Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP.
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.
Hyman
A A
AA
Department of Pharmacology, University of California, San Francisco 94143.
Salser
S
S
Drechsel
D N
DN
Unwin
N
N
Mitchison
T J
TJ
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008854:Microscopy, Electron; D008870:Microtubules / Q000378:metabolism* / Q000648:ultrastructure; D011108:Polymers / Q000378:metabolism
D011108:Polymers; C004805:5'-guanylylmethylenebisphosphonate; D006160:Guanosine Triphosphate