Mitochondrial carnitine palmitoyltransferase 1a (CPT1a) is part of an outer membrane fatty acid transfer complex

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.

FIGURE 1.

Separation of MOM protein complexes by BNE. The MOM was solubilized with Triton X-100 (Triton X-100/protein ratio of 3:1 (w/w)) at 4 °C and analyzed by BNE. A, five different protein complexes greater than 210 kDa but smaller than 980 kDa were separated by BNE. B, the separated complexes were analyzed by Western blotting using affinity-purified anti-CPT1a peptide (raised against amino acids 230–245), anti-ACSL, and anti-VDAC antisera.

FIGURE 2.

Immunoisolation of CPT1a and proteins associated with CPT1a. A, upper panel, SDS-PAGE separation and silver staining of proteins immunoprecipitated (IP) with affinity-purified anti-CPT1a antibodies coupled to protein G beads; lower panel, immunoblot analysis of the starting material (initial MOM solubilized with SDS sample buffer (i-SDS) and initial MOM solubilized with 2% Lubrol PX (i-L-PX)), the pellet after Lubrol PX extraction (Airfuge; P), and the Lubrol PX supernatant (Airfuge; SN) with affinity-purified anti-CPT1a antibodies. Arrows and arrowheads show CPT1a and anti-CPT1a antibodies, respectively. B, immunocapture (IC) of CPT1a and associated proteins with a mixture of five affinity-purified anti-CPT1a antibodies directly coupled to CarboLink. In the eluate, CPT1a, but not anti-CPT1a antibodies, was detected. FT, flow-through with either protein G or CarboLink; E1 and E2, first and second eluents from either protein G- or CarboLink-coupled IgG, respectively.

FIGURE 3.

Co-immunoprecipitation using anti-CPT1a, anti-ACSL, and anti-VDAC1 antisera. Anti-CPT1a, anti-ACSL, or anti-VDAC antiserum was precleared by centrifugation (Airfuge, 10 min, 160,000 × g), and 20-μl aliquots each were mixed with Lubrol PX supernatant from the MOM (300 μg). The immunoprecipitate (IP; Airfuge, 10 min, 30 p.s.i.) was subjected to SDS-PAGE (A) and Western blot analysis (B). B, upper, middle, and lower panels, immunoprecipitation with anti-CPT1a, anti-VDAC1, and anti-ACSL antisera, respectively. Arrowheads, CPT1a; closed circles, VDAC1; arrows, ACSL.

FIGURE 4.

Proposed model of the hetero-oligomeric MOM complex consisting of CPT1a, ACSL, and VDAC. Palmitoyl-CoA formed by ACSL in the cytosol is channeled into the intermembrane space through VDAC, where it is transesterified to palmitoylcarnitine by CPT1a.