The E3 Ubiquitin Ligase Parkin Regulates Mitophagy from the Nucleus

Abstract

Parkin is known for facilitating clearance of damaged mitochondria. We have previously shown that Parkin is important for adaptation to myocardial infarction (MI) and that loss of Parkin leads to accumulation of dysfunctional mitochondria. Parkin enables mitophagy by translocating to depolarized mitochondria; however, it is unclear whether Parkin also functions in other subcellular compartments. Here, we found that wild type (WT) Parkin overexpressed in neonatal myocytes localized to both the cytosol and the nucleus, whereas the pathogenic mutant ParkinR42P was excluded from the nucleus. Western blotting of subcellular fractions from WT mouse hearts confirmed the presence of endogenous Parkin in the nucleus. Additionally, hearts from cardiac-specific Parkin transgenic mice had elevated nuclear Parkin, which correlated with increased ubiquitination of nuclear proteins. Interestingly, WT mice subjected to MI displayed an increase in both Parkin and ubiquitinated proteins in the nuclear fraction of border zone myocytes. To further examine conditions that induce Parkin nuclear translocation, we subjected HeLa cells stably expressing YFP-Parkin to starvation or hypoxia. Under nutrient-limiting conditions, Parkin rapidly exited the nucleus and accumulated in the cytosol. Conversely, exposure to hypoxia caused Parkin to translocate to the nucleus. To evaluate the function of Parkin in the nucleus and its effect on mitophagy, we generated nuclear- and mitochondrial- targeted constructs: NLS-Parkin and Mito-Parkin. Mito-Parkin displayed accelerated mitochondrial clearance compared to WT Parkin in response to treatment with mitochondrial uncoupler FCCP. Unexpectedly, NLS-Parkin also induced mitochondrial clearance, albeit at a slower rate, suggesting that Parkin may regulate mitophagy transcriptionally. In addition to mitochondrial-Parkin, receptors on the outer mitochondrial membrane can also initiate mitophagy. We therefore examined transcription of various mitophagy receptors and found that cells overexpressing Parkin exhibited increased transcript levels of BNIP3 and NIX. These data indicate that Parkin facilitates mitophagy from multiple subcellular locations to ensure efficient mitochondrial clearance.