CYTOSOLIC PROTEIN INTERACTION WITH THE VOLTAGE DEPENDENT ANION CHANNEL OF THE MITOCHONDRIAL OUTER MEMBRANE CONTROLS CELLULAR RESPIRATION
Sheldon, Kely Lee
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The voltage dependent anion channel (VDAC) is a beta-barrel protein that spans the mitochondrial outer membrane and serves as the passageway for the flux of metabolites responsible for mitochondrial function. It was discovered in 2008 that the cytosolic protein dimeric tubulin interacts with VDAC causing highly reversible blockages of the channel (Rostovtseva and Bezrukov 2008). It was later shown that this tubulin-blocked state of VDAC is impermeable for ATP (Gurnev, Rostovtseva et al. 2011; Noskov, Rostovtseva et al. 2013), suggesting that this protein-protein interaction may be responsible for the regulation of respiration. Here, we expand on the current understanding of VDAC demonstrating a possible control mechanism of this tubulin-VDAC interaction. We show that when VDAC is phosphorylated in vitro by both protein kinase A (PKA) or glycogen-synthase kinase (GSK) and reconstituted into a planar lipid bilayer the on-rate of tubulin-VDAC interaction is increased by over two orders of magnitude. Our results were confirmed in subsequent whole cell experiments using microtubule stabilizing or destabilizing agents to alter the concentration of free tubulin in the cytosol. The increase and decrease of local tubulin concentration altered mitochondrial potential as measured by tetramethylrhodamine methyester (TMRM). Moreover, when cells were treated with small synthetic peptide protein kinase inhibitor (PKI) inhibiting the activity of PKA, mitochondrial potential was increased, even after treatment of cells with microtubule destabilizing agent cholchicine. To explore the role that each VDAC isoform plays in the modulation of mitochondrial potential, samples of single VDAC isoforms were isolated from HepG2 cells treated with siRNA against two VDAC isoforms and reconstituted into planar lipid bilayers. Remarkably, although the VDAC isoforms displayed similar single channel characteristics, each isoform exhibited markedly different sensitivities to both voltage induced gating, with VDAC2 being the most voltage sensitive, followed by VDAC1 and VDAC3, respectively, and to tubulin interaction, with VDAC1 and VDAC2 having tubulin-VDAC on-rates nearly two orders of magnitude higher than VDAC3. Taken together, we speculate that cellular respiration may be influenced by the local tubulin concentration, the expression rates of each VDAC isoform, the phosphorylation state of VDAC, and the potential across the MOM.