Microtubule 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-Pi subunits (where Pi 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-Pi subunits can transiently accumulate at microtubule ends. There is no direct evidence that GDP-Pi-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-Pi stabilizes microtubules we sought evidence for a synergism between the effect of Pi and BeFn. We found, however, that Pi antagonizes the effect of BeFn by displacing it from tubulin subunits. The alternate mechanism in which Pi inhibits BeFn stabilization of microtubules by displacing fluoride from beryllium was ruled out from the 9Be and 19F NMR spectra in the presence and absence of Pi. Further evidence that tubulin-GDP-BeFn is not an analogue of tubulin-GDP-Pi and that tubulin-GDP-Pi is not responsible for maintaining the growth phase in microtubules manifesting dynamic instability was provided by our observation that Pi did not decrease the disassembly rate under conditions where tubulin-GDP-Pi subunits are expected to have formed. Results showing that BeFn binds randomly to subunits in microtubules provided evidence that Pi dissociation from the tubulin-GDP-Pi intermediate formed during GTP hydrolysis occurs randomly rather than processively starting at the growing microtubule tip.
Caplow
M
M
Department of Biochemistry, University of North Carolina, Chapel Hill 27599-7260, USA. caplow@med.unc.edul
Shanks
J
J
D000818:Animals; D001608:Beryllium / Q000378:metabolism; D002417:Cattle; D005459:Fluorides / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000737:chemistry / Q000502:physiology*; D008870:Microtubules / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*; D008954:Models, Biological; D010710:Phosphates / Q000737:chemistry / Q000502:physiology*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D014404:Tubulin / Q000737:chemistry / Q000378:metabolism* / Q000502:physiology*
D010710:Phosphates; D014404:Tubulin; D006153:Guanosine Diphosphate; C035982:beryllium fluoride; D001608:Beryllium; D005459:Fluorides