S. pombe Mto1[bonsai]:Mto2 stabilizes γ-tubulin positions within the γ-TuRC

Microtubules are present in all eukaryotes and are nucleated by multiprotein γ-tubulin ring complexes (γ-TuRCs), ring-shaped structures that contain several copies of the γ-tubulin small complex (γ-TuSC). γ-TuRCs nucleate microtubules via a template-based mechanism in which the positions of γ-tubulins in the γ-TuSCs match the position of γ-tubulin dimers in the microtubule lattice. Although γ-TuSC proteins are highly conserved, other components of the γ-TuRC can vary in different organisms, as can their relative importance. Here, we characterize two different simplified γ-TuRCs from fission yeast Schizosaccharomyces pombe, using recombinant purified proteins and negative stain electron microscopy. We show that a nucleation-incompetent γ-TuRC that contains only γ-TuSC proteins plus the microprotein Mzt1 (γ-TuSC:Mzt1) can form rings as efficiently as a large, nucleation-competent complex (MGM) that additionally contains the proteins Mto1 and Mto2. We find that the position of γ-tubulins in γ-TuSC:Mzt1 are less well defined than in MGM. Comparative modelling using our electron microscopy and crosslinking mass spectrometry data, together with structures of γ-TuRCs from other organisms, suggests that Mto1 and Mto2 form a multimeric outer mantle surrounding the γ-TuSC:Mzt1 γ-TuRC. This mantle may stabilize positions of γ-tubulins within MGM, explaining the microtubule-nucleating ability of MGM and highlighting the diversity of non-γ-TuSC proteins in modulating γ-TuRC structure and function.