Tubulin Binding Sites on γ-Tubulin: Identification and Molecular Characterization

Abstract

γ-Tubulin is essential to microtubule organization in eukaryotic cells. It is believed that γ-tubulin interacts with tubulin to accomplish its cellular functions. However, such an interaction has been difficult to demonstrate and to characterize at the molecular level. γ-Tubulin is a poorly soluble protein, not amenable to biochemical studies in a purified form as yet. Therefore basic questions concerning the existence and properties of tubulin binding sites on γ-tubulin have been difficult to address. Here we have performed a systematic search for tubulin binding sites on γ-tubulin using the SPOT peptide technique. We find a specific interaction of tubulin with six distinct domains on γ-tubulin. These domains are clustered in the central part of the γ-tubulin primary amino acid sequence. Synthetic peptides corresponding to the tubulin binding domains of γ-tubulin bind with nanomolar K_ds to tubulin dimers. These peptides do not interfere measurably with microtubule assembly in vitro and associate with microtubules along the polymer length. On the tertiary structure, the γ-tubulin peptides cluster to surface regions on both sides of the molecule. Using SPOT analysis, we also find peptides interacting with γ-tubulin in both the α- and β-tubulin subunits. The tubulin peptides cluster to surface regions on both sides of the α- and β- subunits. These data establish γ-tubulin as a tubulin ligand with unique tubulin-binding properties and suggests that γ-tubulin and tubulin dimers associate through lateral interactions.