Phosphate Release during Microtubule Assembly:  What Stabilizes Growing Microtubules?

Abstract

The molecular mechanism underlying microtubule dynamic instability depends on the relationship between the addition of tubulin-GTP to a growing microtubule and its hydrolysis in the microtubule lattice to tubulin-GDP, with release of inorganic phosphate (Pi). Since this relationship remains controversial, we have re-examined the release of Pi upon microtubule assembly using a fluorometric assay for Pi, based on the phosphate-binding protein of Escherichia coli [Brune M., Hunter, J. L., Corrie, J. E. T., and Webb, M. R. (1994) Biochemistry 33, 8262-8271]. Microtubule assembly and Pi release were monitored simultaneously in a standard fluorimeter as an increase in the turbidity and fluorescence, respectively, in tubulin-GTP solutions assembled under conditions supporting dynamic instability. At the steady state of assembly, Pi release is nonlinear with respect to time, proceeding at a rate determined by the following: (a) the intrinsic GTPase activity of the nonpolymerized tubulin-GTP, and (b) the microtubule number concentration, which decreases progressively. Direct observation of the time course of nucleated microtubule assembly indicates that Pi release is closely coupled to microtubule elongation, even during the initial stages of assembly when uncoupling of tubulin-GTP addition and GTP hydrolysis would be most evident. Studies of the inhibition and reversal of the growth phase by cytostatic drugs show no evidence of a burst of Pi release. We conclude that nucleotide hydrolysis can keep pace with tubulin-GTP addition rates of 200 molecules per second per microtubule and that extended caps of tubulin-GTP or tubulin-GDP-Pi are not generated in normal assembly, nor are they required to stabilize growing microtubules or to support the phenomenon of dynamic instability of microtubules at the steady state.

Intrinsic microtubule GTP-cap dynamics in semi-confined systems: kinetochore–microtubule interface

“…3a). This result is highly comparable with the experimental results [5,[34][35][36] and can explain apparent differences among the experimental results. This result serves as an excellent test of the .…”

Altered tubulin assembly dynamics with N‐homocysteinylated human 4R/1N tau in vitro

“…At the steady state, aliquots of the sample were diluted to different final concentrations of tubulin (C t ), followed by reachievement of the steady state and separation of unassembled tubulin from MTP by ultracentrifugation. After passing through a P11 phosphocellulose column (Whatman, Piscataway, NJ), the tubulin concentration in the supernatant (C s ) was determined by the Bradford reagent (Bio-Rad Laboratories, Hercules, CA), using serum albumin as standard and C c and F i were calculated as the intercept and slope of a plot of C s versus C t , respectively [25].…”

Variations in the colchicine-binding domain provide insight into the structural switch of tubulin

“…To be competent for polymerization, tubulin should be in a GTP state, i.e., with GTP bound to the ␤-subunit (the GTP molecule bound to ␣ is neither hydrolyzed nor exchanged). GTP hydrolysis accompanies microtubule assembly, and GDP-tubulin is released upon disassembly (2). Tubulin also undergoes a structural cycle that is deciphered only in part.…”