The human SKA complex drives the metaphase-anaphase cell cycle transition by recruiting protein phosphatase 1 to kinetochores

We thank the reviewers for critical evaluation of our manuscript and for the useful suggestions below. We agree that PP1 plays an essential role to reverse the spindle checkpoint in mitosis. We are happy that the reviewers find our findings “significant to the field of mitosis”. We have taken the reviewers’ suggestions to heart, and performed additional experiments to establish direct binding between PP1 and Ska1 CTD. We present our results below and have included them in the manuscript. With the new data, our original conclusions are further strengthened. We hope that the reviewers will support acceptance of this manuscript at eLife. Please find a detailed response to reviewers’ comments below.

1) Direct binding between PP1 and Ska1 CTD must be demonstrated. The authors should make recombinant proteins, demonstrate PP1 activity to ensure it is functional, and perform ITC or SEC (in this case, with chromotograms reporting A280 or A215 if the CTD has no Trp residues of each protein alone, the complex and molecular weight standards in addition to gels; ITC is preferable) to show an interaction. We purified PP1 and Ska1 CTD from bacteria (E. coli) and performed Microscale Thermophoresis (MST) assay. Active PP1 is difficult to purify in large quantity, which is a requirement for ITC. MST is a relatively new and sensitive technique to measure protein interactions in vitro. To perform MST, we sought help from an expert, Chad A Brautigam, who has been added as a co-author. We determined that the K_d of the Ska1 CTD-PP1 interaction is 1.5 μM. This is comparable to other protein-protein interactions that allow dynamics. We have presented these data in Figure 3C of the revised manuscript. Note that these data are also supported by the gel filtration (SEC) data that we originally included that shows a direct interaction between PP1 and the Ska complex with recombinant proteins. These SEC data demonstrated a requirement for Ska1 CTD in direct PP1 binding. We could not, however, detect an interaction between PP1 and the isolated Ska1 CTD using SEC, presumably due to the moderate affinity of this interaction. Concentration of the Ska complex at kinetochores may allow cooperative binding, or other components of the Ska complex may contribute to PP1 binding.

2) Given the difficulty of identifying a specific PP1 binding mutant in Ska1, we would like to see that the addition of a short peptide with an RVXF motif on the Ska1 mutant will rescue. This will allow dynamic recruitment of PP1 and be more meaningful than the constitutive PP1 fusion.

We thank the reviewers for this wonderful suggestion. As suggested, we fused the fragment of Knl1 (from amino acids 34 to 81 to human Knl1) that contains the PP1-binding motif (but importantly lacks the Knl1 microtubule-binding domain) to Ska1ΔCTD and performed live cell imaging. As the control, we used the same fragment of Knl1 where the PP1-binding RVSF motif was mutated to AAAA. We found that the Ska1ΔCTD fused to the wild type Knl1 fragmentrescued mitotic defects caused by Ska1 depletion while Ska1ΔCTD fused to Knl1 AAAA did not. We have presented these data in Figure 6 and in the second paragraph of the subsection “Ska-mediated kinetochore recruitment of PP1 promotes anaphase onset”.