VDAC1 Topology in the Outer Mitochondrial Membrane: The Final Answer

摘要: The first two authors equally contributed.Voltage Dependent Anion Channel (VDAC) is a pore forming protein located in outer mitochondrial membrane (1). Its lack lethal human (2) and it involved cellular cross-talk, important the apoptotic cascade (3). structure transmembrane β-barrel (4) organized 19 β-strands N-terminal α-helix probably for gating (5). However sidedness of VDAC inside still remains unresolved (4). This issue essential order to define structural determinants interacting with soluble proteins. We expressed, HeLa cells, recombinant hVDAC1 carrying C-terminal tag including hemagglutinin-tag (HA), specific caspase 3/7 cleavage site (DEVD) 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 are exposed protease digestion after plasma permeabilization. If C-terminus VDAC1 cytosolic side, we expect lose His cleavage. As result only HA detected at level. Conversely if oriented towards IMS, not cleavable both tags will mitochondria. Our results confocal microscopy appropriate controls other membrane-oriented constructs showed that this strategy able pore.1) Shoshan-Barmatz, V. et al (2010) Mol Aspects Med 31, 227-285.2) Huizing, M. (1994) 8924, 762-762.3) Tomasello M.F. (2009) Cell Res. 19, 1363-1376.4) De Pinto (2008) J. Bioenerg Biomemb 40, 139-147.5) (2007) Chembiochem 8, 744-756.Acknowledgements FIRB_PRIN Grants.