title: Demonstration of N- and C-terminal domain intramolecular interactions in rat liver carnitine palmitoyltransferase 1 that determine its degree of malonyl-CoA sensitivity.
abstract: 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 sensitivity 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 (Ser24-->Ala)/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.
authors.affiliation: 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.
authors.forename: Audrey
authors.initials: A
authors.lastname: Faye
authors.forename: Karen
authors.initials: K
authors.lastname: Borthwick
authors.forename: Catherine
authors.initials: C
authors.lastname: Esnous
authors.forename: Nigel T
authors.initials: NT
authors.lastname: Price
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Gobin
authors.forename: Vicky N
authors.initials: VN
authors.lastname: Jackson
authors.forename: Victor A
authors.initials: VA
authors.lastname: Zammit
authors.forename: Jean
authors.initials: J
authors.lastname: Girard
authors.forename: Carina
authors.initials: C
authors.lastname: Prip-Buus
mesh_terms: D000818:Animals; D019905:Benzyl Alcohol / Q000494:pharmacology; D002334:Carnitine O-Palmitoyltransferase / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D003432:Cross-Linking Reagents / Q000378:metabolism / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D003921:Diabetes Mellitus, Experimental / Q000139:chemically induced / Q000201:enzymology / Q000378:metabolism; D004032:Diet; D008099:Liver / Q000201:enzymology*; D008297:Male; D008316:Malonyl Coenzyme A / Q000378:metabolism*; D008560:Membrane Fluidity / Q000187:drug effects; D008930:Mitochondria, Liver / Q000201:enzymology / Q000378:metabolism; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D017354:Point Mutation / Q000235:genetics; D017434:Protein Structure, Tertiary; D051381:Rats; D017208:Rats, Wistar; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D013217:Starvation / Q000201:enzymology / Q000378:metabolism; D013311:Streptozocin; D013379:Substrate Specificity / Q000187:drug effects; D014162:Transfection / Q000379:methods
chemical_list: D003432:Cross-Linking Reagents; D010455:Peptides; D008316:Malonyl Coenzyme A; D013311:Streptozocin; D002334:Carnitine O-Palmitoyltransferase; D019905:Benzyl Alcohol