Cik1 and Vik1 Accessory Proteins Confer Distinct Functions to the Kinesin-14, Kar3

Abstract

ABSTRACTThe budding yeast Saccharomyces cerevisiae has a closed mitosis in which the mitotic spindle and cytoplasmic microtubules (MTs) used to segregate chromosomes remain separated by the nuclear envelope throughout the cell cycle. Kar3, the yeast kinesin-14, has unique roles in both compartments and has been implicated in capturing unattached kinetochores, stabilizing crosslinked interpolar microtubules (MT), and creating intranuclear and cytoplasmic MT arrays at the spindle pole body for kinetochore capture and karyogamy, respectively. Here, we show that two proteins, Cik1 and Vik1, that form heterodimers with Kar3, regulate its localization and function within the cell and along MTs in a cell cycle-dependent manner. Using a cell cycle synchronized, yeast MT dynamics reconstitution assay in cell lysate, we found that Kar3Vik1 induces MT catastrophes in S phase and metaphase and limits MT polymerization in G1 and anaphase. In contrast, Kar3Cik1 is a catastrophe and pause promoter in G1, while increasing catastrophes in metaphase and anaphase. Adapting this assay to track single-molecules, we saw that Kar3Cik1 is necessary for tracking MT plus-ends in S phase and metaphase, but, surprisingly, not during anaphase. These experiments demonstrate how the binding partners of Kar3 modulate its diverse functions both spatially and temporally.SUMMARY STATEMENTWe show through biochemical reconstitution experiments and live-cell imaging that the functions and localization of the budding yeast kinesin-14, Kar3, are dictated by which of its two accessory protein binding partners, Cik1 or Vik1, it binds to and by the cell cycle stage.