Spc7-23 degradation depends on the proteasome-associated DUB Ubp3.

(A) The growth on solid media of spc7-23 cells transformed with either a control plasmid (vector) or expression constructs for ubp3⁺ and ubp3-W466X was compared at different temperatures. (B) The growth on solid media of wild type, ubp3Δ, spc7-23 and the ubp3Δspc7-23 double mutant was compared at the indicated temperatures. (C) Equal (wild type) or unequal DNA segregation was quantified in spc7-23-gfp and spc7-23-gfp ubp3Δ cells at 30°C. ** p<0.01 (Welch test) for the spc7-23-gfp ubp3Δ strain compared to the spc7-23-gfp strain. For spc7-23-gfp (n = 200) and spc7-23-gfp ubp3Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the spc7-23ubp3Δ double mutant. (D) Cells transformed with vector (control) or Ubp3-Flag were used for immunoprecipitation experiments using antibodies to the Flag epitope. The precipitated material was analyzed by blotting for the proteasome subunit Mts4 (upper panel) or as a loading control Ubp3 (lower panel). (E) Wild type or ubp3Δ cells transformed to express 6His-tagged ubiquitin, were lysed and used for precipitation experiments with a Ni²⁺ resin in 8 M urea. The precipitated material was analyzed by blotting with antibodies to the HA-tag on Spc7-23 or the 6His tag on ubiquitin. The arrowhead marks the position where non-ubiquitylated Spc7-23 migrates.