The copy-number and varied strengths of MELT motifs in Spc105 balance the strength and responsiveness of the spindle assembly checkpoint

Abstract

During mitosis, the Spindle Assembly Checkpoint (SAC) maintains genome stability while also ensuring timely anaphase onset. To maintain genome stability, the SAC must be strong to delay anaphase even if just one chromosome is unattached, but for timely anaphase onset, it must promptly respond to silencing mechanisms. How the SAC meets these potentially antagonistic requirements is unclear. Here we show that the balance between SAC strength and responsiveness is determined by the number of ‘MELT’ motifs in the kinetochore protein Spc105/KNL1 and their Bub3-Bub1 binding affinities. Many strong MELT motifs per Spc105/KNL1 minimize chromosome missegregation, but too many delay anaphase onset. We demonstrate this by constructing a Spc105 variant that trades SAC responsiveness for much more accurate chromosome segregation. We propose that the necessity of balancing SAC strength and responsiveness drives the dual evolutionary trend of the amplification of MELT motif number, but degeneration of their functionally optimal amino acid sequence.

Kre28-Spc105 interaction is essential for Spc105 loading at the kinetochore

“…Previous studies show that the C-terminal RWD domain of Spc105 binds directly to the Mtw1 complex and remains in the proximity of Spc24/Spc25 C-termini [14,22,23]. On the other hand, the N-terminus of Spc105 (abbreviated as N-Spc105) consists of a long, disordered phosphodomain that lies somewhere between the Dam1 complex and the C-termini of Ndc80 and Nuf2 within the Ndc80 complex [6,7,24]. To map out the overall organization of the Spc105 phosphodomain, we inserted a GFP at locations within Spc105 that demarcate domains predicted to possess secondary structure (see Fig.…”

“…This experiment was performed as described previously [24, 27]. Starting from 0.1 OD 600 of log phase cultures, we prepared 10-fold serial dilutions and frogged or spotted them on YPD and YPD containing 20 μg/ml and 30 μg/ml benomyl.…”

“…We performed flow cytometry as described previously [24, 27]. For strains expressing Kre28_FL or truncated kre28, we started with overnight inoculums grown in Sd-Trp and shifted to YPD to grow till early to mid-log phase before supplementing the media with Nocodazole (final concentration 15µg/ml) or DMSO control.…”

“…( A ) Left: Line diagram of Spc105 molecule. The illustration was reproduced from our previous study [24]. Red bars represent PP1/Glc7 recruitment site (amino acid 75-79), and six MELT repeats.…”

“…( E ) Localization of Spc105 molecules with respect to other proteins in the KMN network of bioriented kinetochores. The illustration was reproduced from our previous study [24]. Although the C-terminus of this protein (amino acid 709-917) is structured and could be localized in the proximity of C-termini of Spc24/Spc25 and Nsl1, the N-terminal region (amino acid 1-455) is unstructured and could be localized within a 20nm region between C-termini of Dad4 and that of Ndc80.…”