(55) TOPOLOGICAL ORIENTATION OF VDAC IN MITOCHONDRIA

J. Dawson¹, M. Forte² and M. Colombini^1
1Department of Biology, University of Maryland, College Park, College Park, MD 20742
²Vollum Institute, Oregon Health Science University, 3181 SW Sam Jackson Park Road, Portland, OR 97201

The permeability properties of the mitochondrial outer membrane are dominated by a channel-forming protein called VDAC (mitochondrial porin).  This 30 kDA protein acts as a monomer.  Its structure is inherently asymmetrical.  The transmembrane folding pattern of N. crassa VDAC reconstituted in planar phospholipid membranes was previously defined using site-directed mutagenesis.  Here we define the orientation of this protein in the mitochondrial outer membrane.  N. crassa VDAC (naturally lacks cysteine) containing a single engineered cysteine residue was expressed in a strain of S. cerevisiae lacking its native channel-forming VDAC, yeast VDAC1.  Mitochondria with intact outer membranes were isolated and biotinylated.  In a one-step centrifugation procedure, the mitochondria was washed, labeled with neutravidin, and excess neutravidin washed out, all under isotonic conditions.  Western blotting against VDAC was used to identify neutravidin-VDAC complexes.  The formation of these complexes demonstrate the extramitochondrial location of the site mutated to cysteine.  Sites located on the opposite side of the membrane did not form neutravidin complexes.  The orientation of VDAC in mitochondria is one in which both N and C termini face the cytosolic compartment.

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