Peptide-specific antibodies as probes of the topography of the voltage-gated channel in the mitochondrial outer membrane of Neurospora crassa

Abstract

The voltage-dependent anion-selective channel (VDAC) in mitochondrial outer membranes is formed by a polypeptide (M(r) 31,000) coded by a nuclear gene whose cDNA sequence is known for several organisms. Antibodies have been raised against synthetic peptides corresponding to four different regions in the predicted sequence of the VDAC polypeptide of the fungus Neurospora crassa (residues 1-20, amino terminus; 195-210, 251-268, and 272-283, carboxyl terminus). Specificity of the antibodies has been characterized in terms of binding to peptides or fungal mitochondria on microtiter plates and binding to mitochondrial proteins of several species in Western blots. Reactivity of three of the four antibodies with fungal mitochondria in suspension increases with lysis of outer membranes, indicating that the respective epitopes (including those near the amino and carboxyl termini) are exposed on the surface of the outer membrane that faces inside the mitochondrion. Preincubation of mitochondria with a polyanion that modulates VDAC voltage dependence strongly inhibits binding of the antibody against residues 251-268, whose epitopes are on the outer mitochondrial surface.