Figure 9.
Kinetochore localization of KKT3 depends on the central domain in T. brucei.(A) Multiple sequence alignment of KKT3. Residues that are expected to coordinate zinc ions as well as the conserved aspartic acid residue are shown. (B) Percentage of GFP-positive cells that have kinetochore-like dots were quantified at 1 d after induction (n > 22 each). Inducible GFP-NLS fusion proteins were expressed with 10 ng/ml doxycycline. Cell lines: BAP291, BAP359, BAP360, BAP446, BAP447, BAP1721, BAP1722, BAP362, and BAP341. (C) TbKKT3 C668A/C671A and D692A mutants do not localize at kinetochores and fail to support normal cell growth, while TbKKT3 C707A/C710A mutant is functional. One allele of TbKKT3 was mutated and tagged with a C-terminal YFP tag, and the other allele was depleted using RNAi-mediated knockdown by targeting the 3′UTR of the TbKKT3 transcript. Top: Cells were diluted every 2 d, and cell growth was monitored for 8 d upon induction of RNAi. Similar results were obtained for at least three clones of TbKKT3 mutants. Controls are uninduced cell cultures. Bottom: Example of cells expressing the TbKKT3 mutants before RNAi induction, showing that TbKKT3C668A C671A and TbKKT3D692A do not localize at kinetochores while TbKKT3C707A C710A localizes normally (n > 90 each; also see Fig. S5 E). Maximum intensity projections are shown. RNAi was induced with 1 µg/ml doxycycline (Dox). Cell lines: BAP1791, BAP1793, and BAP1783. Scale bars, 5 µm.