Figure 1.

The Ndc10-2 kinetochore protein is a short-lived substrate of the Doa10 pathway. (A) Degradation of Ndc10-2 protein in doa10Δ and cue1Δ cells. Cycloheximide was added 15 min after shifting them to 37°C, and aliquots of cells were removed at the indicated times. Lysates were analyzed by anti-Ndc10 immunoblotting. A strain with the chromosomal NDC10 tagged with the TAP-tag coding sequence (lane 1) was used as a control for antibody specificity. Asterisk, a crossreacting protein that served as a loading control. (B) Two previously isolated suppressors of ndc10-2 are also defective for Deg1-mediated proteolysis. Strains carrying the suppressor mutations kis3–66 and kis4–14 were transformed with a LEU2 plasmid expressing the fusion Deg1-Ura3. Failure to degrade this protein rapidly allows growth on uracil. (C) Complementation analysis demonstrates that kis3–66 has a mutation in DOA10 and kis4–14 a mutation in CUE1. The strains on the upper half of the plate were mated to cue1Δ, while those on the bottom half were mated to doa10Δ cells. Two diploids from each cross were streaked on each plate. (D) Anti-Doa10 immunoblot analysis of kis mutants.