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

Abstract

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