The outer mitochondrial membrane channel, VDAC, is modulated by a protein localized in the intermembrane space

The mitochondrial outer membrane channel, VDAC, provides a pathway for the flux of metabolites between the cytoplasm and mitochondrion. VDAC is voltage-dependent and occupies states of differing conductivity and ion selectivity that are dependent on transmembrane potential. A protein, derived from preparations of mitochondria, has been shown to increase the voltage dependence of VDAC and is called the VDAC modulator. Both VDAC and the VDAC modulator have been extensively characterized by reconstitution into planar lipid bilayers. In order for the VDAC modulator to have physiological significance it must have physical access to VDAC in the cell. This constraint dictates that the modulator be an extrinsic outer mitochondrial membrane protein, occupy the mitochondrial intermembrane space, or be a cytoplasmic constituent. To address the question of subcellular localization, purified mitochondria were selectively lysed with digitonin or treated with trypsin while resuspended in hypo-osmotic or iso-osmotic medium. Marker enzymes and modulator activity were monitored during the various treatments. Results indicate that the integrity of the outer membrane was necessary to prevent modulator release or protection from trypsin digestion. Outer membrane lysis, under conditions where the inner membrane remained intact, resulted in modulator release or inactivation by trypsin. These results suggest an intermembrane space location for the VDAC modulator in the mitochondrion.