Abstract
Mitosis begins with the tethering of chromosomes to the mitotic spindle and their orientation perpendicular to the axis of cell division. In budding yeast, mitotic spindle orientation and the subsequent chromosome segregation are two independent processes. Early spindle orientation is driven by the actin-bound myosin Myo2p, which interacts with the adapter Kar9p. The latter also binds to microtubule-associated Bim1p, thereby connecting both types of cytoskeleton. This study focuses on the interaction between Kar9p and Bim1p and its regulation. We solved the crystal structure of the previously reported Kar9p-binding motif of Bim1p and identified a second, novel Kar9p interaction domain. We further show that two independent post-translational modification events regulate their interaction. Whereas Kar9p sumoylation is required for efficient complex formation with Bim1p, Aurora B/Ipl1p-dependent phosphorylation of Bim1p down-regulates their interaction. The observed effects of these modifications allow us to propose a novel regulatory framework for the assembly and disassembly of the early spindle orientation complex.
Highlights
Mitotic spindle orientation in budding yeast is achieved by a motor complex consisting of Myo2p, Kar9p, and Bim1p
This region is subject to cell cycle-dependent phosphorylation by the Aurora B kinase, which we found to downregulate Bim1p binding to Kar9p
It was further shown that the Bim1p-Kar9p complex assembles before loading onto the microtubules via the spindle pole bodies [37]
Summary
Mitotic spindle orientation in budding yeast is achieved by a motor complex consisting of Myo2p, Kar9p, and Bim1p. The linker region between the CH and EB1-like domains contains no structurally defined domain In yeast, this linker region of Bim1p is the target of cell cycle-dependent phosphorylation by Aurora B/Ipl1p and supports the microtubule binding of the CH domain [16]. Kar9p is subject to sumoylation [18, 19] and phosphorylation [2, 20] Both types of modification are regulated independently, mutations in Kar9p affecting either of them result in impaired spindle orientation [18]. We identified a novel Kar9p interaction region in Bim1p This region is subject to cell cycle-dependent phosphorylation by the Aurora B kinase, which we found to downregulate Bim1p binding to Kar9p
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