Degradation of Ybr138C and Fir1 and their interaction with Cdh1.

<p>(<b>A</b>)Two hybrid assay to detect Cdh1-substrate interactions. The bait in this assay was either wild-type Cdh1-ΔN200 or Cdh1-ΔN200-D12, which contains mutations within Cdh1's WD40 domain previously demonstrated to disrupt Cdh1's interaction with the substrate D-Box <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045895#pone.0045895-Burton3" target="_blank">[37]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045895#pone.0045895-Kraft1" target="_blank">[41]</a>. The prey consisted of the APC/C substrate Hsl1 or the APC/C pseudosubstrate inhibitor Acm1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045895#pone.0045895-Burton3" target="_blank">[37]</a>. (<b>B</b>) Yeast two-hybrid interaction between Cdh1 and Ybr138C. Cells expressing Cdh1 or Cdh1-D12 and Ybr138C, Ybr138C-ΔN200, or Ybr138C-N270 were tested for growth on selective medium, which indicates interaction of the respective proteins. Bottom: Schematic representation of Ybr138C indicating positions of potential D-boxes (DB). (<b>C</b>) Degradation of Ybr138C requires its N-terminal region but not putative D-boxes. Cells expressing wild-type <i>YBR138C</i>, <i>YBR138C-3mdb</i>, and <i>YBR138C-ΔN80</i> from a <i>GAL1</i> promoter were arrested in G1 and induced with galactose. Samples were withdrawn at the indicated times after cycloheximide addition and processed for blotting with PAP antibodies as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0045895#pone-0045895-g002" target="_blank">Fig. 2A</a>. (<b>D</b>) <i>cdc15-2</i> and <i>cdc15-2 cdh1Δ</i> cells carrying endogenous <i>YBR138C-TAP</i> were synchronized in mitosis by incubation at 37°C for 3 hours and released at 23°C at time zero. Samples were withdrawn at the indicated times and processed for immunoblotting. Cdc28 was used as a loading control. (<b>E</b>) Yeast two-hybrid interaction between Cdh1 and Fir1. As in (B) but cells carried Fir1 or Fir1-N200 instead of Ybr138C. Bottom: Schematic of Fir1 indicating its potential D-boxes (DB) and its SUMO-interacting motif (SIM).