γ-Tubulin assembles into the γ-tubulin ring complex (γTuRC) that acts as the primary nucleator of cellular microtubules. γ-Tubulin binds Mg²⁺-associated GTP or GDP in an exchangeable manner; however, the nature of its GTP/GDP-binding properties has remained unclear. Here, we demonstrate the Förster resonance energy transfer (FRET) between γ-tubulin and fluorescently labeled guanine nucleotides, mant-GTP and mant-GDP, and we establish a real-time FRET-based assay to monitor nucleotide binding to γ-tubulin. Using this assay, we found that GTP and GDP associate with and dissociate from γ-tubulin with rapid kinetics, and that γ-tubulin exhibits a much higher affinity for GTP than for GDP. This nucleotide preference is conferred by Mg²⁺, which interacts with γ-tubulin to enhance GTP binding while suppressing GDP binding. Disrupting Mg²⁺ coordination through mutation eliminates this GTP preference and impairs γ-tubulin–dependent microtubule nucleation in cells. Therefore, Mg²⁺ plays a pivotal role in coordinating GTP loading onto γ-tubulin, a process essential for its microtubule-nucleating activity.
Yin et al. (Thu,) studied this question.