Dataset Information

Identification of GRAF1 interactome mediated by GRAF1 phosphorylation in response to Mitophagy

ABSTRACT: Defects in the clearance of dysfunctional mitochondria contribute to the development of heart failure and several neurological disorders including Parkinson’s disease (PD). The mitophagic signaling pathways that control the rapid recognition, ubiquitin-tagging and encasement of dysfunctional mitochondria into protective autophagosomes have been well characterized and a major mechanism involves coordinated control of the serine/threonine kinase PINK1 and the E3 ubiquitin ligase, Parkin. What is less known is how such clearance processes are spatially controlled. Indeed, while recent studies indicate that actin remodeling can facilitate Parkin-dependent mitochondrial clearance, the underlying players that coordinate localized cytoskeletal remodeling to facilitate the trafficking and clearance of damaged mitochondria remain largely unclear. Herein, we demonstrate that the RhoGAP, GRAF1 (Arhgap26), is a PINK1 substrate that controls mitophagy. Cardiomyocyte-restricted GRAF1 depletion led to accumulation of dysfunctional mitochondria and attenuated stress-induced metabolic adaptation in adult hearts. GRAF1 was required for mitochondria release from F-actin anchors, facilitated mitochondria capture by autophagosomes by enhancing Parkin-LC3 interactions, and promoted mitochondria-associated Arp2/3-dependent actin remodeling. This study employs mass spectrometry-based proteomics to elucidate the interactome of GRAF1, focusing on the protein-protein interactions facilitated by GRAF1 phosphorylation.

INSTRUMENT(S): Q Exactive HF

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Cell Culture

SUBMITTER: Christine Mills

LAB HEAD: Joan Taylor

PROVIDER: PXD043212 | Pride | 2024-01-26

REPOSITORIES: Pride

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			Action	DRS
	20210611_PHCOQEHF_PC842-Taylor_1_R1.raw	Raw
	20210611_PHCOQEHF_PC842-Taylor_1_R2.raw	Raw
	20210611_PHCOQEHF_PC842-Taylor_2_R1.raw	Raw
	20210611_PHCOQEHF_PC842-Taylor_2_R2.raw	Raw
	20210611_PHCOQEHF_PC842-Taylor_3_R1.raw	Raw

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Publications

GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis.

Zhu Qiang Q Combs Matthew E ME Liu Juan J Bai Xue X Wang Wenbo B WB Herring Laura E LE Liu Jiandong J Locasale Jason W JW Bowles Dawn E DE Gross Ryan T RT Pla Michelle Mendiola MM Mack Christopher P CP Taylor Joan M JM

Nature communications 20231211 1

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, w ...[more]

PMID: 38081847

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Project description:Clearance of damaged mitochondria via mitophagy is crucial for cellular homeostasis. While the role of ubiquitin (Ub) ligase PARKIN in mitophagy has been extensively studied, increasing evidence suggests the existence of PARKIN-independent mitophagy in highly metabolically active organs such as the heart. Here, we identify a crucial role for Cullin-RING Ub ligase 5 (CRL5) in basal mitochondrial turnover in cardiomyocytes. CRL5 is a multi-subunit Ub ligase comprised by the catalytic RING box protein RBX2 (also known as SAG), scaffold protein Cullin 5 (CUL5), and a substrate-recognizing receptor. Analysis of the mitochondrial outer membrane-interacting proteome uncovered a robust association of CRLs with mitochondria. Subcellular fractionation, immunostaining, and immunogold electron microscopy established that RBX2 and Cul5, two core components of CRL5, localizes to mitochondria. Depletion of RBX2 inhibited mitochondrial ubiquitination and turnover, impaired mitochondrial membrane potential and respiration, and increased cell death in cardiomyocytes. In vivo, deletion of the Rbx2 gene in adult mouse hearts suppressed mitophagic activity, provoked accumulation of damaged mitochondria in the myocardium, and disrupted myocardial metabolism, leading to rapid development of dilated cardiomyopathy and heart failure. Similarly, ablation of RBX2 in the developing heart resulted in dilated cardiomyopathy and heart failure. Notably, the action of RBX2 in mitochondria is not dependent on PARKIN, and PARKIN gene deletion had no impact on the onset and progression of cardiomyopathy in RBX2-deficient hearts. Furthermore, RBX2 controls the stability of PINK1 in mitochondria. Proteomics and biochemical analyses further revealed a global impact of RBX2 deficiency on the mitochondrial proteome and identified several mitochondrial proteins as its putative substrates. These findings identify RBX2-CRL5 as a mitochondrial Ub ligase that controls mitophagy under physiological conditions in a PARKIN-independent, PINK1-dependent manner, thereby regulating cardiac homeostasis.

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