Using VDAC-Tubulin Interaction to Assess VDAC Orientation in the Mitochondrial Membrane

Abstract

Recently we have found that dimeric αβ-tubulin at nanomolar concentrations induces reversible partial blockage of VDAC channel, reconstituted into a planar lipid membrane (Rostovtseva et al., PNAS 2008). Tubulin induces characteristic, well resolved fast blocking events with the highly voltage-dependent binding parameters. Tubulin interaction with VDAC requires the presence of C-terminal tails (CTT) of tubulin. Tubulin with truncated CTT did not induce reversible blockage typical for intact tubulin. We propose a model in which negatively charged CTT of tubulin penetrates into VDAC pore, reaching through the channel at application from both sides of the membrane and interacting with high affinity with the positively charged channel lumen. We used the VDAC-tubulin specific interaction feature to probe orientation of VDAC in a planar membrane and extrapolate the results to the mitochondria outer membrane. We found that after in vitro phosphorylation by PKA or GSK3β cytosolic kinases the tubulin binding to VDAC (from rat liver mitochondria) becomes highly asymmetrical. When tubulin was added to the cis side of the membrane (side of VDAC addition) kon was more than 10 times higher than with tubulin added to the trans side. Untreated VDAC interacts symmetrically with tubulin. Considering putative PKA and GSK3b phosphorylation sites on the cytosolic loops 3, 5 and 7, we conclude that these loops face cis side in the VDAC reconstitution system. Our preliminary data show that some of the specific antibodies raised against different VDAC peptides, compete with tubulin-VDAC binding when added to the membrane bathing solution and therefore could be employed to probe VDAC orientation and positioning of the loops. Recent VDAC three-dimensional structures are compared with the functional data of VDAC-tubulin binding.