Disentangling the molecular determinants for Cenp‐F localization to nuclear pores and kinetochores

Abstract

Cenp-F is a multifaceted protein implicated in cancer and developmental pathologies. The Cenp-F C-terminal region contains overlapping binding sites for numerous proteins that contribute to its functions throughout the cell cycle. Here, we focus on the nuclear pore protein Nup133 that interacts with Cenp-F both at nuclear pores in prophase and at kinetochores in mitosis, and on the kinase Bub1, known to contribute to Cenp-F targeting to kinetochores. By combining structural modeling and yeast two-hybrid assays, we generate an interaction model between a conserved helix within the Nup133 β-propeller and a short leucine zipper-containing dimeric segment of Cenp-F. We thereby create mutants affecting the Nup133/Cenp-F interface and show that they prevent Cenp-F localization to the nuclear envelope, but not to kinetochores. Conversely, a point mutation within an adjacent leucine zipper affecting the kinetochore targeting of Cenp-F KT-core domain impairs its interaction with Bub1, but not with Nup133, identifying Bub1 as the direct KT-core binding partner of Cenp-F. Finally, we show that Cenp-E redundantly contributes together with Bub1 to the recruitment of Cenp-F to kinetochores.

Kinetochore recruitment of CENP-F illustrates how paralog divergence shapes kinetochore composition and function

“…Our identification of a minimal kinetochore-targeting domain of HsCENP-F within residues 2922-2990 agrees with a previous study that made use of CENP-F deletion mutants (Zhu, 1999). It also agrees with another study, currently in press, that identified distinct binding domains in CENP-F for nuclear envelope and for kinetochore localization (Berto et al, 2018). Specifically, residues 2655 to 2860 of mouse CENP-F (corresponding to residues 2866-3072 of HsCENP-F) were sufficient for kinetochore recruitment (Berto et al, 2018).…”

“…The role of Bub1 in CENP-F kinetochore recruitment was already established in previous work (Berto et al, 2018; Johnson et al, 2004; Liu et al, 2006; Raaijmakers et al, 2018), and a very recent study implicated the Bub1 kinase domain in CENP-F kinetochore recruitment (Raaijmakers et al, 2018). Here, we have extended this previous study by showing that the interaction of the Bub1 kinase domain and CENP-F is direct, and by identifying a minimal CENP-F domain involved in this interaction and capable of kinetochore localization.…”

Miro-dependent mitochondrial pool of CENP-F and its farnesylated C-terminal domain are dispensable for normal development in mice

“…CENP-F is an enigmatic protein with a plethora of suggested cellular functions ranging from chromosome segregation to mitochondrial trafficking. Because of the number of its binding partners and involvement in different cellular processes, a careful genetic dissection is needed to study specific roles of CENP-F. Variants of CENP-F have been characterized that separate its functions at the nuclear envelope and kinetochores (Berto et al, 2018;Zhu, 1999).…”

“…Subsequently in prometaphase, CENP-F relocates to kinetochores (Rattner et al, 1993). The molecular mechanism of CENP-F kinetochore binding has been studied in great detail (Berto et al, 2018;Ciossani et al, 2018;Zhu, 1999). CENP-F is targeted to the outer kinetochore via a KT-core domain (residues 2792 to 2887, Fig 1A) via synergistic action of Bub1 kinase and the kinesin Cenp-E (Berto et al, 2018;Zhu, 1999).…”

Nucleoporin 133 deficiency leads to glomerular damage in zebrafish

“…Moreover, mutations affecting the kinetochore protein Cenp-F, an established partner of Nup133 49 were identified in patients affected by severe ciliopathy and microcephaly 50 .…”