Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.
Open Access
- 1 October 1992
- journal article
- Published by American Society for Cell Biology (ASCB) in Molecular Biology of the Cell
- Vol. 3 (10), 1155-1167
- https://doi.org/10.1091/mbc.3.10.1155
Abstract
The role of GTP hydrolysis in microtubule dynamics has been reinvestigated using an analogue of GTP, guanylyl-(alpha, beta)-methylene-diphosphonate (GMPCPP). This analogue binds to the tubulin exchangeable nucleotide binding site (E-site) with an affinity four to eightfold lower than GTP and promotes the polymerization of normal microtubules. The polymerization rate of microtubules with GMPCPP-tubulin is very similar to that of GTP-tubulin. However, in contrast to microtubules polymerized with GTP, GMPCPP-microtubules do not depolymerize rapidly after isothermal dilution. The depolymerization rate of GMPCPP-microtubules is 0.1 s-1 compared with 500 s-1 for GDP-microtubules. GMPCPP also completely suppresses dynamic instability. Contrary to previous work, we find that the beta--gamma bond of GMPCPP is hydrolyzed extremely slowly after incorporation into the microtubule lattice, with a rate constant of 4 x 10(-7) s-1. Because GMPCPP hydrolysis is negligible over the course of a polymerization experiment, it can be used to test the role of hydrolysis in microtubule dynamics. Our results provide strong new evidence for the idea that GTP hydrolysis by tubulin is not required for normal polymerization but is essential for depolymerization and thus for dynamic instability. Because GMPCPP strongly promotes spontaneous nucleation of microtubules, we propose that GTP hydrolysis by tubulin also plays the important biological role of inhibiting spontaneous microtubule nucleation.Keywords
This publication has 39 references indexed in Scilit:
- Buffer conditions and non‐tubulin factors critically affect the microtubule dynamic instability of sea urchin egg tubulinCell Motility, 1992
- Microtubule Dynamics: Mechanism, Regulation, and FunctionAnnual Review of Cell Biology, 1991
- [39] Preparation of modified tubulinsMethods in Enzymology, 1991
- Role of GTP hydrolysis in microtubule polymerization: evidence for a coupled hydrolysis mechanismBiochemistry, 1990
- GTP analogs interact with the tubulin exchangeable site during assembly and upon bindingBiochemistry, 1990
- Role of Nucleotide Hydrolysis in the Dynamics of Actin Filaments and MicrotubulesInternational Review of Cytology, 1989
- Polewards chromosome movement driven by microtubule depolymerization in vitroNature, 1988
- Direct observation of steady-state microtubule dynamics.The Journal of cell biology, 1986
- Examination of tubulin-nucleotide interactions by protein fluorescence quenching measurementsBiochemical and Biophysical Research Communications, 1978
- Microtubule Assembly and NucleationInternational Review of Cytology, 1978