RalA and RalB relocalization to depolarized mitochondria depends on clathrin-mediated endocytosis and facilitates TBK1 activation

Abstract

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. How cells recognize and ultimately respond to depolarized mitochondria remains incompletely understood. Here we show that the small GTPases RalA and RalB both relocalize to mitochondria following depolarization in a process dependent on clathrin-mediated endocytosis (CME). 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. This phenotype was also observed following inhibition of Ral relocalization. Collectively, these data suggest a model in which RalA and RalB inhibit TBK1 and that relocalization of Ral to depolarized mitochondria facilitates TBK1 activation through release of this inhibition.

Mitochondrial respiration restricts Listeria monocytogenes infection by slowing down host cell receptor recycling

“…However, the mitochondrial fragmentation induced by L. monocytogenes is Drp1 independent and does not result in mitochondrial redistribution to polarized structures (Stavru et al, 2011(Stavru et al, , 2013. Interestingly, a recent report showed that endocytosis-dependent relocalization of the small GTPases RalA and RalB to depolarized mitochondria plays an important role in innate immune signaling (Pollock et al, 2019). Whether L. monocytogenes interferes with similar mechanisms is currently unclear, and future work will be needed to characterize the interplay between mitochondrial energy metabolism, endocytic recycling, and infection.…”

Membrane extraction by calmodulin underpins the disparate signalling of RalA and RalB

“…During mitochondrial fission, phosphorylation by the kinase Aurora A leads to activation of RalA and its relocation to the mitochondrial outer membrane, while RalB is unaffected. [3,4] More recently, it was found that mitochondrial depolarisation triggers both RalA and RalB to relocate to mitochondria, [2] releasing RalB inhibition of TANK-binding kinase 1 (TBK1) at the plasma membrane, which is necessary for mitophagy. [17] While the relocation of RalA/B in response to mitochondrial depolarisation is dependent on clathrin mediated endocytosis, [2] no mechanism of transport has been suggested for RalA-specific trafficking to mitochondria during fission.…”

“…[3,4] More recently, it was found that mitochondrial depolarisation triggers both RalA and RalB to relocate to mitochondria, [2] releasing RalB inhibition of TANK-binding kinase 1 (TBK1) at the plasma membrane, which is necessary for mitophagy. [17] While the relocation of RalA/B in response to mitochondrial depolarisation is dependent on clathrin mediated endocytosis, [2] no mechanism of transport has been suggested for RalA-specific trafficking to mitochondria during fission. We speculate that CaM acts as an RalA-specific chaperone, shuttling RalA to the mitochondrial outer membrane.…”

“…Activation of Ral has been shown to be critical for tumour development and progression [1] as well as playing a key role in mitochondrial dynamics. [2][3][4] RalA and RalB have a central, conserved, Ras-like G-domain and are almost identical in their effector-binding regions, but they mediate markedly different cellular processes, especially in the context of ABBREVIATIONS: CaM, calmodulin; CSP, chemical shift perturbation; GAP, GTPase activating protein; GDI, GDP dissociation inhibitor; GEF, guanine nucleotide exchange factor; GSF, GDI-like solubilisation factor; HVR, hypervariable region; PDE-δ, phosphodiesterase δ; PLD1, phospholipase D1; TBK1, TANK-binding kinase 1 F I G U R E 1 NMR data comparing RalA and RalB interaction with CaM. (A) RalA and RalB HVR peptides were titrated into 15 N-labelled CaM and HSQC experiments performed as reported previously.…”

Mitophagy in the Pathogenesis of Liver Diseases

“…TBK1 phosphorylates OPTN at Ser177 to promote its interaction with ATG8/LC3 proteins [370] and at Ser473 and Ser513 to confer it with the ability to bind to the ubiquitin chain [371][372][373]. In addition, TBK1-mediated autophosphorylation, p62/SQSTM1 phosphorylation at Ser403, Rab7 phosphorylation at Ser72, and other phosphorylation events have been shown to regulate mitophagy [266,[374][375][376]. A recent study also noted that the Ca 2+ -binding protein TBC1 domain family member 9 (TBC1D9) may induce mitophagy by activating TBK1 [377].…”