Abstract
Carnitine palmitoyltransferase (CPT) 1A adopts a polytopic conformation within the mitochondrial outer membrane, having both the N- and C-terminal segments on the cytosolic aspect of the membrane and a loop region connecting the two transmembrane (TM) segments protruding into the inter membrane space. In this study we demonstrate that the loop exerts major effects on the sensitivity of the enzyme to its inhibitor, malonyl-CoA. Insertion of a 16-residue spacer between the C-terminal part of the loop sequence (i.e. between residues 100 and 101) and TM2 (which is predicted to start at residue 102) increased the sensitivity to malonyl-CoA inhibition of the resultant mutant protein by more than 10-fold. By contrast, the same insertion made between TM1 and the loop had no effects on the kinetic properties of the enzyme, indicating that effects on the catalytic C-terminal segment were specifically induced by loop-TM2 interactions. Enhanced sensitivity was also observed in all mutants in which the native TM2-loop pairing was disrupted either by making chimeras in which the loops and TM2 segments of CPT 1A and CPT 1B were exchanged or by deleting successive 9-residue segments from the loop sequence. The data suggest that the sequence spanning the loop-TM2 boundary determines the disposition of this TM in the membrane so as to alter the conformation of the C-terminal segment and thus affect its interaction with malonyl-CoA.
Highlights
32946 JOURNAL OF BIOLOGICAL CHEMISTRY membrane and two transmembrane segments (TM1 and TM2) linked by a loop region that protrudes into the intermembrane space of the mitochondria [2]
Expression Levels of Native and Mutant Proteins—To ascertain that the native and mutant proteins were all expressed at the predicted sizes and within the same order of magnitude of expression, we detected the C-segment with an antipeptide antibody raised against residues 428 – 441 of rat Carnitine palmitoyltransferase (CPT) 1A
The native protein is resistant to trypsin in intact mitochondria because of its tightly folded state but loses its stability to trypsin once the mitochondrial outer membrane is ruptured [2, 11]
Summary
32946 JOURNAL OF BIOLOGICAL CHEMISTRY membrane and two transmembrane segments (TM1 and TM2) linked by a loop region that protrudes into the intermembrane space of the mitochondria [2]. Dietary lipid composition is able to modulate the kinetic characteristics of the enzyme [7] These changes in sensitivity are thought to result from a response of the enzyme to the molecular order of the lipids of the mitochondrial outer membrane [8, 9] and can be mimicked in vitro by conditions that alter membrane fluidity [10]. It has been suggested [9] that the membrane-integral nature of the protein enables its tertiary structure (and kinetic properties) to respond to changes in membrane composition induced by different pathophysiological states. Indicated that such an insertion greatly affected the malonyl- AGGCCTCAG-3Ј (reverse) were annealed and extended by
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.