Tubulin GTP hydrolysis influences the structure, mechanical properties, and kinesin-driven transport of microtubules

Abstract

Tubulin is a GTPase that hydrolyzes its bound nucleotide triphosphate after it becomes incorporated into a microtubule.The only known consequence of nucleotide hydrolysis is that it increases the dissociation rate of tubulin from the end of the microtubule by 2 orders of magnitude.In this study, we investigated whether microtubules composed of tubulin-GMPCPP (guanylyl cy,@methylenediphosphate) (a very slowly hydrolyzed GTP analog) or tubulin-GDP exhibit additional structural or functional differences.W e show that tubulin-GMPCPP microtubules are significantly stiffer than tubulin-GDP microtubules and have a 21% shallower protofilament twist angle.W e also find that kinesin, a microtubulebased motor protein, transports tubulin-GMPCPP microtubules at -30% faster rates than tubulin-GDP microtubules.These findings suggest that growing microtubule ends, which are thought to be composed of tubulin-GTP, may have different structural and mechanical properties from the remainder of the microtubule polymer.Microtubules, cylindrical polymers of C U , ~ tubulin subunits, are involved in various forms of cell motility including ciliary beating, movement of chromosomes during mitosis, and or- ganelle transport (1).Microtubules grow and shrink by adding subunits to their ends, and they exhibit considerable fluctuations in length even under steady state conditions.The growing and shrinking phases correspond to the net addition or loss of thousands of tubulin subunits, and transitions between these two states are stochastic and generally abrupt.This unusual behavior, termed dynamic instability (2), has been visualized for microtubules in vitro (3, 4) and in vivo (5)(6)(7).Dynamic instability enables the cell's microtubule network to turnover rapidly and thereby change its configuration.Dynamic instability, which does not occur with polymers that are in a simple binding equilibrium with a pool of monomers (81, is an energy-requiring phenomenon that is thought to be linked to GTP hydrolysis by tubulin.According to the most widely accepted model (the GTP cap hypothesis; reviewed in Ref. 91, microtubules grow when tubulin containing GTP (bound at the exchangeable site on the p subunit) adds to tu-