Sumoylation of Mcm21p is not required for kinetochore localization of the CPC. (A) Sumoylation of CTF19 complex components. Immunoprecipitation/Western blotting was performed from protein extracts of wild-type or ubc9-2 strains expressing Mcm21p-TAP, Ame1p-TAP, Ctf19p-TAP, or Okp1p-TAP. A strain expressing a version of TAP-tagged Mcm21p lacking 32 lysine residues (mcm21-32R) was also analyzed for SUMO modification. The asterisk indicates sumoylated Mcm21p protein. The anti-SUMO antibody detects both sumoylated and unmodified proteins due to the associated TAP tag. (B) Subcellular localization of wild-type Mcm21p-GFP and Mcm21p-32R-GFP. DIC images, fluorescent micrographs, and merged images of representative wild-type or mcm21Δ anaphase cells expressing either wild-type Mcm21p-GFP or Mcm21p-32R-GFP and RFP-Tub1p are shown. Note the formation of a fish hook spindle in the mcm21-32R mutant background. Bar, 5 µm. (C and D) Ipl1p-GFP (C) and Sli15p-GFP (D) localization to the kinetochore in wild-type, mcm21-32R, and mcm21Δ mutant cells. Mid-log phase cells were treated with nocodazole for 2.5 h at room temperature and released into fresh medium before imaging. Fluorescent micrographs and merged images are shown of representative single cells of wild-type, mcm21Δ, or mcm21-32R strains expressing Ipl1p-GFP and RFP-Tub1p. Bar, 5 µm. (E and F) Quantitation of the defect in Ipl1p-GFP and Sli15p-GFP localization to the kinetochore during metaphase in wild-type, mcm21Δ, and mcm21-32R mutant cells. Cells with spindles between <2 and 2–4 µm were quantified for Ipl1p-GFP (E) or Sli15p-GFP localization to the kinetochore (F) as indicated. n > 200 cells in each spindle length category.