Ndc80 CH-domain function is essential in budding yeast and highly conserved
To gain insight into the function of the kinetochore–microtubule interface, we first tested the contribution of the Ndc80 CH-domain based on available structural information (; Wilson-Kubalek et al., 2008
; Alushin et al., 2010
). Although recent functional studies in human cell lines have demonstrated a critical importance of this element for kinetochore–microtubule attachments (Ciferri et al., 2008
; Sundin et al., 2011
; Tooley et al., 2011
), it remains to be shown whether they play a similar role in the attachment configuration of budding yeast. We systematically mutated conserved lysine residues (K122, K152, K160, K181, K192, and K204) previously shown to be critical for binding of the human Ndc80 complex to microtubules in vitro (Ciferri et al., 2008
). Viability assays on 5′FOA plates, counter-selecting against wild-type NDC80
, demonstrated that all six single lysine mutants could compensate for the loss of the NDC80
wild-type allele. We found, however, that the K122E K204E
double mutation was lethal and consistently all other combinations that involved K122E
were inviable (). In sequence alignments, the position of these residues corresponds precisely to the lysine residues (K89 and K166) that most effectively cripple microtubule binding of the human Ndc80 complex in vitro (Ciferri et al., 2008
). In stable haploid integrations a single point mutation, K204E
, sensitized cells to the presence of benomyl, suggesting that this residue makes the strongest contribution to the microtubule-binding ability of the Ndc80 complex (). Intriguingly, a negative charge in the equivalent human residue K166 results in severe chromosome alignment defects in human cells, suggesting that the functional hierarchy among individual lysine residues located in the Ndc80 CH-domain is precisely conserved from yeast to humans (Sundin et al., 2011
; Tooley et al., 2011
). We conclude that the autonomous microtubule-binding activity of the Ndc80 complex, provided by the CH-domain, is an absolute requirement for a functional kinetochore–microtubule interface, arguing for the existence of an evolutionary conserved binding mode.
Biochemical identification of an Ndc80–Dam1 interaction mutant
Design of candidate mutations that would interfere with the Ndc80–Dam1 interaction is facilitated by the crystal structure of human Ndc80 bonsai complex (Ciferri et al., 2008
), which shows that the Ndc80 CH-domain is connected via a short disordered segment (human residues 203–211, located between helices αG and αH) to the αH-αI helical hairpin preceding the coiled-coil shaft (). Two reasons make this part of the molecule an attractive target for structure–function analysis. First, many Saccharomyces
species contain large insertions in this region, rich in polar residues that may protrude from the surface, and thus can provide a possible interface for protein–protein interactions. Second, the proximity to the microtubule surface and the location on top of the Nuf2 CH-domain may position it closely to microtubule-bound Dam1 complexes. We tested several candidate mutations for the Dam1–Ndc80 interaction in vitro using microtubule cosedimentation assays. The ndc80 Δ256-273
mutation, further characterized in this study, removes part of the insertion but leaves the CH-domain and the helical αH-αI hairpin unchanged. Bacterially expressed Ndc80 Δ256-273 reconstituted into the full Ndc80 complex (Fig. S1 A
) and displayed robust microtubule binding, but in contrast to the wild-type Ndc80, addition of the Dam1 complex was not able to enhance the affinity of Ndc80 Δ256-273 for microtubules ().
Figure 2. Biochemical identification of an Ndc80–Dam1 interaction mutant. (A) Crystal structure of human Ndc80 bonsai complex with secondary structure elements labeled. (B) Gels of microtubule cosedimentation assays and corresponding binding curves of the (more ...)
To further test this effect, we performed cosedimentation assays at a fixed microtubule concentration and titrated the amount of the Dam1 complex. For both wild-type Ndc80 and the Δ1-116 N-tail truncation the addition of Dam1 complex enhanced the association of Ndc80 with microtubules in a dose-dependent manner with the fraction of cosedimenting Ndc80 increasing linearly with the concentration of Dam1. By contrast, deletion of aa 256–273 rendered both Ndc80 and Ndc80 Δ1-116 insensitive to the addition of the Dam1 complex () but left basal autonomous microtubule binding of the complexes unchanged ().
Charge-reversing CH-domain mutants (K122E, K204E, or K122E, K152E, K160E, K181E, K192E, K204E) abolished the autonomous microtubule-binding activity of the Ndc80 complex even under low ionic strength. Addition of the Dam1 complex was not able to recruit these mutants to microtubules (Fig. S1 B). These experiments strengthen the notion that the Ndc80–Dam1 interaction occurs on the microtubule and strictly requires the intrinsic microtubule-binding activity of the Ndc80 complex.
Ndc80 mutants display differential effects on cellular fitness
To quickly remove the endogenous Ndc80 protein from the nucleus and test various biochemically defined rescue alleles, we adapted the anchor-away (AA) technique for kinetochore proteins (Fig. S2 A
; Haruki et al., 2008
). As anticipated, the NDC80-FRB
fusion was inviable on rapamycin plates, but could be rescued by expression of wild-type NDC80
from a centromeric plasmid (). After 90 min rapamycin exposure the NDC80-FRB-GFP signal became diffuse and was depleted by roughly 90% (Fig. S2 B). This removal of Ndc80 from the kinetochore resulted in a simultaneous reduction of the Nuf2 signal, whereas Mtw1 levels were only slightly affected (). Consistent with previously reported Ndc80 phenotypes (De Wulf et al., 2003
), Mtw1 localization was perturbed in these cells, as it appeared fragmented (). Importantly, expression of an Ndc80 wild-type rescue allele restored clustered kinetochores with normal Nuf2 and Mtw1 levels ().
Figure 3. Impairing the Dam1–Ndc80 interaction affects cell growth. (A) Ndc80-FRB is lethal on rapamycin plates but rescued by expression of wild-type Ndc80 from a CEN plasmid. (B) Shuttling Ndc80 into the cytoplasm leads to a drop in Nuf2 levels and dispersion (more ...)
We next addressed the consequences of removing Ndc80 from the nucleus on the subcellular distribution of the Dam1 complex. Cells were arrested in G1
, released into rapamycin, and monitored after 30 min. In control cells expressing the wild-type NDC80
rescue allele, we found that the majority of Dam1-3xGFP colocalized with Nuf2 to kinetochores and a minor subpopulation bound to metaphase or anaphase spindles ( and Videos 1
). In the absence of the rescue allele cells arrested with large buds, short spindles, diminished Nuf2-mCherry signal, and a substantial fraction of Dam1 distributed along nuclear microtubules ( and Videos 3
). The observed displacement of Dam1 from the kinetochore is in line with previous findings suggesting that kinetochore loading of the Dam1 complex depends on microtubules as well as on Ndc80 (Janke et al., 2002
We next introduced the ndc80 ΔN1-116
, and K122E K204E
mutants into the NDC80-FRB
background and analyzed their phenotypes on rapamycin plates. Confirming previous reports (Kemmler et al., 2009
), the N-tail deletion mutant ndc80 ΔN1-116
grew indistinguishably from a wild-type control strain, whereas the CH-domain mutant (K122E K204E
) was lethal on rapamycin (). Further, we observed a slow growth phenotype of the ndc80 Δ256-273
mutant that was apparent at all temperatures. Importantly, additional removal of the N-tail from these cells led to lethality in the presence of rapamycin (), strongly suggesting that these two structural elements are partially redundant and share an essential function. Analyzing cell cycle progression after synchronization in G1 confirmed the results from the plating assay: the ndc80 Δ256-273
mutant was slow to progress through mitosis, whereas ndc80 ΔNΔ256-273
, the K122E K204E
mutant, and the strain lacking a rescue allele arrested as large budded cells ().
Altering Dam1–Ndc80 cooperation causes chromosome segregation defects
We quantitatively assessed the effects of various Ndc80 mutants on Dam1 localization using live-cell microscopy. We noticed that fusing 3xGFP to Dam1 aggravated the growth defect of ndc80 Δ256-273
on rapamycin plates (Fig. S2 D), possibly due to a direct involvement of the Dam1 C terminus in regulating the interaction between Dam1 and Ndc80 complexes (Cheeseman et al., 2002
; Lampert et al., 2010
). In support of this notion, microtubule co-pelleting assays revealed a reduced Ndc80 recruitment efficiency for the Dam1 ΔC complex (Fig. S3
). Live-cell microscopy showed that 90 min after rapamycin treatment, when wild-type cells had already entered anaphase, both ndc80 Δ256-273
and ndc80 K122E K204E
cells exhibited short spindles with high levels of Dam1 decorating the area between the poles. By contrast, the levels of Nuf2 at kinetochores were not significantly affected ().
Figure 4. Ndc80 mutants provoke Dam1 complex mislocalization and chromosome segregation defects. (A) Stills of rapamycin-treated cells (90 min) expressing wild-type Ndc80 or ndc80 Δ256-273 and ndc80 K122E K204E mutant complexes. Mutants arrest with Dam1 (more ...)
As an independent experimental approach to assess the kinetochore association of Dam1 we performed ChIP/qPCR analysis. In an Ndc80 wild-type background, Cse4-13xMyc and Dad1-13xMyc were efficiently bound to CEN3 but not to a control GAL2 locus. In ndc80 Δ256-273 and ndc80 Δ1-116 Δ256-273 strains, however, localization of Dad1-13xmyc to CEN3 was largely abolished (), whereas Nuf2 was retained at normal centromeric levels. This supports the conclusion that ndc80 Δ256-273 separates the functions of the Ndc80 complex: complex integrity and recruitment to the centromere remain intact, but promotion of Dam1 localization to the kinetochore and thus assembly of a correct outer kinetochore structure are compromised.
The inability to form a correct kinetochore–microtubule interface might lead to chromosome segregation defects. To test this, we monitored the behavior of GFP-tagged chromosome V in the presence of rapamycin after release from α-factor arrest. Although the control strain and the ndc80 ΔN1-116 mutant segregated chromosome V correctly (98.8% and 95% of large budded cells containing two GFP dots, respectively), this was not the case in ndc80 ΔNΔ256-273 or ndc80 K122E K204E cells. The sister chromatids in these cells remained unresolved and were predominantly localized at the bud-neck. An intermediate phenotype was observed in the ndc80 Δ256-273 strain, which partially distributed chromosome V to the daughter cells (32.1%; ). Abrogation of the mitotic checkpoint by deleting MAD2 allowed all mutants to enter anaphase and resulted in roughly 40–50% chromosome mis-segregation within a single cell cycle for ndc80 ΔNΔ256-273 and ndc80 K122E K204E, respectively, with the latter sometimes showing a nondisjunction phenotype. This effect was more frequent in the NDC80-FRB strain lacking any rescue allele (70.8%). For the ndc80 ΔN1-116 and Δ256-273 alleles we observed milder mis-segregation defects of 9.5% and 20.8%, respectively.
Given the slow growth phenotype and the high rate of chromosome mis-segregation in the ndc80 Δ256-273 mutant, we asked whether the mitotic checkpoint was essential to keep this strain viable. Growth assays on rapamycin plates demonstrated that the ndc80 Δ256-273 mutation was inviable in a mad2 deletion background (). To a lesser extent, elimination of the checkpoint also compromised growth of the ndc80 ΔN1-116 mutant.
In summary, we conclude that interference with the Dam1–Ndc80 interaction in vivo severely compromises chromosome segregation and creates a dependency on the mitotic checkpoint for viability.
Our work provides novel mechanistic insights into the integration of multiple microtubule-binding activities at the kinetochore. We draw three main conclusions from our study.
Kinetochore–microtubule attachments in budding yeast rely critically on the CH-domain of Ndc80.
Our analysis shows that charge-altering mutations in conserved lysine residues abolish the microtubule-binding ability of the yeast complex in vitro and are detrimental for viability (). The residues localize to the so-called “toe” region of the Ndc80 CH-domain directly contacting the “toe-print” binding motif formed at the interface between tubulin monomers on the microtubule surface (Alushin et al., 2010
Figure 5. Model for kinetochore–microtubule attachments. (A) Current working model of the yeast kinetochore–microtubule interface. The Ndc80 coiled-coil is modeled as a stick with the crystal structures of the Ndc80-Nuf2 CH domains and Spc24-25 (more ...) The Ndc80–Dam1 interaction is critical for cell cycle progression and shares an essential function with the N-tail of Ndc80.
Specific interference with Ndc80–Dam1 coupling delays cell cycle progression, creates a dependency on the mitotic checkpoint, and is synthetic-lethal in combination with removal of the Ndc80 N terminus (). These results support the notion that, unlike in humans, deletion of the N terminus in yeast Ndc80 is tolerated because its absence is sufficiently balanced by Dam1 complex activity (Lampert et al., 2010
; Demirel et al., 2012
Residues critical for the Ndc80–Dam1 interaction map in close proximity to the CH-domain of Nuf2.
Our biochemical experiments show that residues close to the CH-domains support the Ndc80–Dam1 interaction. This conclusion is independently supported by a recent EM analysis of isolated yeast kinetochores (Gonen et al., 2012
). In this study the contact point between the Ndc80 complex and Dam1 rings appeared to be in close proximity to the CH-domains. Combined with our analysis, this challenges the recently proposed role for the evolutionary conserved flexible “loop” (Ndc80 aa 480–520; Maure et al., 2011
). Though loop mutants were specifically defective in the establishment of stable end-on attachments, in vitro Dam1-mediated Ndc80 recruitment to microtubules was not affected (Maure et al., 2011
). Thus, we speculate that mutations within this domain might indirectly affect Dam1 binding in vivo by lowering the degree of intramolecular Ndc80 flexibility (Maiolica et al., 2007
; Ciferri et al., 2008
; Wang et al., 2008
; Joglekar et al., 2009
). The fact that the hinge domain is conserved throughout evolution might suggest a more general role for this element in establishment of attachments (Hsu and Toda, 2011
; Schmidt and Cheeseman, 2011
Combining the available structural data on Ndc80 and Dam1 complexes and the biochemical insights presented in this study allows us to propose one possible model for the Dam1–Ndc80 kinetochore interface (). We note that in the future, cryo-EM studies of both complexes simultaneously bound to microtubules have the potential to precisely establish their spatial arrangement and answer whether amino acids 256–273 are directly in contact with Dam1 or whether their deletion impairs Ndc80–Dam1 coupling by a different mechanism. Despite the evolutionary restriction of the Dam1 complex to yeasts, similar concepts for the integration of Ndc80 complex function at the microtubule plus-end are likely also operative in higher eukaryotes.