(A) Schematic diagram showing Sgo1-associated complexes and their functions at the pericentromere. (B) Sgo1-100 and Sgo1-3A, but not Sgo1-700, retain association with Brn1. Cells carrying BRN1-6HA and SGO1-SZZ(TAP) (AM9266), SGO1-100-SZZ(TAP) (AM9149), SGO1-700-SZZ(TAP) (AM9264), SGO1-3A-SZZ(TAP) (AM9262) or no TAP (AM5708) were arrested in nocodazole for 2 hr before treating with the cross-linker DSP. Prepared extracts were incubated with IgG-coupled beads and immunoprecipitates analyzed by immunoblotting with the indicated antibodies. (C) Brn1 is maintained at the centromere in metaphase-arrested sgo1-3A, but not sgo1-100 or sgo1-700 cells. Wild-type (AM5708), sgo1Δ (AM8834), sgo1-100 (AM9442), sgo1-700 (AM9291) and sgo1-3A (AM9276) cells carrying BRN1-6HA as well as a no tag control (AM1176) were arrested in nocodazole for 2 hr before harvesting for anti-HA ChIP. The levels of Brn1-6HA were measured at the indicated sites by qPCR. (D–F) Tethered Sgo1, Sgo1-100 or Sgo1-3A, but not Sgo1-700 can enrich Brn1-6HA at CEN4 in otherwise sgo1Δ cells. SGO1-tetR-GFP (AM14012), sgo1-100-tetR-GFP (AM13902), sgo1-700-tetR-GFP (AM13907), and sgo1-3A-tetR-GFP (AM13904) were introduced into cells carrying tetOs integrated at CEN4, producing Brn1-6HA and with SGO1 deleted from its endogenous locus. A strain carrying just tetOs integrated at CEN4 but otherwise wild type was used as a no tag control (AM11060). All strains were arrested in mitosis by treatment with nocodazole for 3 hr before harvesting for ChIP and immunoblotting. (D) Schematic diagram of the tethering locus. (E) Levels of Brn1 recruited adjacent to the tethering site (CEN4) when the indicated proteins are fused to TetR-GFP, as measured by ChIP-qPCR. The mean of four independent repeats is shown except for sgo1-3A-tetR-GFP where six repeats are included. Error bars are standard error, significance was calculated using the student t test (*p<0.05). (F) Total cellular levels of the Sgo1-tetR-GFP fusion proteins, Brn1-6HA and Pgk1 (loading control) were analyzed by immunoblot using the indicated antibodies. (G) The bias to sister kinetochore biorientation is absent in sgo1-700 cells. Wild-type (AM4643), sgo1-100 (AM8924), sgo1-700 (AM8925) and sgo1-3A (AM8923) cells were released from G1 and treated with nocodazole after SPB separation as in Figure 7A. The percentage of cells that separated CEN4-GFP foci at least once during the observation period is shown for the indicated strains. p values indicate significance (chi-square test).