Figure 1. Topology prediction, expression pattern, and in vitro E3 ligase activity of RNF-121. (A) Schematic representation of the predicted membrane topology of RNF-121. Six TMDs are predicted, the RING finger domain faces the cytosol. The amino acid sequence of the RING finger domain is shown. The cysteine and histidine residues of the RING finger domain are shown in red. The predicted E2 binding site (V224) is labeled in yellow (B and C) RNF-121::GFP is localized to the ER in body wall muscles (mu), hypodermis (hyp), and seam cells (SE). Bar, 10 um. (D) RNF-121::GFP colocalizes with the mRFP::SP12 reporter. Bar, 5 um. (E and F) RNF-121::GFP is expressed in the somatic gonad and vulva. Lateral view of midL4 animal. Expression is detected in the vulval cells (vul), spermathecal cells (sp), uterine cells (ut), and the distal tip cell (DTC). Bar, 10 um. (G) RNF-121::GFP colocalizes with the mRFP::mans reporter. Bar, 5 um. (H) In vitro self-ubiquitination assays were performed using wild-type GST-RNF-121RING or RING mutant forms of bacterially expressed and purified protein. In lane 4 (-), the reaction includes E1, E2, Ub, and ATP without the E3 ligase. GST-RNF-5 served as a positive control. Arrow indicates the E1 enzyme. Arrowhead indicates the 5% stacking
gel-10% running gel boundary. High-molecular-weight smears generated by autoubiquitination of the indicated E3 are labeled with parenthesis. Coomassie-stained gel of the purified glutathione transferase (GST)-tagged expressed proteins is shown (bottom).