EVIDENCE THAT A SINGLE MONOLAYER TUBULIN-GTP CAP IS BOTH NECESSARY AND SUFFICIENT TO STABILIZE MICROTUBULES

Evidence that 13 or 14 contiguous tubulin-Gm subunits are sufficient t o cap and stabilize a microtubule end and that loss of only one of the se subunits results in the transition to rapid disassembly (catastroph e) was obtained using the slowly hydrolyzable GTP analogue guanylyl-(a ,b)-methylene-diphosphonate (GMPCPP). The minus end of microtubules as sembled with GTP was transiently stabilized against dilution-induced d isassembly by reaction with tubulin-GMPCPP subunits for a time suffici ent to cap the end with an average of 40 subunits. The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was estimated from an observed 25- to 2000-s lifetime of the GMPCPP-stabilized micro tubules following dilution with buffer and from the time required for loss of a single tubulin-GMPCPP subunit from the microtubule end (foun d to be 15 s). Rather than assuming that the 25- to 2000-s dispersion in cap lifetime results from an unlikely 80-fold range in the number o f tubulin-GMPCPP subunits added in the 25-s incubation, it is proposed that this results because the minimum stable cap contains 13 or 14 tu bulin-GMPCPP subunits. As a consequence, a microtubule capped with 13- 14 tubulin-GMPCPP subunits switches to disassembly after only one diss ociation event (in about 15 s), whereas the time required for catastro phe of a microtubule with only six times as many subunits (84 subunits ) corresponds to 71 dissociation events (84-13). The minimum size of a tubulin-GMPCPP cap sufficient to prevent disassembly was also estimat ed with microtubules in which a GMPCPP-cap was formed by allowing chan ce to result in the accumulation of multiple contiguous tubulin-GMPCPP subunits at the end, during the disassembly of microtubules containin g both GDP and GMPCPP. Our observation that the disassembly rate was i nhibited in proportion to the 13-14th power of the fraction of subunit s containing GMPCPP again suggests that a minimum cap contains 13-14 t ubulin-GMPCPP subunits. A remeasurement of the rate constant for disso ciation of a tubulin-GMPCPP subunit from the plus-end of GMPCPP microt ubules, now found to be 0.118 s(-1), has allowed a better estimate of the standard free energy for hydrolysis of GMPCPP in a microtubule and release of Pi: this is +0.7 kcal/mol, rather than -0.9 kcal/mol, as p reviously reported.