RESUMO
Mfn2 is a mitochondrial outer membrane fusion protein with the additional role of tethering mitochondria to the ER. Here, we describe a novel connection between Mfn2 and calcium release from mitochondria. We show that Mfn2 controls the mitochondrial inner membrane sodium-calcium exchange protein NCLX, which is a major source for calcium release from mitochondria. This discovery was made with the fungal toxin Phomoxanthone (PXA), which induces calcium release from mitochondria. PXA-induced calcium release is blocked by a chemical inhibitor of NCLX, while NCLX and Mfn2 deletions both also prevent PXA-induced calcium release. CETSA experiments show that PXA directly targets Mfn2, which likely controls NCLX through physical interactions since co-immunoprecipitation and proximity ligation assays show increased association between Mfn2 and NCLX upon treatment with PXA. Interactions between Mfn2 and NCLX also increase when cells are treated with mitochondrial ROS-inducing conditions, such as oligomycin treatment of respiring cells, while the interactions do not increase in Oma1 -/- cells. It seems likely that opening of cristae by Oma1-mediated cleavage of Opa1 promotes translocation of NCLX from cristae to the rim where it can come into contact with Mfn2 thus promoting PXA-induced calcium release from mitochondria. These results therefore delineate a pathway that connects ROS produced inside mitochondria with calcium release and signaling in the cytosol.
RESUMO
In addition to mitochondrial DNA, mitochondrial double-stranded RNA (mtdsRNA) is exported from mitochondria. However, specific channels for RNA transport have not been demonstrated. Here, we begin to characterize channel candidates for mtdsRNA export from the mitochondrial matrix to the cytosol. Down-regulation of SUV3 resulted in the accumulation of mtdsRNAs in the matrix, whereas down-regulation of PNPase resulted in the export of mtdsRNAs to the cytosol. Targeting experiments show that PNPase functions in both the intermembrane space and matrix. Strand-specific sequencing of the double-stranded RNA confirms the mitochondrial origin. Inhibiting or down-regulating outer membrane proteins VDAC1/2 and BAK/BAX or inner membrane proteins PHB1/2 strongly attenuated the export of mtdsRNAs to the cytosol. The cytosolic mtdsRNAs subsequently localized to large granules containing the stress protein TIA-1 and activated the type 1 interferon stress response pathway. Abundant mtdsRNAs were detected in a subset of non-small-cell lung cancer cell lines that were glycolytic, indicating relevance in cancer biology. Thus, we propose that mtdsRNA is a new damage-associated molecular pattern that is exported from mitochondria in a regulated manner.