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Centriole duplication begins with the assembly of a pre-procentriole at a single site on a mother centriole and proceeds with the hierarchical recruitment of a conserved set of proteins, including Polo-like kinase 4 (Plk4)/ZYG-1, Ana2/SAS-5/STIL, and the cartwheel protein Sas6. During assembly, Ana2/STIL stimulates Plk4 kinase activity, and in turn, Ana2/STIL’s C-terminus is phosphorylated, allowing it to bind and recruit Sas6. The assembly steps immediately preceding Sas6-loading appear clear, but the mechanism underlying the upstream pre-procentriole recruitment of Ana2/STIL is not. In contrast to proposed models of Ana2/STIL recruitment, we recently showed that Drosophila Ana2 targets procentrioles independent of Plk4-binding. Instead, Ana2 recruitment requires Plk4 phosphorylation of Ana2’s N-terminus, but the mechanism explaining this process is unknown. Here, we show that the amyloid-like domain of Sas4, a centriole surface protein, binds Plk4 and Ana2, and facilitates phosphorylation of Ana2’s N-terminus which increases Ana2’s affinity for Sas4. Consequently, Ana2 accumulates at the procentriole to induce daughter centriole assembly.

The yeast microtubule polymerase Stu2’s C-terminal domain is a 15-nm parallel, homodimeric coiled coil with two spatially distinct conserved regions. Determinants in these conserved regions optimally position Stu2 on the mitotic spindle to drive proper spindle structure and dynamics., XMAP215/Dis1 family proteins are potent microtubule polymerases, critical for mitotic spindle structure and dynamics. While microtubule polymerase activity is driven by an N-terminal tumor overexpressed gene (TOG) domain array, proper cellular localization is a requisite for full activity and is mediated by a C-terminal domain. Structural insight into the C-terminal domain’s architecture and localization mechanism remain outstanding. We present the crystal structure of the Saccharomyces cerevisiae Stu2 C-terminal domain, revealing a 15-nm parallel homodimeric coiled coil. The parallel architecture of the coiled coil has mechanistic implications for the arrangement of the homodimer’s N-terminal TOG domains during microtubule polymerization. The coiled coil has two spatially distinct conserved regions: CRI and CRII. Mutations in CRI and CRII perturb the distribution and localization of Stu2 along the mitotic spindle and yield defects in spindle morphology including increased frequencies of mispositioned and fragmented spindles. Collectively, these data highlight roles for the Stu2 dimerization domain as a scaffold for factor binding that optimally positions Stu2 on the mitotic spindle to promote proper spindle structure and dynamics.