Abstract
CPT1a (carnitine palmitoyltransferase 1a) in the liver mitochondrial outer membrane (MOM) catalyzes the primary regulated step in overall mitochondrial fatty acid oxidation. It has been suggested that the fundamental unit of CPT1a exists as a trimer, which, under native conditions, could form a dimer of the trimers, creating a hexamer channel for acylcarnitine translocation. To examine the state of CPT1a in the MOM, we employed a combined approach of sizing by mass and isolation using an immunological method. Blue native electrophoresis followed by detection with immunoblotting and mass spectrometry identified large molecular mass complexes that contained not only CPT1a but also long chain acyl-CoA synthetase (ACSL) and the voltage-dependent anion channel (VDAC). Immunoprecipitation with antisera against the proteins revealed a strong interaction between the three proteins. Immobilized CPT1a-specific antibodies immunocaptured not only CPT1a but also ACSL and VDAC, further strengthening findings with blue native electrophoresis and immunoprecipitation. This study shows strong protein-protein interaction between CPT1a, ACSL, and VDAC. We propose that this complex transfers activated fatty acids through the MOM.
Highlights
The mitochondrial outer membrane (MOM) was considered to be a sieve through which small compounds passed unimpeded
The important question is whether the hexameric CPT1a channel acylcarnitines or CPT1a forms hetero-oligomeric complexes with other metabolically relevant enzymes and channel proteins such as ACSL and voltage-dependent anion channel (VDAC) that are responsible for channeling activated fatty acids through the MOM into the mitochondrial intermembrane space
MOM Multiprotein Complexes Revealed by blue native electrophoresis (BNE)—BNE was coupled with mass spectrometry to determine the oligomeric state of native CPT1a and proteins interacting with CPT1a
Summary
Animals—Male Sprague-Dawley rats (200 – 400 g; Charles River, Wilmington, MA) had free access to food and water. Preparation of Rat Liver Mitochondria and MOMs—Rat liver mitochondria were isolated by differential centrifugation in 220 mM mannitol, 70 mM sucrose, and 5 mM MOPS (pH 7.4) (containing one mini tablet of protein phosphatase inhibitor/100 ml) and purified by Percoll gradient centrifugation [17]. The pellet was resuspended in BNE buffer supplemented with Triton X-100 at a detergent/protein ratio of 3, kept on ice for 30 min with intermittent mixing with a micropipette, and centrifuged using the Airfuge as described above. All data files were searched against a rat subset taken from the Swiss-Prot Database using the Mascot database search engine (version 2.1.04, Matrix Science, London, United Kingdom) For these searches, the mass tolerance for the precursor and fragment ions was set to Ϯ15 ppm and Ϯ1 Da, respectively. The results of the searches were filtered by removing all peptide matches at an ion score of Ͻ25
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