CM1-driven assembly and activation of yeast γ-tubulin small complex underlies microtubule nucleation

Microtubule (MT) nucleation is regulated by the gamma-tubulin ring complex (gamma TuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, gamma TuRC is composed of seven identical gamma-tubulin small complex (gamma TuSC) sub-assemblies, which associate helically to template MT growth. gamma TuRC assembly provides a key point of regulation for the MT cytoskeleton. Here, we combine crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures of both monomeric and dimeric gamma TuSCs, and open and closed helical gamma TuRC assemblies in complex with Spc110p to elucidate the mechanisms of gamma TuRC assembly. gamma TuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent gamma TuSCs. By providing the highest resolution and most complete views of any gamma TuSC assembly, our structures allow phosphorylation sites to be mapped, surprisingly suggesting that they are mostly inhibitory. A comparison of our structures with the CM1 binding site in the human gamma TuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan gamma TuRC.

Automated summary

This study investigates the mechanisms of γ TuRC assembly using a combination of crosslinking mass spectrometry, X-ray crystallography, and cryo-EM structures. The results highlight the crucial role of the evolutionarily conserved CM1 motif in Spc110p in aiding γ TuRC assembly, showing that phosphorylation sites are mostly inhibitory. Comparison with the human γ TuRC structure reveals significant structural changes arising from CM1 helix binding in metazoan γ TuRC.