Holden M J, Colombini M
Department of Zoology, University of Maryland, College Park 20742.
Biochim Biophys Acta. 1993 Oct 4;1144(3):396-402. doi: 10.1016/0005-2728(93)90126-z.
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.
线粒体外膜通道孔蛋白（VDAC）为细胞质与线粒体之间的代谢物通量提供了一条途径。VDAC是电压依赖性的，占据着不同电导率和离子选择性的状态，这些状态取决于跨膜电位。一种源自线粒体提取物的蛋白质已被证明可增加VDAC的电压依赖性，被称为VDAC调节剂。VDAC和VDAC调节剂都已通过重组到平面脂质双分子层中进行了广泛的表征。为了使VDAC调节剂具有生理意义，它必须能够在细胞中实际接触到VDAC。这一限制表明调节剂是一种外在的线粒体外膜蛋白，占据线粒体膜间隙，或者是一种细胞质成分。为了解决亚细胞定位问题，用洋地黄皂苷选择性地裂解纯化的线粒体，或在低渗或等渗培养基中重悬时用胰蛋白酶处理。在各种处理过程中监测标记酶和调节剂活性。结果表明，外膜的完整性对于防止调节剂释放或免受胰蛋白酶消化是必要的。在内膜保持完整的条件下进行外膜裂解，会导致调节剂释放或被胰蛋白酶灭活。这些结果表明线粒体中的VDAC调节剂位于膜间隙。
