Kinetochore clustering is mediated by Mps1 phosphorylation of conserved MELT motifs in Stu1.

Unattached kinetochores promote microtubule capture while preventing cell cycle progression during mitosis. The Mps1 kinase controls these events by mediating kinetochore assembly of the fibrous corona in animal cells and by triggering the spindle checkpoint. In budding yeast, which does not assemble a fibrous corona, the Stu1 and Slk19 spindle proteins promote microtubule capture by clustering unattached kinetochores, but the underlying mechanism is unclear. Here, we show that Mps1 controls this pathway. We identify two conserved MELT motifs in Stu1 that are directly phosphorylated by Mps1 to recruit Slk19 and mediate kinetochore clustering. Structural analysis of the Stu1:Slk19 complex reveals long, string-like filaments and offers mechanistic insight into how kinetochores might cluster. Our findings reveal parallels between the Mps1-Stu1-Slk19 pathway and the fibrous corona and suggest the regulation of kinetochore capture is a conserved Mps1 function across eukaryotes.