Ret Finger Protein: An E3 Ubiquitin Ligase Juxtaposed to the XY Body in Meiosis
Figure 5
RFP has an E3 ubiquitin ligase activity. (a)–(c) Streptavidin peroxidase revelation of substrate-independent in vitro ubiquitination reactions using immunoprecipitated RFP (a) or GST-RING-B-box of RFP (GST-RING/RFP) (b), (c) as potential E3 ubiquitin ligase enzyme. Dark smears and signals higher than the biggest protein in the assay (monoubiquitinated E1 enzyme indicated by the asterisk) reflected polyubiquitinated products. E2 enzymes are indicated on the top part of each figure. M: master mix containing all the components of the system except E2 enzymes. Sizes (kDa) are indicated. (a) RFP protein immunoprecipitated from 15P-1 cell overexpressing Myc-RFP. Polyubiquitination is observed when UbcH1, UbcH5a, 5b, 5c, UbcH6, or HR6B are used as E2 enzymes. (b) Reactions are performed in presence or in absence of ATP. As expected mono- and poly-ubiquitinations are only observed in presence of ATP. (c) Prior to the substrate-independent in vitro ubiquitination reaction, GST-RING/RFP was pretreated with TPEN (left panel) in order to chelate Zn2+ and destroy RING finger architecture, or pretreated with TPEN, washed, and incubated with an excess of Zn2+ (right panel). As expected, when GST-RING/RFP was treated with TPEN, only mono-ubiquitination can be observed (left panel) and incubation of TPEN treated GST-RING/RFP with an excess of Zn2+ restores E3 ubiquitin ligase activity (right panel).
