Stabilization of microtubules by inorganic phosphate and its structural analogues, the fluoride complexes of aluminum and beryllium.
In order to elucidate how the elementary reactions of GTP cleavage and subsequent inorganic phosphate (Pi) release, which accompany microtubule assembly, regulate microtubule dynamics, the effect of Pi and of its structural analogues AlF4- and BeF3- on the stability of GDP-microtubules has been investigated. Inorganic phosphate binds to microtubules with a low affinity (KD = 25 mM) and slows down the rate of GDP-subunit dissociation by about 2 orders of magnitude. AlF4- and BeF3- exhibit phosphate-like effects with 1000-fold higher affinity. Evidence has been obtained for direct binding of BeF3- to microtubules with a stoichiometry of 1 mol of BeF3- per mole of GDP-subunit and an equilibrium dissociation constant of 12-15 microM. AlF4- and Pi compete for this site. Phosphate analogues abolish oscillatory polymerization kinetics and slow down microtubule turnover at steady state. In view of these results, we propose that Pi and its structural analogues bind to the site of the gamma-phosphate of GTP in the E site and reconstitute a GDP-Pi-microtubule, from which tubulin subunits dissociate very slowly. We therefore understand that, following GTP cleavage on microtubules, Pi release in the medium is accompanied by a structural change resulting in a large destabilization of the polymer. A cap of slowly dissociating GDP-Pi-subunits prevents depolymerization of the microtubule GDP-core at steady state. The similarity with the actin system [Carlier, M.-F., & Pantaloni, D. (1988) J. Biol. Chem. 263, 817-825] is underlined.
Carlier
M F
MF
Laboratoire d'Enzymologie, CNRS, 91198 Gif-sur-Yvette, France.
Didry
D
D
Melki
R
R
Chabre
M
M
Pantaloni
D
D
D000535:Aluminum / Q000494:pharmacology*; D017607:Aluminum Compounds; D000818:Animals; D001608:Beryllium / Q000494:pharmacology*; D001921:Brain / Q000378:metabolism; D005459:Fluorides / Q000494:pharmacology*; D005461:Fluorine / Q000494:pharmacology*; D020558:GTP Phosphohydrolases / Q000378:metabolism; D007700:Kinetics; D008870:Microtubules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure*; D010710:Phosphates / Q000494:pharmacology*; D011485:Protein Binding; D013552:Swine; D014404:Tubulin / Q000378:metabolism
D017607:Aluminum Compounds; D010710:Phosphates; D014404:Tubulin; C056920:beryllium trifluoride; C050992:tetrafluoroaluminate; D005461:Fluorine; D000535:Aluminum; D020558:GTP Phosphohydrolases; D001608:Beryllium; D005459:Fluorides