Role of GTP hydrolysis in microtubule treadmilling and assembly.

GTP hydrolysis accompanies addition of tubulin to microtubules. I find that hydrolysis is a requirement for the opposite-end assembly/disassembly of microtubules and consequent subunit treadmilling from one end to the other of the polymer. Neither GDP nor guanosine 5'-[beta, gamma-imido]triphosphate allows or participates in the treadmilling reaction. Therefore, there is a requirement for hydrolysis in the addition of subunits to the favored assembly end of the microtubule. Podophyllotoxin, an assembly inhibitory drug, "caps" the microtubule assembly end, preventing subunit loss from that site to equilibrium. Continued hydrolysis of GTP is required to maintain the podophyllotoxin cap. A corollary of this finding is that GTP hydrolysis is required for cap formation. Microtubules assembled in GTP enter a metastable state when all remaining GTP is hydrolyzed. This state is characterized by its ability to maintain indefinitely a subunit/polymer distribution ratio that is arbitrary and that can be altered at will by brief chilling or by addition of small amounts of GTP. This metastable state is labile to podophyllotoxin. Use of podophyllotoxin allows measurement of the microtubule treadmilling rate; use of podophyllotoxin in the absence of GTP allows measurement of the overall rate of dimer dissociation from the microtubule. Measurement of these rates has permitted determination of the efficiency with which adding dimers incorporate into the microtubule treadmill and are not lost to assembly end equilibrium. The efficiency varies with GTP concentration for unknown reasons, being high at 0.1 mM GTP and low at higher GTP concentrations.