Promiscuous Binding of Microprotein Mozart1 to γ-Tubulin Complex Mediates Specific Subcellular Targeting to Control Microtubule Array Formation

How gamma-tubulin ring complex (gamma-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 gamma-TuRC at different cell cycle stages. Crystal structures of protein complexes demonstrate that Mzt1 promiscuously interacts with the N-terminal domains of multiple gamma-tubulin complex protein subunits in gamma-TuRC via an intercalative binding mode. Genetic- and microscopy-based analyses show that promiscuous binding of Mzt1 in gamma-TuRC controls specific subcellular localization of gamma-TuRC to modulate microtubule nucleation and stabilization in fission yeast. Moreover, we find Mzt1-independent targeting of gamma-TuRC to be crucial for mitotic spindle assembly, demonstrating the cell-cycle-dependent regulation and function of gamma-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 gamma-TuRC.