Spc7-23 is degraded via the ubiquitin-proteasome pathway.
2014-01-30T02:45:57Z (GMT) by
<p>(A) The growth on solid media of wild type (lower panel) and <i>spc7-23</i> cells (upper panel) transformed with either a control vector (vector) or PstI digested genomic DNA construct encoding <i>mts2-219X</i> was compared at different temperatures. (B) The growth of wild type, <i>mts2-1</i>, <i>spc7-23</i> and the <i>mts2-1spc7-23</i> double mutant was compared at the indicated temperatures. (C) The growth on solid media of wild type, <i>nas6</i>Δ, <i>spc7-23</i> and the <i>nas6</i>Δ<i>spc7-23</i> double mutant was compared at the indicated temperatures. (D) Equal (wild type) or unequal DNA segregation was quantified in <i>spc7-23-gfp</i> and <i>spc7-23-gfp nas6</i>Δ cells at 30°C. ** p<0.01 (Welch test) for the <i>spc7-23-gfp nas6</i>Δ strain compared to the <i>spc7-23-gfp</i> strain. For <i>spc7-23-gfp</i> (n = 200) and <i>spc7-23-gfp nas6</i>Δ (n = 100) late anaphase cells. Note that wild type DNA segregation is re-established in the <i>spc7-23nas6</i>Δ 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 <i>spc7-23</i> 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 Ni<sup>2+</sup> 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 <i>bag102</i>Δ mutants.</p>