Redox-dependent gating of VDAC by mitoNEET

Abstract

MitoNEET is an outer mitochondrial membrane protein essential for sensing and regulation of iron and reactive oxygen species (ROS) homeostasis. It is a key player in multiple human maladies including diabetes, cancer, neurodegeneration, and Parkinson's diseases. In healthy cells, mitoNEET receives its clusters from the mitochondrion and transfers them to acceptor proteins in a process that could be altered by drugs or during illness. Here, we report that mitoNEET regulates the outer-mitochondrial membrane (OMM) protein voltage-dependent anion channel 1 (VDAC1). VDAC1 is a crucial player in the cross talk between the mitochondria and the cytosol. VDAC proteins function to regulate metabolites, ions, ROS, and fatty acid transport, as well as function as a "governator" sentry for the transport of metabolites and ions between the cytosol and the mitochondria. We find that the redox-sensitive [2Fe-2S] cluster protein mitoNEET gates VDAC1 when mitoNEET is oxidized. Addition of the VDAC inhibitor 4,4'-diisothiocyanatostilbene-2,2'-disulfonate (DIDS) prevents both mitoNEET binding in vitro and mitoNEET-dependent mitochondrial iron accumulation in situ. We find that the DIDS inhibitor does not alter the redox state of MitoNEET. Taken together, our data indicate that mitoNEET regulates VDAC in a redox-dependent manner in cells, closing the pore and likely disrupting VDAC's flow of metabolites.

Keywords: CISD1; VDAC1; direct coupling; ferroptosis; mitoNEET.

Fig. 1.

mNT binding to VDAC is redox state dependent. MST analysis used to assess the affinity of mNT to fluorescently labeled VDAC reconstituted in 1% DMPC/CHAPSO bicelles under oxidizing conditions results in measured K_d of 76 ± 2 nM (black). When mNT is reduced with DTT, binding is not observed (red).

Fig. 2.

VDAC channel conductance is inhibited by mNT. VDAC was reconstituted into a planar lipid bilayer and channel conductance was measured as a function of applied voltage. The recordings were taken before and after the addition of 5 μg/mL mNT (A) or NAF-1 (B). Conductance measurements were normalized to the conductance at 10 mV using the following formula: G/G_max = (Conductance/Maximal Conductance of Control).

Fig. 3.

Regions of mNT with increased protection upon interaction with VDAC. Selected plots of deuterium incorporation into peptides from regions with significant increases in protection upon complex formation are shown. An additional plot is given (peptide 93 to 108) that is representative of a region with no significant change in deuterium incorporation upon complex formation. Regions that exhibit significant increases in HD-exchange protection are highlighted on the mNT crystal structure (PDB ID code 2QH7) in blue, while regions with similar protection factors are shown in beige.

Fig. 4.

Regions of VDAC with increased protection upon interaction with mNT. Selected plots of deuterium incorporation into peptides from regions with significant increases in protection upon complex formation are shown. An additional plot is given (peptide 2 to 25) that is representative of peptides with no significant change in deuterium incorporation upon complex formation. This unaffected peptide corresponds to the N-terminal α-helix inside the VDAC barrel. Regions that exhibit significant increases in HD-exchange protection are highlighted on the VDAC crystal structure (PDB ID code 5XDO) in blue, and those without changes are in beige.

Fig. 5.

DIDS inhibits mNT–VDAC interaction. (A) MST analysis of the effect of DIDS on the binding of mNT to fluorescently labeled VDAC in 1% DMPC/CHAPSO bicelles under oxidizing conditions. The final DIDS concentration following mNT addition was 700 μM. (B and C) The effect of DIDS on mNT-induced changes in mitochondrial iron levels. H9c2 cells were preincubated with and without DIDS (100 µM) for 1 h, labeled with RPA, and permeabilized with digitonin to allow the entry of mNT into cells. The change in RPA fluorescence was followed every 5 min. Twenty micromolar mNT was added after 10 min. Five micromolar ferrous ammonium sulfate, complexed to equimolar hydroxyquinoline (FeHQ), which is a siderophore that allows iron to pass the membrane, was added after 45 min. RPA fluorescence is expressed in relative units (r.u.) obtained by analyzing individual cell fluorescence with ImageJ (open software), by averaging 5 cells per field.

Fig. 6.

Combined experimental and computational model of mNT docked to VDAC. Data from HDX-MS experiments and Fd-DCA calculations were combined to generate a model for the docking of the mNT dimer inside the VDAC pore. Increased protection mapping is indicated by darker blues, minimal protection by light blues, and no protection by tan. VDAC PDB ID code, 5XDO; MNT PDB ID code, 2QH7.