Microtubule Dynamics of S. Pombe Tubulin

Abstract

We have developed a fission yeast system for expression and purification of single isoform tubulin. Replacing the non-essential S. pombe α2 tubulin gene with a second copy of the α1 encoding gene creates a strain expressing single α1 and β tubulin isoforms. We obtain yields of about 10 mg of highly purified (>99%) single isoform tubulin from an 80 l culture. Mass spectroscopy of the purified tubulin detects no post-translational modifications. Dynamic microtubules were nucleated from axonemes, recorded by video enhanced DIC microscopy and microtubule lengths measured. Microtubule growth rates increase linearly with tubulin concentration at both the fast and slow growing microtubule ends. Rate constants were determined using a simple bimolecular model. α1β tubulin has fast end kon 5.5 uM−1 s−1 and koff 6.7 s−1, and slow end kon 3.1 uM−1 s−1 and koff 2.8 s−1. Mixed isoform α1α2β has fast end kon 7.6 uM s−1 and koff 15.1 s−1 and slow end kon 2.6 uM−1 s−1 and koff 5.0 s−1. The Kd for both microtubule ends was ∼2uM for α1α2β and ∼1.1 uM for α1β suggesting similar binding affinities for tubulin heterodimers at fast and slow ends, but different kinetics. Following catastrophe the rate of rapid shrinkage was independent of the free tubulin concentration and about 2x faster at the fast compared to the slow ends: 218 ± 16 s−1 compared to 124 ± 23 s−1 for α1α2β and 263 ± 26 s−1 compared to 111 ± 29 s−1 for α1β. We conclude that S. pombe microtubule dynamics are qualitatively similar to those of brain tubulin microtubules but the kinetic rates are different, consistent with S. pombe tubulin assembling at lower concentrations than brain tubulin. We are now comparing S. pombe microtubule growth with that of brain tubulin at higher resolution.