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

Abstract

Microtubules are nucleated in vivo by γ-tubulin complexes. The 300 kDa γ-tubulin small complex (γTuSC), consisting of two molecules of γ-tubulin and one copy each of the accessory proteins Spc97p and Spc98p, is the conserved, essential core of the microtubule nucleating machinery1,2. In metazoa multiple γTuSCs assemble with other proteins into γ-tubulin ring complexes (γTuRCs). The structure of γTuRC suggested that it functions as a microtubule template2–5. Because each γTuSC contains two molecules of γ-tubulin, it was assumed that the γTuRC-specific proteins are required to organize γTuSCs to match thirteen-fold microtubule symmetry. Here, we show that γTuSC forms rings even in the absence of other γTuRC components. The yeast adaptor protein Spc110p stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8Å cryo-EM reconstruction of the filament reveals thirteen 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 Spc97p and Spc98p suggest functions for conserved sequence motifs, with implications for the γTuRC-specific proteins. The γTuSC 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.