Regulation of S. pombe microtubule dynamics by Alp14, Alp7 and Dis1

• Main Author: Hussmann, Frauke
• Published: Institute of Cancer Research (University Of London) 2011
• Subjects: 571.654
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.551112

TOG-family polymerases track microtubule plus ends and modulate dynamics in vivo. In vitro XMAP215 and Stu2 have been shown to exhibit opposing activities, which led to conflicting proposals for molecular mechanisms. To understand more about these apparently contradictory findings and to test candidate models in more detail, I used an in vitro system in which all the components derived from the same organism, Sacchammyce pombe. Deletion mutants of Alp14 and Dis1, the XMAP215 orthologues in S. pombe, show defects in microtubule assembly, consistent with an effect on microtubule dynamics. I have expressed and purified Alp14 and Disl and the TACC-protein Alp7 using a baculovirus system and reconstituted their activities in vitro using dynamically unstable single micro tubules built from purified single isoform (cd and (3)) S. pombe tubulin. In this work I show that Alp14 accelerates the shrinkage of GMP-CPP stabilized microtubules 2x while it accelerates the assembly of GTP-S. pombe tubulin into micro- tubules J Ox. Growth rates change within a growth phase, corresponding to different amounts of Alp14 at the tip. The growth rate acceleration by Alp14 causes a decrease in catastrophe frequency consistent with the extension of the GTP-cap. Remark- ably, although Alp14 binds to mammalian brain tubulin, it does not accelerate the growth rate of mammalian brain micro tubules, and instead is competitively inhibited by 10% mammalian brain tubulin, indicating a catalytic mechanism for Alp14. Catalytic activity and tip tracking are tightly coupled, but separable. Alp14 loses its tip tracking ability upon the addition of 10% mammalian brain tubulin. The TACC-protein Alp7 can restore the tip-tracking ability of Alp14 in the presence of mammalian brain tubulin but not its activity, consistent with reports that Alp7 is a localization factor of AJp14 in vivo. On dynamic S. pombe microtubules Alp7 increases the processivity of Alp14 and suppresses the catastrophe frequency. Growth rates are not further elevated by the addition of Alp7. In contrast, Disl has no TACC-protein interacting partner. Like Alp14 it disassembles GMP-CPP microtubules and increases the growth rate of S. pombe microtubules. Likewise it binds to mammalian brain tubulin without the ability to increase the growth rate of mammalian brain microtubules. However, unlike Alp14, Dis1 is able to tip-track on dynamic mammalian brain microtubules. As both Alp14 and Disl function as S. pombe microtubule polymerases, but not as mammalian brain microtubule polymerases, I concluded that not only the association of tubulin to XMA P215-family members is important for proper function but also that the ready dissociation of tubulin is just as important. Slow dissociation inhibits the activity of XMAP215-family member, supporting a catalytic model. Furthermore I found that higher affinity to the microtubule lattice did not enhance the catalytic activity of Alp14. Instead the higher affinity to the microtubule lattice of Alp14 in the presence of Alp7 increased the residence time of Alp14 at the tip. In return this decreased the catastrophe frequency, enhancing the effectiveness of Alp14 as a polymerase at the microtubule tip. Disl possesses an intrinsic high affinity to the microtubule lattice, tracking the microtubule end readily, but does not exhibit higher polymerase activity than Alp14. On the basis of these data, I propose an assembler model for the molecular mechanism of Alp14-Alp7 and Dis l , in which both XMAP215- family members enhance the growth rate of S. pombe microtubules by accelerating the exchange reaction of GTP-tubulin. High processivity and in return high effectiveness of the catalysts are mediated by Alp7 in the case of Alp14 and by internal microtubule lattice binding regions in the case of Disl.