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Updated: Sep 18, 2025

Comparing the Affinity of GTPase-binding Proteins using Competition Assays
Published on: October 8, 2015
A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange
Wesley J Yon1, Taekjip Ha2, Yixian Zheng1
1Department of Embryology, Carnegie Institution for Science, Baltimore, Maryland, USA; Cell, Molecular, Developmental Biology, and Biophysics Program, Johns Hopkins University, Baltimore, Maryland, USA.
Abstract:
α- and β-tubulin form GTPase heterodimers and assemble into microtubules. Like other GTPases, the tubulin heterodimer's nucleotide-bound state regulates its activity. In the dimer, α-tubulin is constitutively bound to GTP, while β-tubulin can bind to either GDP (GDP-tubulin) or GTP (GTP-tubulin). Following assembly into microtubules, GTP-tubulin hydrolyzes GTP to GDP, triggering microtubule disassembly. This generates free GDP-tubulin, which must exchange GDP for GTP to undergo assembly again. Tubulin dimers undergo rapid nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary for microtubule assembly in cells. Here, we use quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. We further show that BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells.
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