How γ-tubulin ring complex (γ-TuRC), a master template for microtubule nucleation, is spatially and temporally regulated for the assembly of new microtubule arrays remains unclear. Here, we report that an evolutionarily conserved microprotein, Mozart1 (Mzt1), regulates subcellular targeting and microtubule formation activity of γ-TuRC at different cell cycle stages. Crystal structures of protein complexes demonstrate that Mzt1 promiscuously interacts with the N-terminal domains of multiple γ-tubulin complex protein subunits in γ-TuRC via an intercalative binding mode. Genetic- and microscopy-based analyses show that promiscuous binding of Mzt1 in γ-TuRC controls specific subcellular localization of γ-TuRC to modulate microtubule nucleation and stabilization in fission yeast. Moreover, we find Mzt1-independent targeting of γ-TuRC to be crucial for mitotic spindle assembly, demonstrating the cell-cycle-dependent regulation and function of γ-TuRC. Our findings reveal a microprotein-mediated regulatory mechanism underlying microtubule cytoskeleton formation, whereby Mzt1 binding promiscuity confers localization specificity on the multi-protein complex γ-TuRC.

γ-微管蛋白环复合物（γ-TuRC），微管成核的主模板，如何在空间和时间上调节新微管阵列的组装尚不清楚。在这里，我们报道进化上保守的微蛋白Mozart1（Mzt1）在不同的细胞周期阶段调节γ-TuRC的亚细胞靶向和微管形成活性。蛋白质复合物的晶体结构表明，Mzt1通过插入结合模式与γ-TuRC中多个γ-微管蛋白复合蛋白亚基的N末端结构域混杂相互作用。基于遗传和显微镜的分析表明，Mzt1在γ-TuRC中的混杂结合控制γ-TuRC的特定亚细胞定位，以调节裂变酵母中的微管成核和稳定化。此外，我们发现不依赖Mzt1的γ-TuRC靶向对于有丝分裂纺锤体组装至关重要，证明了γ-TuRC的细胞周期依赖性调节和功能。我们的发现揭示了微蛋白介导的调节机制，其基础是微管细胞骨架的形成，从而使Mzt1结合的混杂性赋予了多蛋白复合物γ-TuRC的定位特异性。