulphosuccinimide ester; TM, transmembrane.

¹To whom correspondence should be addressed (email prip-buus@cochin.inserm.fr).

Received 7 September 2004; accepted 21 October 2004

Published as BJ Immediate Publication 21 October 2004, DOI 10.1042/BJ20041533

The Biochemical Society, London ©2005

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