A novel role for Ral in response to mitochondrial depolarization

Healthy mitochondria use an electrochemical gradient across the inner mitochondrial membrane (IMM) to generate energy in the form of ATP. A variety of endogenous and exogenous factors can lead to transient or sustained depolarization of the IMM, including mitochondrial fission events, expression of uncoupling proteins, electron transport chain (ETC) inhibitors, or chemical uncouplers. This depolarization in turn leads to a variety of physiological responses, ranging from selective mitochondrial clearance (mitophagy) to cell death. There are multiple types of mitophagy including PINK1-Parkin mitophagy in which the kinase, PINK1 accumulates on outer mitochondrial membrane (OMM) and results in Parkin (an E3 ubiquitin ligase) translocation from the cytosol to the OMM to recruit the autophagy machinery. How cells recognize and ultimately respond to depolarized mitochondria remains incompletely understood. We found that the small Ras-like GTPases, RalA and RalB relocalize to depolarized mitochondria in a clathrin-dependent process. Furthermore, both genetic and pharmacologic inhibition of RalA and RalB leads to an increase in the activity of the atypical IκB kinase TBK1 both basally and in response to mitochondrial depolarization. We also observed this phenotype following inhibition of Ral relocalization. Collectively, these data suggest a model in which RalA and RalB inhibit TBK1 and that relocalization of Ral (both RalA and RalB) to depolarized mitochondria facilitates TBK1 activation through release of this inhibition. We also found that RalA, but not RalB, participates in Parkin recruitment during PINK1-Parkin mitophagy as RalA knockdown or knockout results in impaired Parkin translocation from the cytosol to depolarized mitochondria. Since the mechanism underlying Parkin recruitment remains unclear, our findings underscoring the importance of RalA provide new insight into Parkin relocalization. Additionally, pharmacologic and genetic impairment of clathrin-mediated endocytosis (CME) decreases Parkin recruitment, suggesting that CME not only regulates Ral relocalization, but also affects Parkin trafficking. Collectively, our findings point to a novel role for RalA and RalB in response to mitochondrial depolarization.