Regulation of Mitochondrial Respiration by Different Tubulin Isoforms in Vivo

Abstract

Studies of regulation of mitochondrial outer membrane selective permeability of cardiac and brain cells in vitro (isolated mitochondria) and in situ (permeabilized cells) condition has revealed remarkable differences between kinetic parameters of respiration activation. This is probably a result of the specific control of voltage dependent anion channel (VDAC) permeability by some cytoskeleton proteins, among them may be some isoforms of tubulin. Our recent results with immunofluorescence and Western blot analysis have shown that possible candidates could be β-tubulin isoforms 2c (IVb) or 4 (IVa). The aim of this study was to construct, express and purify different β-tubulin isoforms and their C-terminal tail truncated recombinants and to investigate their effect on regulation of mitochondrial respiration.Therefore we have designed a GST-tagged β-tubulin isoform constructs to express the recombinant proteins in BL21(DE3)pLys cell line. The proteins were purified in native conditions and utilized to study the kinetics of mitochondrial respiration regulation. The reconstitution experiments with isolated brain mitochondria and GST-TUBB2c or with GST-TUBB4 were carried out. The preliminary results showed that using GST-TUBB2c there were two populations of mitochondria with different properties; the Km value for ADP of the second population of mitochondria was significantly higher, compared to the first population. Therefore, it's possible to conclude that β2c-tubulin is one possible candidate of factor X, which is able to restore the low permeability of VDAC. As the β-tubulin C-terminal tail has been shown to be responsible for selective regulation of VDAC permeability, we have also produced truncated β-tubulin isoforms which lack the C-terminal tail and investigated their effect on mitochondrial respiration. Our results show that β-tubulin 2c isoform is able to restore the diffusion restrictions for ADP which is common for permeabilized cells with oxidative metabolism.