Externalization of Cardiolipin as an “Eat-Me” Mitophageal Signal is Facilitated by NDPK-D

Abstract

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).