Activation of the γ-Tubulin Complex by the Mto1/2 Complex

The multisubunit gamma-tubulin complex (gamma-TuC) is critical for microtubule nucleation in eukaryotic cells [1, 2], but it remains unclear how the gamma-TuC becomes active specifically at microtubule-organizing centers (MTOCs) and not more broadly throughout the cytoplasm [3, 4]. In the fission yeast Schizosaccharomyces pombe, the proteins Mto1 and Mto2 form the Mto1/2 complex, which interacts with the gamma-TuC and recruits it to several different types of cytoplasmic MTOC sites [5-10]. Here, we show that the Mto1/2 complex activates gamma-TuC-dependent microtubule nucleation independently of localizing the gamma-TuC. This was achieved through the construction of a "minimal'' version of Mto1/2, Mto1/2[bonsai], that does not localize to any MTOC sites. By direct imaging of individual Mto1/2[bonsai] complexes nucleating single microtubules in vivo, we further determine the number and stoichiometry of Mto1, Mto2, and gamma-TuC subunits Alp4 (GCP2) and Alp6 (GCP3) within active nucleation complexes. These results are consistent with active nucleation complexes containing similar to 13 copies each of Mto1 and Mto2 per active complex and likely equimolar amounts of gamma-tubulin. Additional experiments suggest that Mto1/2 multimers act to multimerize the fission yeast gamma-tubulin small complex and that multimerization of Mto2 in particular may underlie assembly of active microtubule nucleation complexes.