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. 2017 Sep 21;171(1):72–84.e13. doi: 10.1016/j.cell.2017.08.017

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© 2017 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

Ctf19 Phosphorylation Is Required for Scc2 Localization at Centromeres

(A and B) Model for the mechanism of Scc2/4 (gray) recruitment by the Ctf19 complex (blue) and DDK (red). Ctf3 is shown in green, and phosphate modifications are depicted as yellow circles. (A) Ctf3 recruits DDK, which phosphorylates Ctf19 to provide a docking site for Scc4. (B) Cells lacking the Ctf19 phosphorylation sites lack the Scc4 docking site, despite normal DDK recruitment.

(C and D) Ctf19 phosphorylation is required for efficient localization of Scc2 to centromeres. Strains of the indicated genotypes expressing Scc2-GFP and Mtw1-tdTomato (SMH353, SMH361, SMH354, SMH355, SMH451, SMH452, SMH459, SMH460, SMH416, and SMH417 listed in the order shown) were imaged during unperturbed vegetative growth and scored for the presence of centromeric Scc2-GFP foci. Values are the averages of at least three independent experiments. At least 25 cells were scored per strain per experiment (error bars, +/−SD for the independent experiments; ^∗∗∗p < 0.001; ^∗∗p < 0.01; ns, not significant; Student’s t test versus CTF19 strain, two tails, unequal variance). (D) Representative images from experiments quantified in (C) (scale bar, 3 μm). Time after the beginning of image acquisition is shown.

(E and F) Scc2 and Scc1 localization require Ctf19 phosphorylation. Cells were arrested in metaphase following treatment with nocodazole and benomyl for 2.5 hr (AM1176, untagged control strain). Values are means from four independent experiments (error bars, SE; ^∗p < 0.05; ^∗∗p < 0.01; Student’s t test for indicated comparisons, unpaired, two-tails). (E) ChIP-qPCR for Scc2 performed using wild-type (AM6006), CTF19 (AM20613), ctf19-9A (AM20619), ctf19-6A (AM20622), and ctf19-2A (AM21908) cells, carrying SCC2-6His-3FLAG. (F) ChIP-qPCR for Scc1 performed using wild-type (AM1145), CTF19 (AM20629), ctf19-9A (AM23456), and ctf19-2A (AM21958) cells, carrying SCC1-6HA.

(G and H) Immunoblots showing levels of Scc2-6His-3FLAG, Scc1-6HA, and Pgk1 (loading control) from a representative experiment.