Sequence diversity of tubulin isotypes in regulation of the mitochondrial voltage-dependent anion channel

Tatiana K Rostovtseva,Philip A Gurnev,David P Hoogerheide,Amandine Rovini,Minhajuddin Sirajuddin,Sergey M Bezrukov

doi:10.1074/jbc.ra117.001569

Abstract

The microtubule protein tubulin is a heterodimer comprising α/β subunits, in which each subunit features multiple isotypes in vertebrates. For example, seven α-tubulin and eight β-tubulin isotypes in the human tubulin gene family vary mostly in the length and primary sequence of the disordered anionic carboxyl-terminal tails (CTTs). The biological reason for such sequence diversity remains a topic of vigorous enquiry. Here, we demonstrate that it may be a key feature of tubulin's role in regulation of the permeability of the mitochondrial outer membrane voltage-dependent anion channel (VDAC). Using recombinant yeast α/β-tubulin constructs with α-CTTs, β-CTTs, or both from various human tubulin isotypes, we probed their interactions with VDAC reconstituted into planar lipid bilayers. A comparative study of the blockage kinetics revealed that either α-CTTs or β-CTTs block the VDAC pore and that the efficiency of blockage by individual CTTs spans 2 orders of magnitude, depending on the CTT isotype. β-Tubulin constructs, notably β3, blocked VDAC most effectively. We quantitatively described these experimental results using a physical model that accounted only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. Based on these results, we speculate that the effectiveness of VDAC regulation by tubulin depends on the predominant tubulin isotype in a cell. Consequently, the fluxes of ATP/ADP through the channel could vary significantly, depending on the isotype, thus suggesting an intriguing link between VDAC regulation and the diversity of tubulin isotypes present in vertebrates.

Highlights

The microtubule protein tubulin is a heterodimer comprising ␣/␤ subunits, in which each subunit features multiple isotypes in vertebrates
Using recombinant yeast ␣/␤-tubulin constructs with ␣-carboxyl-terminal tails (CTTs), ␤-CTTs, or both from various human tubulin isotypes, we probed their interactions with voltage-dependent anion channel (VDAC) reconstituted into planar lipid bilayers
To evaluate molecular details of the CTT–VDAC interaction, we examined five previously described yeast tubulin-human CTT recombinant constructs [3] for their ability to interact with VDAC reconstituted into planar lipid membranes formed from diphytanoyl-phosphatidylcholine

Summary

Introduction

The microtubule protein tubulin is a heterodimer comprising ␣/␤ subunits, in which each subunit features multiple isotypes in vertebrates. ␤-Tubulin constructs, notably ␤3, blocked VDAC most effectively We quantitatively described these experimental results using a physical model that accounted only for the number and distribution of charges in the CTT, and not for the interactions between specific residues on the CTT and VDAC pore. VDAC was shown to be involved in a wide variety of mitochondria-associated pathologies, from various forms of cancer to neurodegeneration [17,18,19] The uniqueness of this large, weakly selective ␤-barrel channel arises mainly from its location at the interface between the mitochondria and the cytosol [20], where it serves as a pathway for all mitochondrial water-soluble respiratory substrates, such as ATP and ADP, and for small ions [21,22,23,24]. The ionic current through the VDAC channel is induced by the applied transmembrane potential and is monitored for changes induced by the addition of dimeric tubulin, as illustrated in Fig. 1, A and B

Results

Discussion

Conclusion