Cardiolipin bound to mitochondrial ADP/ATP carrier supports the structure andtransport-related function

Nanami Senoo (1), Matthew G. Baile (1), Bodhisattwa Saha (2), Dinesh Chinthapalli (2), Oluwaseun Ogunbona (1), Jake Saba (1), Teona Munteanu1, Dror Chorev (2), Carol Robinson (2), Steven M. Claypool (1)

(1) Department of Physiology, Johns Hopkins University School of Medicine,
(2) Physical and Theoretical Chemistry Laboratory, University of Oxford.

The mitochondrial phospholipid cardiolipin (CL) ensures cellular energy production via oxidative phosphorylation (OXPHOS). Our previous studies have shown that CL is vital for the structure and function of the ADP/ATP carrier (AAC in yeast/ANT in mammals) which resides in the inner mitochondrial membrane and exchanges ADP and ATP to enable OXPHOS. Three tightly bound CL molecules have been evolutionarily identified in AAC/ANT structures. Here, we used yeast Aac2 as a model and investigated whether the buried CL molecules within Aac2 are essential for its structure and/or function. We designed mutations to introduce negatively charged amino acids into each CL-binding pocket of Aac2 to disrupt the interaction with the CL phosphate headgroup via electrostatic repulsion. Native mass spectrometry analysis verified that CL molecules were dissociated from each Aac2 mutant. We found that the monomeric structure of each mutant was destabilized when challenged by blue native-PAGE. Critically, most mutants had impaired ADP/ATP transport activity. These results suggest that the three tightly bound CL molecules within yeast Aac2 support the carrier’s structure and activity through overlapping and yet distinct mechanisms. We also found that a recently identified patient mutation in human ANT1, L141F, corresponds to a CL-binding pocket residue mutated in Aac2. Engineering this patient mutation in a human cell line revealed that ANT1 L141F resulted in reduced OXPHOS capacity, indicating the pathological significance of one of the three conserved CL-binding pockets in AAC/ANT. Our findings highlight the conserved structural and functional significance of tightly-bound CL in mitochondrial carrier proteins.