Spc7-23 is degraded via the ubiquitin-proteasome pathway.

(A) The growth on solid media of wild type (lower panel) and spc7-23 cells (upper panel) transformed with either a control vector (vector) or PstI digested genomic DNA construct encoding mts2-219X was compared at different temperatures. (B) The growth of wild type, mts2-1, spc7-23 and the mts2-1spc7-23 double mutant was compared at the indicated temperatures. (C) The growth on solid media of wild type, nas6Δ, spc7-23 and the nas6Δspc7-23 double mutant was compared at the indicated temperatures. (D) Equal (wild type) or unequal DNA segregation was quantified in spc7-23-gfp and spc7-23-gfp nas6Δ cells at 30°C. ** p<0.01 (Welch test) for the spc7-23-gfp nas6Δ strain compared to the spc7-23-gfp strain. For spc7-23-gfp (n = 200) and spc7-23-gfp nas6Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the spc7-23nas6Δ double mutant. (E) The amount of Spc7-23 protein was followed in cultures at 27°C and 30°C where protein synthesis was inhibited with 100 µg/mL cycloheximide (CHX) for 4 hours. To some cultures 1 mM of the proteasome inhibitor Bortezomib (BZ) was also added. Equal loading was checked using antibodies to tubulin. (F) The growth on solid media of wild type and spc7-23 cells was compared at the indicated temperatures in the absence (control) or presence of 100 µM BZ. (G) Strains with the indicated genetic backgrounds and 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 to the 6His tag on ubiquitin. The arrowhead marks the position where non-ubiquitylated Spc7-23 migrates. Note that ubiquitylated Spc7-23 species are visible in proteasome and bag102Δ mutants.