Controlling the Mitochondrial Protonmotive Force with Light to Impact Cellular Stress Resistance

Abstract

28Mitochondrial respiration generates an electrochemical proton gradient across the mitochondrial 29 inner membrane called the protonmotive force (PMF) to drive diverse functions and make ATP. 30 Current techniques to manipulate the PMF are limited to its dissipation; there is no precise, 31 reversible method to increase the PMF. To address this issue, we used an optogenetic 32 approach and engineered a mitochondria-targeted light-activated proton pumping protein we 33 called mitochondria-ON (mtON) to selectively increase the PMF. Here, mtON increased the 34 PMF light dose-dependently, supported ATP synthesis, increased resistance to mitochondrial 35 toxins, and modulated energy-sensing behavior in Caenorhabditis elegans. Moreover, transient 36 mtON activation during hypoxia prevented the well-characterized adaptive response of hypoxic 37 preconditioning. Our novel optogenetic approach demonstrated that a decreased PMF is both 38 necessary and sufficient for hypoxia-stimulated stress resistance. Our results show that 39 optogenetic manipulation of the PMF is a powerful tool to modulate metabolic and cell signaling 40 outcomes. 41 42 76organism Leptosphaeria maculans (Chow et al., 2010, Waschuk, Bezerra et al., 2005 to 77 3 mitochondria and selectively increase the PMF. We call this optogenetic tool mitochondria-ON 78 (mtON) due to its ability to mimic the proton pumping activity of the ETC in response to light, 79 independent of oxygen or substrate availability. 80 We validated mtON using the well-characterized genetic model organism, C. elegans 81 (Butler, Ventura et al., 2010, Dingley et al., 2010, Tsang & Lemire, 2003. Using hypoxia and 82 reoxygenation, we tested the hypothesis that hypoxia adaptation through preconditioning 83 requires a decreased PMF. Our data demonstrate that transient loss of PMF during 84 preconditioning is necessary and sufficient for resistance to hypoxia. By probing the 85 evolutionarily conserved hypoxia adaptation response (Pena, Sherman et al., 2016, Wang, Lim 86 et al., 2019, Wojtovich, Nadtochiy et al., 2012b, Wojtovich, Nadtochiy et al., 2013, we show that 87 tools like mtON allow precise determination of cause and effect in physiologic models. 88 89 RESULTS & DISCUSSION 90 Light-activated proton pump mitochondria-ON (mtON) is expressed in mitochondria. 91 Using a ubiquitously-expressed gene promoter (Peft-3), we directed expression of a 92 light-activated proton pump to the mitochondrial IM in C. elegans. Mitochondrial localization was 93 achieved by fusion of the proton pump to an N-terminal mitochondrial targeting sequence of the 94 IMMT1 protein (Fischer, Igoudjil et al., 2011, John, Shang et al., 2005 in an orientation that 95 allows proton pumping from the mitochondrial matrix towards the intermembrane space to 96 increase the PMF in response to light (Fig. 1A). Using a C-terminal GFP fusion for subcellular 97 visualization, and MitoTracker TM CMXRos, we observed overlap of green and red fluorescence 98 in C. elegans tissues, indicating the intended mitochondrial...

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