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Mitochondria are vulnerable to damage, particularly by oxidative stress-induced injury imposed by many genetic and environmental factors. These malfunctioning mitochondria have to be eliminated to prevent an increasing generation of reactive oxygen species (ROS) that could trigger cell injury and death. This selective elimination of damaged mitochondria is executed via initiation of a specific type of mitochondrial autophagy - mitophagy. Our previous work has established that a mitochondria-specific phospholipid, cardiolipin (CL) - normally asymmetrically distributed between the mitochondrial inner (IMM) and outer (OMM) membranes - undergoes translocation to the OMM where it becomes externalized to the mitochondrial surface. This externalized CL serves as recognition signal for the autophageal machinery leading to the elimination of these mitochondria. The recognition is achieved through the selective binding of externalized CL with microtubule-associated protein light chain 3 (LC3). The mechanisms driving CL redistribution/externalization remain unknown. By using LC-MS analysis of mono-lyso-cardiolipins (mL-CL) formed by phospholipase A2 exogenously added and impermeable to mitochondria, we established that treatment of HeLa cells with a protonophoric uncoupler, CCCP, triggers mitophagy of depolarized mitochondria accompanied by CL externalization. We further found that CL externalization is dependent on an intermembrane space enzyme, nucleoside diphosphate kinase, NDPK-D. The latter, upon interaction with CL, loses its kinase function and turns into a CL-translocase. CL externalization and mitophagy were stimulated by transfecting HeLa cells with w/type but not mutant R90A NDPK-D, incapable of CL binding. Identification of NDPK-D as a pro-mitophageal CL translocase may be used in drug discovery paradigms for regulation of “mitochondrial health.” Supported by NIH U19AIO6802, Fondation pour la Recherche Medicale France (US, DPM20121125557) and Fulbright scholarship program (VEK).

Molecular functions of mitochondrial nucleoside diphosphate kinase (NDPK-D) were studied using different biophysical and biochemical techniques. Subfractionation of rat liver and HEK 293 cell mitochondria revealed that NDPK-D is essentially bound to the inner membrane. The kinase interacted electrostatically with anionic phospholipids, showing highest affinity for cardiolipin as quantified by surface plasmon resonance. NDPK-D was also able to cross-link anionic phospholipid-containing liposomes as seen in light scattering assays, suggesting that the hexameric kinase could promote intermembrane contacts. Mutation of the central arginine (R90) in a surface exposed basic RRK motif unique to NDPK-D strongly reduced these membrane interactions. In a model using HeLa cells naturally almost devoid of NDPK-D, wt protein and R90D mutant were stably expressed, but only wt protein was found attached to membranes. Respiration was significantly stimulated by the NDPK substrate TDP only in mitochondria containing wt NDPK-D, but not in those expressing R90D mutant that is catalytically equally active. This indicates local ADP regeneration in the mitochondrial intermembrane space and a tight functional coupling of NDPK-D with oxidative phosphorylation that depends on the membrane-bound state of the kinase. A model is proposed for a mitochondrial NDPK microcompartment.