VDAC1 topology in the outer mitochondrial membrane: the final answer.

Voltage Dependent Anion Channel (VDAC) is a pore forming protein located in outer mitochondrial membrane (1). Its lack is lethal in human (2) and it is involved in cellular cross-talk, important in the apoptotic cascade (3). Its structure is a transmembrane β-barrel (4) organized in 19 β-strands and a N-terminal α-helix probably important for gating (5). However the sidedness of VDAC inside the membrane still remains unresolved (4). This issue is essential in order to define the pore structural determinants interacting with soluble proteins. We expressed, in HeLa cells, a recombinant hVDAC1 carrying C-terminal tag including the hemagglutinin-tag (HA), the specific caspase 3/7 cleavage site (DEVD) and 7 histidine residues (7xHis). DEVD amino acidic sequence can be cleaved upon apoptosis induction by staurosporine. A complementary Fluorescence Protease Protection assay was performed where cell protein are exposed to protease digestion after plasma membrane permeabilization. If the C-terminus of VDAC1 is exposed to the cytosolic side, we expect to lose the His tag upon DEVD cleavage. As result only the HA can be detected at mitochondrial level. Conversely if the C-terminus is oriented towards the IMS, the DEVD is not cleavable and both tags will be detected in mitochondria. Our results by confocal microscopy and appropriate controls with other membrane-oriented constructs showed that this strategy is able to define the sidedness of the VDAC pore.