Fig. 2
From: Slx5-Slx8 ubiquitin ligase targets active pools of the Yen1 nuclease to limit crossover formation
Yen1 interacts with Slx5–Slx8 in the nucleus. a Diploid strains carrying one allele of galactose inducible VC-Slx5 and VN-Yen1 with wild-type copies of YEN1 and SLX5 in the homologous chromosomes were observed by live microscopy. BiFC (white arrows) signal denotes an interaction between the two BiFC (Venus) epitopes. Control diploids lacking one or both of the epitope-tagged proteins (ϕ) were used to substract background signal. A plasmid carrying Nup49-mCherry was transformed on the diploid strain harboring VC-Slx5/VN-Yen1 to visualize the nuclear perimeter. BiFC interactions were only detected in the nuclear compartment. b Cells carrying either an empty vector or a pYES2 plasmid expressing GST-Slx5 under galactose control were grown in selective media and induced with galactose for 3 h. Lysates were then applied to a glutathione-sepharose column. After washing, the bound proteins were eluted and immunoblotted with α-HA (upper) or α-GST (lower). c Two-hybrid assays were performed with strains carrying the indicated activating domain (AD) or DNA binding domain (BD) fusions. Strains were grown in selective media lacking leucine (L) and tryptophan (W) prior to spotting on media lacking histidine (H) to detect a positive interaction. Experiments were independently replicated three times and images are representative of the reproducible results obtained
