Stu2:Ndc80c-binding details, stu2 ^T866A mutant, and Bik1-binding phenotypes. (A) The crystal structure of Stu2 C-terminus bound to Ndc80^Dwarf (PDB 7KDF) showing electrostatic potential surface of Ndc80^Dwarf. Stu2 illustrated as ribbon. (B) Zoom in of crystal structure (PDB 7KDF) showing hydrophobic interaction between gamma carbon of Stu2^T866 and Spc24^F14. This carbon is lost in an stu2^T866A mutation but preserved in stu2^T866V. (C) Exponentially growing stu2-AID pMET-CDC20 cultures with an ectopically expressed STU2-GFP allele (STU2^WT-GFP, M2599; stu2^T866E-GFP, M2600; and stu2^T866A-GFP, M2601) that also contained SPC110-mCherry (spindle pole) were cultured in methionine- and auxin-containing media to arrest cells in metaphase. Cells were fixed and imaged to determine kinetochore-proximal Stu2-GFP. Bars represent the mean of n = 102–126 individual measurements. Error bars are SEM. P values from two-tailed unpaired t tests. (D) Exponentially growing stu2-AID cultures expressing an ectopic copy of STU2-3V5 (STU2^WT, M622; stu2^T866E, M1448; and stu2^T866A, M2108) as well as DSN1-6His-3Flag from the genomic locus were treated with auxin 30 min prior to harvesting. Kinetochore particles were purified from lysates by anti-Flag immunoprecipitation (IP) and analyzed by immunoblotting. (E) WT (M3) and stu2-AID cells expressing various STU2-3HA alleles from an ectopic locus (STU2^WT, M2898; stu2^T866E, M2829; stu2^T866A, M2830; and stu2^T866V, M4444) were serially diluted (fivefold) and spotted on plates containing DMSO, 500 μM auxin, 5 μg/ml benomyl, and 500 μM auxin + 5 μg/ml benomyl. (F) To analyze expression levels, exponentially growing stu2-AID strains with various T866 mutations and fusion tags (STU2^WT-V5, M622; stu2^T866E-V5, M1448; stu2^T866A-V5, M2108; stu2^T866V-V5, M4398; STU2^WT-HA, M2898; stu2^T866E-HA, M2829; stu2^T866A-HA, M2830; stu2^T866V-HA, M4444; STU2^WT-GFP, M3774; stu2^T866E-GFP, M6524; stu2^T866A-GFP, M3775; and stu2^T866V-GFP, M4429) were lysed and subjected to immunoblot analysis. (G) Exponentially growing stu2-AID BIK1-3FLAG cells expressing STU2-3V5 variants (STU2^WT, M1035; stu2^Δ855–888, M1037; stu2^{L869E I873E M876E}, M2285; and stu2^T866E, M2286) were treated with auxin 30 min prior to harvesting. Bik1 complexes were purified from lysates by anti-Flag immunoprecipitation (IP) and analyzed by immunoblotting. The loss of Stu2:Bik1 interaction in stu2^T866E mutant cells implies that phosphorylation of Stu2^T866 may alter interaction with Bik1 and potentially other Stu2 interactors that bind to Stu2’s C-terminus. (H) AlphaFold 3 prediction of full-length Bik1 dimer bound to Stu2^(855–888). Zoom in shows hydrophobic residues on Stu2 interacting with hydrophobic Bik1 surface. (I) Structure prediction as in F but colored to show plDDT confidence score. (J)fpr1Δ TOR1-1 cells (M1375) and stu2-AID fpr1Δ TOR1-1 cells expressing STU2-FRB alleles at an ectopic locus with and without NUF2-FKBP12 or BIK1-FKBP12 (STU2^WT, M1513; stu2^Δ855–888, M1587; STU2^WTNUF2-FKBP12, M1505; stu2^Δ855–888NUF2-FKBP12, M6473; STU2^WTBIK1-FKBP12, M4516; and stu2^Δ855–888BIK1-FKBP12, M6474) were serially diluted (fivefold) and spotted on plates containing the indicated drugs. Tethering Stu2^Δ855–888-FRB to Nuf2-FKBP has been previously shown to restore cell viability; however, tethering Stu2^Δ855–888-FRB to other cellular interactors of the Stu2 CTS does no rescue growth defects (Zahm et al., 2021; Abouelghar et al., 2022, Preprint). These and prior results suggest that the Ndc80c:Stu2 interaction is the most important CTS interaction for cell viability, and we thus primarily considered T866 phosphorylation in the context of Stu2:Ndc80c binding. Source data are available for this figure: SourceData FS2.