The Mub1/Ubr2 Ubiquitin Ligase Complex Regulates the Conserved Dsn1 Kinetochore Protein
Figure 2
CENP-C recruits Mub1/Ubr2 onto Dsn1-derived kinetochore particles.
(A) Dsn1 association with Ubr2 requires CENP-CMif2. Dsn1-His-Flag was immunoprecipitated from cells with either MIF2 (SBY8253) or mif2-3 (SBY8405) and analyzed via SDS-PAGE and silver staining. Note that CENP-CMif2 runs at the same position as background bands (indicated by asterisk). (B) Dsn1 association with Mub1 requires CENP-CMif2. Dsn1-His-Flag was immunoprecipitated from cells containing Mub1-Myc and either MIF2 (SBY8550) or mif2-3 (SBY8551). (C) CENP-CMif2 associates with Ubr2. Dsn1-His-Flag (SBY8253) and CENP-CMif2-Flag (SBY8519) were immunoprecipitated and analyzed via SDS-PAGE and silver staining. (D) CENP-CMif2-Flag MS summary table. See Table S1 for all proteins identified by MS. (E) CENP-CMif2 association with Ubr2 requires Mub1. CENP-CMif2-Flag was immunoprecipitated from cells in the presence (SBY8519) or absence (SBY8911) of MUB1 and analyzed via SDS-PAGE and silver staining. (F) The association between CENP-CMif2 and Mub1 does not require Ubr2. CENP-CMif2-Flag was immunoprecipitated from cells containing Mub1-Myc in the presence (SBY8546) or absence (SBY8572) of UBR2 and analyzed via immunoblot. The Mub1 protein level in the input is higher in ubr2Δ due to the lack of Ubr2-dependent proteolysis [26]. Note that the CENP-CMif2-Flag band overlaps with a background signal in the input.