Abstract
SummaryFor proper chromosome segregation in mitosis, sister kinetochores must interact with microtubules from opposite spindle poles (chromosome bi-orientation) [1, 2]. To promote bi-orientation, Aurora B kinase disrupts aberrant kinetochore-microtubule interactions [3, 4, 5, 6]. It has long been debated how Aurora B halts this action when bi-orientation is established and tension is applied across sister kinetochores. A popular explanation for it is that, upon bi-orientation, sister kinetochores are pulled in opposite directions, stretching the outer kinetochores [7, 8] and moving Aurora B substrates away from Aurora-B-localizing sites at centromeres (spatial separation model) [3, 5, 9]. This model predicts that Aurora B localization at centromeres is required for bi-orientation. However, this notion was challenged by the observation that Bir1 (yeast survivin), which recruits Ipl1-Sli15 (yeast Aurora B-INCENP) to centromeres, can become dispensable for bi-orientation [10]. This raised the possibility that Aurora B localization at centromeres is dispensable for bi-orientation. Alternatively, there might be a Bir1-independent mechanism for recruiting Ipl1-Sli15 to centromeres or inner kinetochores [5, 9]. Here, we show that the COMA inner kinetochore sub-complex physically interacts with Sli15, recruits Ipl1-Sli15 to the inner kinetochore, and promotes chromosome bi-orientation, independently of Bir1, in budding yeast. Moreover, using an engineered recruitment of Ipl1-Sli15 to the inner kinetochore when both Bir1 and COMA are defective, we show that localization of Ipl1-Sli15 at centromeres or inner kinetochores is required for bi-orientation. Our results give important insight into how Aurora B disrupts kinetochore-microtubule interaction in a tension-dependent manner to promote chromosome bi-orientation.
Highlights
The COMA complex promotes robust sister chromatid cohesion at peri-centromere regions by recruiting Dbf4-dependent kinase (DDK) to kinetochores [15,16,17,18]
How does COMA promote Ipl1 localization at centromeres or kinetochores, independently of Bir1? COMA may physically interact with Ipl1-Sli15 to enable their recruitment to the inner kinetochore, and we investigated possible physical interactions between Mcm21 and Sli15 using the yeast two-hybrid method (Figure 2D)
We addressed whether Sli15 directly interacts with COMA, using purified recombinant proteins
Summary
The COMA complex promotes robust sister chromatid cohesion at peri-centromere regions by recruiting Dbf4-dependent kinase (DDK) to kinetochores [15,16,17,18]. Robust peri-centromere cohesion is important for bi-orientation [15, 16, 19]. We addressed whether the above effects of Mcm depletion are due to a defect in peri-centromere cohesion. A C-terminus-tagged DBF4 (dbf4-myc), which impairs DDK recruitment to kinetochores [17], should show a similar defect to Mcm depletion. Dbf4-myc produced a defect in peri-centromere cohesion in similar extent to (or slightly greater than) mcm deletion (Figures S1C and S1D).
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