Assembly of microtubule protein: role of guanosine di- and triphosphate nucleotides

Abstract

A quantitative analysis of the interplay between guanosine 5'-triphosphate (GTP) and guanosine 5'-diphosphate (GDP) in microtubule assembly and accompanying GTP hydrolysis has been performed when tubulin was polymerized in the presence of microtubule-associated proteins (MAPs) which display an interfering GTPase activity. The use of adenylyl beta-imidodiphosphate, which specifically inhibits the MAPs GTPase activity, and of vinblastine (or podophyllotoxin), which specifically inhibits GTP hydrolysis due to tubulin, made possible a study of the extensive GTP hydrolysis associated to microtubule assembly. The results indicate that GDP binds to microtubule ends with an affinity comparable to GTP, thus strongly inhibiting both the elongation process and the steady-state GTP hydrolysis at microtubule ends. GDP shifts the equilibrium between tubulin and microtubules toward disassembly. The MAPs which are released from the microtubules during the GDP-driven depolymerization cluster on the remaining microtubules. The resulting increased stability of microtubules is quantitatively consistent with the decrease in the critical concentration of the polymerizing species GTP-tubulin.

Interference of GTP hydrolysis in the mechanism of microtubule assembly: an experimental study.

“…Previous experiments (18) have indicated that this ratio is close to zero because tubulin does not bind to a GDP-bound end.…”

“…These data (18) suggest that not only is GTP hydrolysis not necessary for polymerization but it inhibits polymerization. It is likely that the rate of dissociation of tubulin subunits also depends on whether GTP or GDP is bound at MT ends.…”

“…An approximate analytical formulation of the properties of this cap has been reported (16). (ii) Tubulin present at the very end of MTs can bind either GTP or GDP, and this affects the rate of growth; typically an elongating site having GDP bound is blocked and cannot bind tubtilin subunits (18). Relaxation studies (19) lead to the same conclusion.…”

Fluorescence stopped‐flow study of the binding of S6‐GTP to tubulin

“…In such a system at steady state, a net growth at one end is compensated by a net dissociation at the opposite end. The efficiency of this treadmilling depends on the nucleotide composition of at least one end [6,7].…”

Polymerization and Calcium Binding of the Tubulin-colchicine Complex in the GDP State

“…20,21) These facts mean that tubulin and the tubulin-colchicine complex have GTPase activity. 22,23) The GTPase rate is not in proportion to the rate of microtubule assembly and polymerization, 21,22,24) while the hydrolysis occurs at microtubule ends. 24) The addition of GDP to the microtubule assembly system induces the inhibition of the reaction.…”