GCP5 and GCP6: Two New Members of the Human γ-Tubulin Complex

Abstract

The γ-tubulin complex is a large multiprotein complex that is required for microtubule nucleation at the centrosome. Here we report the purification and characterization of the human γ-tubulin complex and the identification of its subunits. The human γ-tubulin complex is a ring of ∼25 nm, has a subunit structure similar to that reported for γ-tubulin complexes from other species, and is able to nucleate microtubule polymerization in vitro. Mass spectrometry analysis of the human γ-tubulin complex components confirmed the presence of four previously identified components (γ-tubulin and γ-tubulin complex proteins [GCPs] 2, 3, and 4) and led to the identification of two new components, GCP5 and GCP6. Sequence analysis revealed that the GCPs share five regions of sequence similarity and define a novel protein superfamily that is conserved in metazoans. GCP5 and GCP6, like other components of the γ-tubulin complex, localize to the centrosome and associate with microtubules, suggesting that the entire γ-tubulin complex takes part in both of these interactions. Stoichiometry experiments revealed that there is a single copy of GCP5 and multiple copies of γ-tubulin, GCP2, GCP3, and GCP4 within the γ-tubulin complex. Thus, the γ-tubulin complex is conserved in structure and function, suggesting that the mechanism of microtubule nucleation is conserved.