Figure 7

SUB can physically interact with AN. (A) Results of a yeast two-hybrid assay are depicted. The top panel shows that only cells carrying both plasmids encoding SUBICD and AN can grow on drop-out medium lacking leucine, tryptophan, and histidine (−LWH). Interaction depends on the presence of a functional SUBICD as indicated by the absence of signal in the variant SUBICD-R599C which mimics a strong sub mutation [6]. As a control all transformants can grow on medium lacking leucine and tryptophan (−LW) (bottom panel). Assays were done in the presence of 2.5 mM 3-amino-1,2,4-triazole (3-AT). GAD and GBD denote empty vector controls. (B) Western blot analysis of in vitro pull-down experiments to test MBP:SUB (full length) or MBP:SUBICD binding to GST:AN. AN was detected using a specific anti-AN antibody. The antibody detects two bands, both of which represent intact AN, as shown by MALDI-TOF analysis, suggesting two different conformations. GST:AN does not interact with MBP alone.