A molecular biosensor reveals regulation of mitochondrial protein import by the PINK1 kinase-driven Parkin ubiquitin ligase

The Parkinson’s disease (PD) related proteins PINK1 and Parkin act jointly as a mitochondrial quality control system, linking loss of mitochondrial import efficiency to the autophagy-dependent degradation of dysfunctional mitochondria. The aim of this study was to evaluate a possible involvement of the PINK1/Parkin system in the regulation of mitochondrial protein import. We engineered an inducible biosensor for monitoring the main presequence-mediated import pathway in living cells with a quantitative bioluminescence-based readout. Validation of this probe in HEK293T cells showed that it was appropriately targeted to mitochondria and sensitive to import impairment caused by pharmacological or genetic approaches. By using complementary tools to mimic or antagonize the activation of Parkin by PINK1 on mitochondria (siRNA-mediated downregulation of PINK1 or Parkin; overexpression of Parkin, PD-causing Parkin variants, phosphomimetic or non-phosphorylatable Parkin ; co-expression of ubiquitin, phosphomimetic, non-phosphorylatable or lysine-less ubiquitin), we show that the PINK1/Parkin system stimulates mitochondrial import in a kinase- and ubiquitin ligase-dependent manner. Use of the biosensor in primary skin fibroblasts from PD patients revealed lower levels of import compared to controls in a subset of PARK2 (Parkin) and PINK1 patients. Altogether, our results suggest that mitochondrial import defects may contribute to PD pathogenesis.