Fig. 4

Analysis of UFD-2 substrate specificity. a UFD-2-mediated ubiquitination of UNC-45_ΔUCS, UNC-45_GST-UCS, UNC-45_ΔTPR, and UNC-45_E781K in comparison to the wild-type, full-length protein. b Plotted lane profiles of ubiquitination reactions shown in panel a. Mono- and di-ubiquitinated species are indicated for the deletion constructs. c Pull-down studies showing the interaction of UFD-2 with different UNC-45 proteins. d Cartoon representation of UNC-45 (PDB code: 4i2z) with TPR, central, neck, and UCS domains colored in green, orange, yellow, and gray, respectively. Ubiquitination sites identified in MS experiments are shown as colored spheres (green: residues bordering the myosin-binding canyon; magenta: residues near the C terminus of the UCS domain; and yellow: residues close to the flexible UCS loop). e Upper panel: 60 and 75 min time points of UNC-45 KR ubiquitination reactions with proteins that have been pre-incubated for 60 min at 30 °C were analyzed by anti-UNC-45 western blot. Lower panel: plotted lane profiles of the 60 min time point of the ubiquitination reactions, presented in two graphs for clarity