Abstract
Kinetochores perform an essential role in eukaryotes, coupling chromosomes to the mitotic spindle. In model organisms they are composed of a centromere-proximal inner kinetochore and an outer kinetochore network that binds to microtubules. In spite of universal function, the composition of kinetochores in extant eukaryotes differs greatly. In trypanosomes and other Kinetoplastida, kinetochores are extremely divergent, with most components showing no detectable similarity to proteins in other systems. They may also be very different functionally, potentially binding to the spindle directly via an inner-kinetochore protein. However, we do not know the extent of the trypanosome kinetochore, and proteins interacting with a highly divergent Ndc80/Nuf2-like protein (KKIP1) suggest the existence of more centromere-distal complexes. Here we use quantitative proteomics from multiple start-points to define a stable 9-protein kinetoplastid outer kinetochore (KOK) complex. This complex incorporates proteins recruited from other nuclear processes, exemplifying the role of moonlighting proteins in kinetochore evolution. The outer kinetochore complex is physically distinct from inner-kinetochore proteins, but nanometer-scale label separation shows that KKIP1 bridges the two plates in the same orientation as Ndc80. Moreover, KKIP1 exhibits substantial elongation at metaphase, altering kinetochore structure in a manner consistent with pulling at the outer plate. Together, these data suggest that the KKIP1/KOK likely constitute the extent of the trypanosome outer kinetochore and that this assembly binds to the spindle with sufficient strength to stretch the kinetochore, showing design parallels may exist in organisms with very different kinetochore composition.
Highlights
Kinetochores are complex multi-protein machines that ensure the faithful segregation of eukaryotic chromosomes by coupling them to the mitotic spindle and coordinating their movement
A tenth outer kinetochore protein, KKIP5, which binds to the kinetochore only until anaphase (D’Archivio and Wickstead, 2017), clearly binds via kinetoplastid outer kinetochore (KOK) complex components
As our model of the trypanosome kinetochore improves, it is very possible that additional components will be discovered – especially if such components bind transiently or form interactions that are not stable under conditions used for immuno-purification
Summary
Kinetochores are complex multi-protein machines that ensure the faithful segregation of eukaryotic chromosomes by coupling them to the mitotic spindle and coordinating their movement. The Kinetoplastid Outer Kinetochore Complex kinetochore KMN network consisting of the Knl1-Mis12-Ndc complexes [for reviews see (Varma and Salmon, 2012; Nagpal and Fukagawa, 2016; Musacchio and Desai, 2017)] These major networks approximately correspond to the inner and outer plates of electron-opaque material seen at kinetochores by electron microscopy (Pesenti et al, 2016). Microtubule binding activity is mediated by calponin homology (CH) domains near the Ntermini of Ndc and Nuf (Wei et al, 2007; Ciferri et al, 2008), which are positioned at the outer kinetochore, while Spc and Spc bound to the C-termini of Ndc80/Nuf interact with components of the CCAN (Schleiffer et al, 2012; Malvezzi et al, 2013; Nishino et al, 2013), indirectly connecting the spindle to the centromeres
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