Csi1 interacts with Sad1. (A) Schematic diagrams of Csi1 and two mutants. Two asterisks represent mutations of two amino acids from L to P. (B) Live-cell imaging of mutant forms of Csi1-GFP together with Sid4-mRFP. (C) ChIP analysis of mutant Csi1 protein levels at centromeres. Error bars represent standard deviations of three experiments. (D) Yeast two-hybrid analysis of Csi1 with Sad1. Csi1 or Δ2–29 was fused with GAL4 DNA-binding domain (BD), and Sad1 was fused with an activation domain (AD). Interaction between Sad1 and Csi1 results in the activation of a HIS3 reporter gene, allowing cells to grow in the absence of histidine. (E and F) Cell lysate from strains expressing Sad1-HA or Spc7-HA and indicated forms of Csi1-myc were immunoprecipitated with a myc antibody, and Western blot analyses were performed with a HA antibody. Molecular masses are given in kilodaltons. (G) Live-cell imaging analysis of Mis6-GFP and Sid4-mRFP in Csi1 mutants. (H) Csi1 mutant cells containing Ch16 were grown into single colonies to measure loss rate. DIC, differential interference contrast; IP, immunoprecipitation; WT, wild type. Bars, 1 µm.