Structural Basis of Stu2 Recruitment to Yeast Kinetochores

Abstract

Accurate chromosome segregation during cell division requires engagement of the kinetochores of sister chromatids with microtubules emanating from opposite poles of the mitotic spindle. In yeast, these bioriented metaphase sister chromatids experience tension as the corresponding microtubules (one per sister chromatid) shorten. Spindle-assembly checkpoint signaling appears to cease from a kinetochore under tension, which also stabilizes kinetochore-microtubule attachment in single-kinetochore experiments in vitro. The microtubule polymerase, Stu2, the yeast member of the XMAP215/ch-TOG protein family, associates with kinetochores in cells and contributes to tension-dependent stabilization, both in vitro and in vivo. We show here that a C-terminal segment of Stu2 binds the four-way junction of the Ndc80 complex (Ndc80c) and that amino-acid residues conserved both in yeast Stu2 orthologs and in their metazoan counterparts make specific contacts with Ndc80 and Spc24. Mutations that perturb this interaction prevent association of Stu2 with kinetochores, impair cell viability, produce biorientation defects, and delay cell-cycle progression. Ectopic tethering of the mutant Stu2 species to the Ndc80c junction restores wild-type function. These findings show that the role of Stu2 in tension sensing depends on its association with kinetochores by binding with Ndc80c.

chTOG is a conserved mitotic error correction factor

“…First, we tested whether the basic pair mutant affected chTOG localization to the kinetochore using quantitative fluorescence microscopy and biochemical analysis in nocodazole-treated cells ( Figure 4c , Figure 4—figure supplement 1b,c ). There was no change in localization, consistent with our previous findings in yeast that the C-terminus of Stu2 is necessary and sufficient for stable association with the Ndc80 complex and kinetochores ( Miller et al, 2019 ; Zahm et al, 2020 ). Thus, defects in the regulation of kinetochore-microtubule attachments in cells expressing the basic pair mutant are not due to altered protein localization.…”

“…In this experiment, chTOG was still detected on kinetochores, consistent with a kinetochore-bound pool that is separate from microtubule tips ( Figure 1a,b , Figure 1—figure supplement 1a ). chTOG and its budding yeast ortholog, Stu2, physically interact with the Ndc80 kinetochore complex in vitro ( Miller et al, 2016 ; Zahm et al, 2020 ). To test whether they associate in human cells, we immunopurified FLAG-tagged chTOG from HEK-293T cells under conditions refractory to microtubule formation and found that the endogenous Hec1 protein co-purifies ( Figure 1c ).…”

“…We therefore took advantage of yeast genetic tools to identify mutants that potentially inactivate its kinetochore function. Previous cross-linking mass-spectrometry with yeast proteins revealed that both the Stu2 basic linker and C-terminus interact with the Ndc80 complex, but only the C-terminus was required for kinetochore association ( Miller et al, 2019 ; Zahm et al, 2020 ). This suggested the basic linker may instead interact with the Ndc80 complex to regulate kinetochore-microtubule attachments.…”

“…We suggest this because the basic pair mutant retains the ability to bind to kinetochores and microtubule tips, yet it fails to destabilize erroneous kinetochore-microtubule attachments, similar to depletion of chTOG ( Figure 5c,d ). Thus, we suggest a model where the C-terminus of Stu2/chTOG is required for stable association with the kinetochore ( Miller et al, 2019 ; Zahm et al, 2020 ) while the basic linker modulates Hec1’s microtubule-binding behavior through competitive or allosteric means ( Figure 7 ). While it is also possible the basic linker inhibits the recruitment of other microtubule-binding factors to the Nc80 complex, this is less likely because many of these factors are unique to budding yeast (Dam1/DASH complex) or metazoans (SKA and Astrin-SKAP complexes) ( van Hooff et al, 2017a ; van Hooff et al, 2017b ), yet the basic linker functions similarly in both species.…”