NDPK-D (NM23-H4)-mediated externalization of cardiolipin enables elimination of depolarized mitochondria by mitophagy

Abstract

Mitophagy is critical for cell homeostasis. Externalization of the inner mitochondrial membrane phospholipid, cardiolipin (CL), to the surface of the outer mitochondrial membrane (OMM) was identified as a mitophageal signal recognized by the microtubuleassociated protein 1 light chain 3. However, the CL-translocating machinery remains unknown. Here we demonstrate that a hexameric intermembrane space protein, NDPK-D (or NM23-H4), binds CL and facilitates its redistribution to the OMM. We found that mitophagy induced by a protonophoric uncoupler, carbonyl cyanide m-chlorophenylhydrazone (CCCP), caused externalization of CL to the surface of mitochondria in murine lung epithelial MLE-12 cells and human cervical adenocarcinoma HeLa cells. RNAi knockdown of endogenous NDPK-D decreased CCCP-induced CL externalization and mitochondrial degradation. A R90D NDPK-D mutant that does not bind CL was inactive in promoting mitophagy. Similarly, rotenone and 6-hydroxydopamine triggered mitophagy in SH-SY5Y cells was also suppressed by knocking down of NDPK-D. In situ proximity ligation assay (PLA) showed that mitophagy-inducing CL-transfer activity of NDPK-D is closely associated with the dynamin-like GTPase OPA1, implicating fission-fusion dynamics in mitophagy regulation.

NME4/nucleoside diphosphate kinase D in cardiolipin signaling and mitophagy

“…9 Very much like phosphatidylserine externalized to the cellular surface serves for elimination of damaged cells via apoptosis, CL externalized to the mitochondrial surface acts as a signal for the elimination of damaged mitochondria via mitophagy. [11][12][13][14] Increased CL at MOM has been observed under different conditions that favor mitophagy like chemical models of Parkinson disease 10 and caloric restriction. 15 Externalization of CL to the mitochondrial surface is also part of other damage-related signaling pathways, including early pro-apoptotic signaling [16][17][18] and inflammatory reactions in intracellular compartments and extracellular environments.…”

“…We found in different cellular model systems that a collapse of ΔΨ (eg, using the protonophoric uncoupler CCCP) is triggering CL externalization and induction of mitophagy, and that this requires the presence of CL-binding competent NME4/NDPK-D that exposes triads of basic amino acids. 11 In fact, the CL movement requires a CL-transfer-competent NME4/NDPK-D topology, where the enzyme localized in the mitochondrial intermembrane space binds simultaneously to MIM and MOM, and is NDP kinase-inactive ( Figure 1, right part). It has long been known that such MIM-MOM contact sites are enriched in NDPK and CL.…”

“…8 We have recently identified a novel pathway that describes for the first time a role of lipid signaling in mitophagy. [9][10][11] Different mitophageal stimuli such as rotenone, staurosporine, 6-hydroxydopamine, and others are able to trigger migration of the mitochondria-specific phospholipid cardiolipin (CL), from its site of synthesis, MIM, to the opposed MOM, and further to the mitochondrial surface. 9 This exposed CL directly serves as an 'eat me' signal, as it is recognized by two CL-binding pockets within the LC3 receptor.…”

Lipid-mediated signals that regulate mitochondrial biology

“…This MOM-localized cardiolipin appears to have very important signaling functions. Decreased delivery of mitochondria to autophagosomes was observed upon genetic deletion or RNAi knockdown of cardiolipin synthase or upon knockdown of two enzymes that translocate cardiolipin to the MOM, phospholipid scramblase-3 or nucleoside diphosphate kinase D (NDPK-D) (8,10,11). Through a combination of molecular docking analyses, in vitro binding assays, and fluorescence microscopy, Chu et al identified sites on LC3, a component of the autophagy machinery, required for direct interaction with MOM cardiolipin and for mitophagy ( Figure 1) (8).…”

“…In healthy mitochondria, cardiolipin is found almost exclusively in the MIM, where it helps to stabilize cristae and support the assembly and function of electron transport chain complexes (8,9). However, recent studies have shown that mitochondrial injury, depolarization, and other damage signals cause externalization of cardiolipin to the mitochondrial outer membrane (MOM) where it is exposed to the cytosol (8,10). This MOM-localized cardiolipin appears to have very important signaling functions.…”