Abstract 3044: The ability of tubulin to close mitochondrial VDAC pores depends on beta tubulin isotype
Authors: Sheldon Kely L. , Sackett Dan L. 
Published: August 2015

Abstract
Tubulin heterodimers can contain any of a number of beta-tubulin isotypes, and differential beta isotype expression has been found to have prognostic significance in human cancer and in chemotherapy response. We have shown that tubulin dimers can bind to and close the pore of the mitochondrial outer membrane protein VDAC (Voltage Dependent Anion Channel). VDAC acts as the “Gatekeeper” of metabolite flux into or out of the mitochondria, including all movement of ADP and ATP (and hence is a regulator of oxidative phosphorylation), and is also involved in early steps of apoptosis. We wished to test if all beta isotypes are equivalent in their ability to close VDAC, or if there are differences which may correlate to isotype expression in patient tumors. We previously showed that tubulin closure of VDAC blocks channel traffic and reduces oxidative metabolism, and that this requires the unstructured anionic tail peptides found on both the alpha- and beta-tubulin subunits. These intrinsically disordered tails contain only ∼3% of the mass but ∼40% of the net charge on the tubulin heterodimer. While all of the tails share some features, there are significant differences in length and sequence between alpha- and beta-tubulin tails, and between beta tubulin isotypes. Our earlier studies left unclear if the alpha- and beta-tails contribute equally to VDAC blockade, whether tails from different beta isotypes are all equivalent, and the role, if any, of tubulin posttranslational modifications (PTM), which mostly occur on the tails.
Here we use single molecule binding studies to show that channel closure is due to the tails alone, and not the tubulin body, since grafting the tails to albumin gives the same VDAC closure extent as tubulin. The method involves electrophysiological recording from single VDAC molecules inserted in lipid bilayers and exposed to controlled concentrations of tubulin or BSA-tail constructs. We show that the beta-I tail is ∼100-fold more potent than the alpha-tail, and that this difference is largely due to the presence or absence of a terminal tyrosine on alpha. The activation of the alpha-tail by detyrosination is reversed by subsequent removal of the next residue. We also show that BSA-tail constructs from different beta isotypes differ widely in their VDAC closure activity. In particular, beta-I tails are the most potent of those tested, while beta-III is at least 100-fold less active, although the sequence differences between the two isotypes are not great. Other beta isotypes show intermediate levels of closure activity.
Our results demonstrate that small changes in sequence or PTM of the disordered tails of tubulin result in substantial changes in VDAC binding and closure. Sequence differences between beta tubulin isotypes have major consequences on VDAC closure activity. In particular switching from the common epithelial beta-I to the beta-III isotype results in at least 100-fold reduction of VDAC closure.
Citation Format: Kely L. Sheldon, Dan L. Sackett. The ability of tubulin to close mitochondrial VDAC pores depends on beta tubulin isotype. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3044. doi:10.1158/1538-7445.AM2015-3044