Collective effect of Vigna sp. (mung) tubulin GTP hydrolysis rate divergence on microtubule filament assembly.

Abstract

Microtubules (MTs) are dynamic cytoskeletal filaments with highly conserved sequences across evolution, polymerizing by the GTP-dependent assembly of tubulin subunits. Despite the sequence conservation, MT polymerization kinetics diverge quantitatively between vertebrate brain, the model plant Arabidopsis and the protozoan Plasmodium. Previously, tubulin purified from seedlings of the plant Vigna sp. (mung) by temperature cycling was found to have a very low critical concentration. However, the lengths of MTs were sub-micron, much shorter than brain tubulin filaments. This was explained in simulations to be the result of the collective effect of high nucleation and GTP hydrolysis rates. Here, we test the effect of GTPase rates of affinity-purified Vigna sp. tubulin on microtubule polymerization and elongation. Affinity-purified mung tubulin is active and has a critical concentration of .37 μM. The GTP-dependent polymerization kinetics are transient, consistent with previous results. Polymerization is stabilized in the presence of either GTP analog GMPPNP (non-hydrolyzable) or GMPCPP (slow-hydrolyzable). Using interference reflection microscopy (IRM) we find polymerization with the non-hydrolysable analog significantly increases filament numbers, while lengths are unaffected for both GTP analogs. However, prolonged incubation with slow-hydrolyzable GMPCPP results in long filaments, pointing to GTP hydrolysis as a key factor determining MT length. We find the average GTPase turnover number of mung tubulin is 22.8 min-1, compared to 2.04 min-1 for goat brain tubulin. Thus modulating GTPase rates affects both nucleation and elongation. This quantitative divergence in kinetics despite high sequence conservation in the GTPase domains of α- and β-tubulin could help better understand the roles of selective pressure and function in the diverse organisms.