Abstract
Stu2 is the budding yeast member of the XMAP215/Dis1 family of microtubule polymerases. It is essential in cell division, allowing proper spindle orientation and metaphase chromosome alignment, as well as spindle elongation during anaphase. Despite Stu2 having a phenotype that suggests it promotes microtubule growth, like the other members of the XMAP215/Dis1 family, previous studies with purified Stu2 indicate only that it antagonizes microtubule growth. One potential explanation for these contradictory findings is that the assays were performed with mammalian brain tubulin, which may not be the right substrate to test the activity of Stu2 given that yeast and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications. To test this possibility, we reconstituted the activity of Stu2 with purified budding yeast tubulin. We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin. Furthermore, Stu2 accelerated polymerization in yeast tubulin to a much greater extent than in porcine brain tubulin, and the concentration of Stu2 required to reach 50% maximum activity in yeast tubulin was nearly 2 orders of magnitude lower than that in porcine brain tubulin. We conclude that Stu2 is a microtubule polymerase, like its relatives, and that its activity is considerably higher in yeast tubulin compared with mammalian brain tubulin. The biochemical properties of Stu2 reported here account for many of the phenotypes of Stu2 observed in cells.
Highlights
The reported inhibition of microtubule growth by Stu2 is difficult to reconcile with its cellular phenotypes
We found that Stu2 accelerated microtubule growth in yeast tubulin by severalfold, similar to the acceleration reported for XMAP215 in porcine brain tubulin
Stable microtubules were grown from porcine brain tubulin, 25% of which was labeled with rhodamine, in the presence of GMP-CPP, a slowly hydrolyzable GTP analog [21]
Summary
The reported inhibition of microtubule growth by Stu is difficult to reconcile with its cellular phenotypes. Studies with purified Stu have shown that it slows, rather than accelerates, elongation of microtubules grown in brain tubulin [12] These authors reported that Stu promotes catastrophe, the transition of growing microtubules to shrinking ones, which often correlates with slower growth rates. One interpretation of these results is that not all members of the XMAP215/Dis family are polymerases and that the shared protein domains, notably the TOG domains [13, 14], may have divergent activities. Stu Is a Microtubule Polymerase and brain tubulins are quite divergent in sequence and that the vertebrate tubulins are subject to many post-translational modifications (see “Discussion”) To test this possibility, we reconstituted the polymerization activity of Stu with budding yeast tubulin
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