Abstract
Voltage-Dependent Anion selective Channels (VDAC) are pore-forming mitochondrial outer membrane proteins. In mammals VDAC3, the least characterized isoform, presents a set of cysteines predicted to be exposed toward the intermembrane space. We find that cysteines in VDAC3 can stay in different oxidation states. This was preliminary observed when, in our experimental conditions, completely lacking any reducing agent, VDAC3 presented a pattern of slightly different electrophoretic mobilities. This observation holds true both for rat liver mitochondrial VDAC3 and for recombinant and refolded human VDAC3. Mass spectroscopy revealed that cysteines 2 and 8 can form a disulfide bridge in native VDAC3. Single or combined site-directed mutagenesis of cysteines 2, 8 and 122 showed that the protein mobility in SDS-PAGE is influenced by the presence of cysteine and by the redox status. In addition, cysteines 2, 8 and 122 are involved in the stability control of the pore as shown by electrophysiology, complementation assays and chemico-physical characterization. Furthermore, a positive correlation between the pore conductance of the mutants and their ability to complement the growth of porin-less yeast mutant cells was found. Our work provides evidence for a complex oxidation pattern of a mitochondrial protein not directly involved in electron transport. The most likely biological meaning of this behavior is to buffer the ROS load and keep track of the redox level in the inter-membrane space, eventually signaling it through conformational changes.
Highlights
The voltage-dependent anion selective channels (VDAC) are a family of proteins whose primary role is to transport hydrophilic metabolites and molecules through the mitochondrial outer membrane (MOM)
The exposure of the human VDAC3 (hVDAC3) cysteines suggests that they are highly accessible to soluble oxidative molecules and for this reason they are possibly linked to some specific biological function
While hVDAC1 showed the expected electrophoretic migration corresponding to the estimated Mr (35 kDa), in the various conditions, hVDAC3 was almost completely present in the top of the gel, most probably in an aggregated form, even though minor bands were visible at the expected Mr of the monomeric form and at intermediate migration levels
Summary
The voltage-dependent anion selective channels (VDAC) are a family of proteins whose primary role is to transport hydrophilic metabolites and molecules through the mitochondrial outer membrane (MOM). The swapping chimera hN1-VDAC3 restored the growth of Δpor yeast cell on glycerol at 37°C [20] and, upon reconstitution in planar bilayers, inserted into the membrane, showing a channel conductance comparable to VDAC1 [21]. This result prompted us to analyze in detail the N-terminal sequence of VDAC3. We have investigated the redox state of cysteines both in a recombinant human VDAC3 protein and in rat mitochondrial VDAC3. We studied the results of cysteine removal in terms of the modification of the biological and biophysical features of the protein
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