Supplementary Figure 7: Potential Cdk1 phosphorylation site T1011 in Cut7 is not involved in Cut7-Mad1 interaction.
From: Mad1 promotes chromosome congression by anchoring a kinesin motor to the kinetochore
(a) Alignment of conserved BimC motif in Cut7 family motor proteins. Cdk1 phosphorylation site in human46 is highlighted in red. Identical or similar residues are highlighted in blue. Alignments of 997-1019 from S. pombe Cut7[gene bank number x57513], 912-934 from H.sapiense Eg5[x85137], aa.923-945 from X.laevis Eg5[x71864], 992-1014 A.nidulans BimC [M32075]. (b) A yeast two-hybrid assay shows that the potential Cdk1 phosphorylation sites (T1011) in Cut7 is not important for Mad1 interaction. (c) The GFP signals were measured in the indicated mitotic (nda3-KM311) cells expressing Cut7-GFP or Cut7-T1011A-GFP, Mis6-2mCherry (kinetochore) and Sfi1-CFP (SPB). Note that kinetochore localization of Cut7-T1011A mutant was intact. Error bars, s.e.m. for n = 30 kinetochores detached from SPB from 10 cells. (d) Serial dilution assay (28 °C, 34 °C, TBZ 15 μg ml−1 at 28 °C). Note that cut7-T1011A mutant cells show temperature sensitivity at 34 °C, and does not show TBZ sensitivity unlike mad1-KAKA. Scale bar, 3 μm.
