Microtubule nucleating γ-TuSC assembles structures with 13-fold microtubule-like symmetry

Abstract

Microtubules are an important component of the cytoskeleton, generally organized in vivo in groups of 13 protofilaments around a core of γ-tubulin complexes. How this precise geometry is controlled remains unclear. The structure of higher-order assemblies of the universally conserved core microtubule nucleating complex, γ-tubulin small complex (γTuSC) — isolated from the yeast Saccharomyces cerevisiae — has now been determined using cryo-electron microscopy. The structure provides insight into how γTuSC establishes thirteen-fold tubulin symmetry. XXXMicrotubules are nucleated in vivo by γ-tubulin complexes and comprise 13 protofilaments. How this precise geometry is controlled remains unclear. These authors report the cryo-electron microscopic structure of the universally conserved, core microtubule nucleating complex, γ-tubulin small complex. The structure provides insight into how this complex establishes thirteen-fold tubulin symmetry. Microtubules are nucleated in vivo by γ-tubulin complexes. The 300-kDa γ-tubulin small complex (γ-TuSC), consisting of two molecules of γ-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery1,2. In metazoa multiple γ-TuSCs assemble with other proteins into γ-tubulin ring complexes (γ-TuRCs). The structure of γ-TuRC indicated that it functions as a microtubule template2,3,4,5. Because each γ-TuSC contains two molecules of γ-tubulin, it was assumed that the γ-TuRC-specific proteins are required to organize γ-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae γ-TuSC forms rings even in the absence of other γ-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-Å cryo-electron microscopic reconstruction of the filament reveals 13 γ-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the γ-TuRC-specific proteins. The γ-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.