Alp7 Potentiates Microtubule Tip Tracking by the Processive Plus End Polymerase Alp14

TOG-family polymerases track microtubule plus ends in vivo and modulate microtubule dynamics. Several different model mechanisms for the molecular mechanism of action of these proteins have been proposed (Kerssemakers et al. (2006) Nature 442:709; Al-Bassam et al (2006) JCB 172:1009; Brouhard et al. (2008) Cell 132:79). To test candidate models in more detail, we have expressed and purified Alp14 and Alp7 using a baculovirus system and reconstituted their activities in vitro using dynamically unstable microtubules built from purified single isoform (a1b) S.pombe tubulin. Alp14 deletion mutants show an in vivo defect in microtubule assembly (Sato et al. (2004) MCB 15:1609), consistent with an effect on microtubule dynamics. We find that in vitro, Alp14 accelerates the shrinkage of GMPCPP microtubules 2x and the growth of GTP-microtubules up to 10x. Changes in growth rate within a growth phase correspond to changes in the amount of Alp14 at the tip. The acceleration of growth by Alp14 causes a decrease in catastrophe frequency consistent with extension of the GTP-cap. Remarkably, although Alp14 binds to mammalian brain tubulin, it does not accelerate the growth of mammalian brain microtubules. Instead, Alp14 is competitively inhibited by mammalian brain tubulin. Tip tracking by Alp14 is tightly linked to the catalysis of microtubule growth: Alp14 loses its tip tracking ability upon the addition of 10% mammalian brain tubulin. The addition of the TACC-protein Alp7 restores the tip-tracking ability of Alp14, but not its ability to enhance the microtubule growth rate. This result is consistent with reports that Alp7 is a localization factor of Alp14 in vivo (Sato et al. (2004) MCB 15:1609). On dynamic S.pombe microtubules, Alp7 enhances the processivity of Alp14, causing sustained fast growth and correspondingly reduced catastrophe.