Micro tubule dynamic instability does not result from stabilization of microtubules by tubulin-GDP-Pi subunits

The proposal that microtubule dynamic instability results from stabilization of microtubule ends by tubulin-GDP-P-i subunits (where P-i is inorganic phosphate) [Melki et al. (1996) Biochemistry 35, 12038] was based on studies of GTP hydrolysis and microtubule assembly that showed that tubulin-GDP-P-i subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP P-i-subunits can stabilize microtubules under conditions where dynamic instability is observed and this has been inferred from the observation that tubulin-GDP-BeFn subunits stabilize microtubules. To test if tubulin-GDP-P-i stabilizes microtubules we sought evidence for a synergism between the effect of P-i and BeFn. We found, however, that P-i antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which P-i inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the Be-9 and F-19 NMR spectra in the presence and absence of P-i. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-P-i and that tubulin-GDP-P-i is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that P-i did not decrease the disassembly rate under conditions where tubulin-GDP-P-i subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that P-i dissociation from the tubulin-GDP-P-i intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.