(A) Effect of mutations at the binding interface. The Stu2 constructs contain the Stu2 coiled-coil domain, a glycine-serine linker, and either native or mutated C-terminal segment (CTS). Ndc80cdwarf was immobilized on Co-NTA agarose, incubated with Stu2, washed, and eluted with 400 mM imidazole. (B) Exponentially growing STU2-AID cultures expressing an ectopic copy of STU2 (STU2WT, M622; stu2Δ855-888, M653; stu2L869E,I873E,M876E or stu2EEE, M1444; stu2L869A,I873A,M876A or stu2AAA, M1576) and also expressing from the genomic locus DSN1-6His-3Flag were treated with auxin 30 min prior to harvesting. Kinetochore particles were purified from lysates by anti-Flag immunoprecipitation (IP) and analyzed by immunoblotting. (C) Exponentially growing STU2-AID CDC20-AID cultures with an ectopically expressed STU2-GFP allele (STU2WT-GFP, M1757; stu2EEE-GFP, M1985) that also contained SPC110-mCherry (spindle pole) were treated with auxin for 2.5 hr to arrest cells in metaphase. Left: Representative micrographs for each are shown. White bars represent 2 μm. Right: Line scan plots through spindle axis (encompassing maximum Stu2-GFP signal) show Stu2-GFP and Spc110-mCherry intensity from example cells shown on left. (D) Spindle-Proximal Stu2-GFP signal from cells (n = 100) and line scan plots described in (C). Area under the curve for each line scan was measured using Fiji (p-value from a two-tailed unpaired t-test). (E) Spindle length (distance between Spc110-mCherry foci) was measured for cells (n = 100) described in (C) (p-value from a two-tailed unpaired t-test; n.s. = not significant). (F) Wild-type (M3), STU2-AID (no covering allele, M619), and STU2-AID cells expressing various STU2-3V5 alleles from an ectopic locus (STU2WT, M622; stu2EEE, M1444; stu2AAA, M1576; stu2Δ855-888, M653) were serially diluted (fivefold) and spotted on plates containing DMSO, 500 μM auxin, or 500 μM auxin + 5 μg/mL benomyl. (G) Exponentially growing STU2-AID fpr1Δ TOR1-1 cultures expressing an ectopic copy of STU2-FRB with wild-type NUF2 (STU2WT, M1513; stu2EEE, M1515) or with NUF2-FKBP12 (STU2WT, M1505; stu2EEE, M1507) were treated with rapamycin and auxin 30 min prior to harvesting. Protein lysates prepared, subjected to α-Flag IP, and analyzed as in (B). (H) fpr1Δ TOR1-1 cells (M1375), STU2-AID fpr1Δ TOR1-1 cells (no covering allele, M1476), and STU2-AID fpr1Δ TOR1-1 cells expressing STU2-FRB alleles at an ectopic locus with and without NUF2-FKBP12 (M1513; M1515; M1505; M1507 – same genotypes as in D) were serially diluted (fivefold) and spotted on benomyl plates containing DMSO, 50 ng/mL rapamycin, 500 μM auxin, or 500 μM auxin + 50 ng/mL rapamycin.