FIGURE 7.

Cps1 NTD mutant E7R specifically impacts Rsp5 C-lobe binding. A, GFP-PVEKAPR-DPAP B and GFP-PVrKAPR-DPAP B chimeras were expressed in the bsd2Δ background and visualized by fluorescence microscopy. B, ubiquitination status of DPAP B or the indicated DPAP B chimeras was assessed as described in Fig. 5C, with the exception that immunoprecipitation and detection was using anti-GFP monoclonal antibody. C, ability of Cps1^E7R NTD-GST to recruit His-MBP-C-lobe was compared with Cps1^WT NTD-GST and DPAP B NTD. Detection of bound Rsp5 was by anti-MBP Western blot, bait was visualized by Coomassie Brilliant Blue staining following SDS-PAGE.