Cm1-driven assembly and activation of yeast g-tubulin small complex underlies microtubule nucleation

Abstract

Microtubule (MT) nucleation is regulated by the g-tubulin ring complex (gTuRC), conserved from yeast to humans. In Saccharomyces cerevisiae, gTuRC is composed of seven identical g-tubulin small complex (gTuSC) sub-assemblies, which associate helically to template MT growth. gTuRC 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 gTuSCs, and open and closed helical gTuRC assemblies in complex with Spc110p to elucidate the mechanisms of gTuRC assembly. gTuRC assembly is substantially aided by the evolutionarily conserved CM1 motif in Spc110p spanning a pair of adjacent gTuSCs. By providing the highest resolution and most complete views of any gTuSC 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 gTuRC structure at the interface between GCP2 and GCP6 allows for the interpretation of significant structural changes arising from CM1 helix binding to metazoan gTuRC.