PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria

Abstract

Senescent and damaged mitochondria undergo selective mitophagic elimination through mechanisms requiring two Parkinson's disease factors, the mitochondrial kinase PINK1 (PTEN-induced putative kinase protein 1; PTEN is phosphatase and tensin homolog) and the cytosolic ubiquitin ligase Parkin. The nature of the PINK-Parkin interaction and the identity of key factors directing Parkin to damaged mitochondria are unknown. We show that the mitochondrial outer membrane guanosine triphosphatase mitofusin (Mfn) 2 mediates Parkin recruitment to damaged mitochondria. Parkin bound to Mfn2 in a PINK1-dependent manner; PINK1 phosphorylated Mfn2 and promoted its Parkin-mediated ubiqitination. Ablation of Mfn2 in mouse cardiac myocytes prevented depolarization-induced translocation of Parkin to the mitochondria and suppressed mitophagy. Accumulation of morphologically and functionally abnormal mitochondria induced respiratory dysfunction in Mfn2-deficient mouse embryonic fibroblasts and cardiomyocytes and in Parkin-deficient Drosophila heart tubes, causing dilated cardiomyopathy. Thus, Mfn2 functions as a mitochondrial receptor for Parkin and is required for quality control of cardiac mitochondria.

Fig. 1. Interaction of Mfn2 with Parkin in a PINK1-dependent manner and its requirement for Parkin translocation to depolarized mitochondria

(A and B) Fibroblasts were transfected with Flag-Mfn1 (A) or Mfn2 (B), PINK1, and/or HA-Parkin, immunoprecipitated with anti-Flag, and immunoblotted (IB). Right panels show IB of input homogenates. (C) Subcellular Parkin redistribution (green) induced by mitochondrial depolarization with FCCP in wild-type, Mfn1-deficient, and Mfn2-deficient mouse cardiomyocytes.

Fig. 2. Defective ubiquitination and mitophagy in Mfn2-deficient mouse hearts

(A) Mfn2 ubiquitination in PINK1/Parkin-containing HEK cell immune complexes (left). Input proteins are on right. (B) Protein ubiquitination stimulated by FCCP in Mfn2/Parkin immune complexes from mouse heart. (C) Mitochondrial ubiquitination (green) induced by FCCP in wild-type, Mfn1-, and Mfn2-deficient mouse cardiomyocytes. Cell borders are outlined in white. (D) Parallel studies of mitophagy-adaptor protein p62/SQSTM1 (green).

Fig. 3. PINK1 Phosphorylation of Mfn2 T111 and S442 dictates Parkin binding

(A) Mfn2 electrophoretic mobility shifts (arrowheads) induced by PINK1 in SDS-PAGE (top) and Phos-Tag (bottom) gels. Exploded view of lanes 4 and 5 is shown to the right. (B) PINK1-mediated Mfn2 phosphorylation (arrowheads) by anti-phosphoserine immunoblot (IB). (C) (left) Mfn2-Parkin co-immunoprecipitation study with functional and kinase-defective (KD) PINK1; (right) Mfn2 Phos-Tag phosphorylation study with functional and KD PINK1. (D) Effects of Mfn2 T111A and S442A mutations on PINK-stimulated Mfn2-Parkin binding. (E) Abrogation of PINK1-stimulated Mfn2-Parkin binding by Mfn2 T111A/S442A mutation and induction of PINK1-independent Mfn2-Parkin binding by Mfn2 T111E/S442E mutation.

Fig. 4. Progressive cardiomyopathy of Mfn2-deficient mouse hearts and Parkin-deficient Drosophila heart tubes

(A) Transmission electron microscopic examination of mouse cardiomyocyte mitochondria (5,000x). (B) Whole cell O₂ consumption studies of cadiomyocytes isolated from wild-type (black) or Mfn2-deficient (red) mouse hearts. (C) M-mode echocardiograms of mouse hearts. (D) Confocal imaging of Drosophila cardiomyocyte mitochondria. (E) O₂ consumption studies of heart tubes isolated from wild-type (black) or Parkin-deficient (red) Drosophila. (F) Optical coherence tomography of Drosophila heart tubes. (G) Mitochondrial ubiquitination (green) induced by FCCP in wild-type and Parkin-deficient Drosophila cardiomyocytes.