Using Engineered Recombinant Tubulin Sotypes to Study VDAC Regulation

Abstract

Highly variable anionic C-terminal tails (CTTs) of tubulin had been implicated in regulation of permeability of the mitochondrial voltage-dependent anion channel (VDAC). Tubulin, a structural protein of microtubules, is a heterodimer comprising α/β subunits. In cells tubulin possesses significant variability due to the expression of several tubulin genes. Seven α- and eight β-tubulin isoforms in the human tubulin gene family vary mostly in the length and primary sequence of the CTTs. This inherent non-homogeneity of the endogenous tubulin CTTs hindered the analysis of their interaction with VDAC reconstituted into planar lipid bilayers. A recent study (Sheldon et al., 2015) with peptide-protein chimeras evaluated the frequency of the VDAC blockage events to demonstrate that the on-rate is highly dependent on the tail chemical composition. In that study CTT-like peptides were grafted to albumin, a protein that does not bind to the membrane surface, though it is now conclusively established that membrane binding of the tubulin body is an essential step in tubulin/VDAC interaction. Here we took an advantage of recently generated recombinant yeast α/β-tubulin constructs with CTTs from various human tubulin isotypes (Sirajuddin et al., 2014). The effects of each of the engineered constructs on the reconstituted VDAC were compared to those of w.t. yeast tubulin and recombinant tubulin without CTTs. Single-molecule studies of the tubulin-VDAC interaction showed that length and total anionic charge of the individual CTTs correlate strongly with the average duration of the blockage. In particular, at 30-mV applied voltage the characteristic time of VDAC blockage by the longer and more highly charged β3-CTT is up to hundredfold greater than that observed for the α1-CTT, with the apparent gating charge doubled. These results suggest novel implications in VDAC regulation by the diversity of tubulin isotypes found in vertebrates.