Human Disease-Related Mutation at R262 of β3-Tubulin Critical for Kinesin Motility and ATP Hydrolysis

Abstract

Kinesin-microtubule interaction plays essential roles in brain development and function. In human neuronal cells, a point mutation, R262C/H, in β3-tubulin causes a disrupton in the interaction of microtubules with kinesin, leading to developmental disorders collectively termed TUBB3 syndrome (Tischfield, 2010). To clarify the molecular mechanism underlying the disease, in vitro functional analysis of mutant microtubules is essential. Aiming to examine the effect of tubulin mutation on kinesin motility and ATPase activity, here we developed a method to express and purify recombinant human tubulin using a baculovirus-insect-cell expression system. The method yields >1 mg of human α1β3-tubulin with a purity of >95% from 1 L culture of High Five cell. In a single-molecule motility assay, while kinesin moved along wild type (WT) human microtubules at a velocity comparable to that on porcine brain microtubules, it scarcely bound to β3-R262A microtubules. In parallel with this observation, in β-R262A mutants, the maximum ATPase rate (kcat) was reduced four-fold and the Michaelis constant (kM,MT) was 40 times larger than that of WT. These results clearly demonstrate the involvement of β-R262 in kinesin-microtubule interaction. Compared with other critical residues previously identified using yeast mutant microtubules (Uchimura 2010), β-R262 is unusual because it is the only positively charged residue critical for kinesin-microtubule interaction. Whether the β-R262 residue directly binds to a putatively negatively charged residue in kinesin or modulates kinesin-microtubule interaction through repulsion will be discussed at the poster session. Our new method provides a powerful tool to perform molecular level analyses on the function of tubulin/microtubules associated with human diseases.