The thiol switch C684 in Mitofusin-2 mediates redox-induced alterations of mitochondrial shape and respiration

The Charcot Marie Tooth Disease Mutation R94Q in MFN2 Decreases ATP Production but Increases Mitochondrial Respiration under Conditions of Mild Oxidative Stress

“…Non-mitochondrial oxygen consumption, ROX, which represents oxygen consumption of auto-oxidation reactions and other cellular oxygen-consuming enzymes such as oxidase and peroxidase [19] was recorded after inhibition of complex I with rotenone and of complex III with antimycin A and subtracted from the routine, leak or ETS values. We observed that oxygen flow per cell at routine respiration and the ETS was significantly increased in KO cells and in KO cells expressing EV as previously reported [6,7] whereas overexpression of both, WT and R94Q MFN2, similarly rescued the respiration phenotype to levels indistinguishable from the parental wildtype cells (Fig. 1C).…”

“…This, however, revealed no difference (Fig. 3B) suggesting that the similar increase in respiration of C684A [7] and R94Q upon exposure to 100 µM H2O2 must be caused by a different mechanism. We next assessed whether this increase in respiration corresponds to an increased ATP production and transfected the genetically-encoded reporter BTeam [18] targeted to mitochondria or the cytosol into our cells.…”

“…Its downregulation attenuates the mitochondrial membrane potential, cellular respiration and the mitochondrial proton leak [4] while overexpression increased the activity of the respiratory chain even when expressed as truncated, fusion-inactive protein [5]. Others, including us, found that Mfn2-/-mouse embryonic fibroblasts (MEFs) exhibit increased respiration compared to wildtype controls [6,7]. These discrepancies might be due to differences in redox conditions sensed by the thiol switch cysteine 684 [7].…”

“…As this very much resembled the phenotype we recently described for KO cells stably transfected with the mutant C684A, a mutant that removes a cysteine involved in the process of mitochondrial hyperfusion [21], we repeated a key experiment of this publication which demonstrated that cysteine 684 renders MFN2 more susceptible to alterations of the redox environment. In these experiments, we found that treating digitonin-permeabilized cells with 1 mM GSH for 10 min greatly reduced respiration in C684A but not in WT MFN2 cells [7]. To clarify whether R94Q, has the same effect we repeated this experiment and permeabilized the cells with digitonin to investigate mitochondrial respiration in the presence or absence of GSH and GSSG.…”

“…Others, including us, found that Mfn2-/-mouse embryonic fibroblasts (MEFs) exhibit increased respiration compared to wildtype controls [6,7]. These discrepancies might be due to differences in redox conditions sensed by the thiol switch cysteine 684 [7]. MFN2 is also a key determinant of so-called mitochondria-ER contact sites (MERCS) also known as mitochondria-associated membranes (MAMs), hot spots of interactions between the ER and mitochondria and important signaling hubs.…”