Fig. 1: Screening E3 ligases with condensation and self-degradation capacities for the Targeted Condensation-prone-protein Degradation system (TCD). | Nature Communications

Fig. 1: Screening E3 ligases with condensation and self-degradation capacities for the Targeted Condensation-prone-protein Degradation system (TCD).

From: Genetically-encoded targeted protein degradation technology to remove endogenous condensation-prone proteins and improve crop performance

Fig. 1

See also Supplementary Figs. 1, 2. a The schematic diagram of TCD. X, an endogenous target protein X. E3, an E3 ligase from the UPS system. X–E3, a genetically engineered protein degrader by fusing X with E3. b The heatmap to show the distribution of E3 ligases among the RING subfamilies during screening. Plants possess three families of E3 ligases: RING, HECT, and U-box. A total of 508 RING E3 ligases were analyzed using PLAAC to predict their status as intrinsically disordered proteins (IDPs). Of these, 51 were identified as potential IDPs (E3IDPs). Among these E3IDPs, 44 were successfully cloned and expressed in N. benthamiana as YFP fusion proteins (E3IDP–YFP). 17 E3IDPs formed visible condensates, 17 did not form visible condensates, and 10 exhibited no detectable fluorescence signals. Deleting the RING domain (ΔRING) restored YFP fluorescence as condensates for five of the ten fluorescenceless E3IDP–YFP. c Exemplifying E3IDPs with (E3IDP1) or without (E3IDP6) visible condensates. d Exemplifying E3IDPs with or without RING domains (ΔRING). ΔLCD, deleting the LCD domain. e The domain organization of E3IDP45. The SMART website predicts low-complexity regions (red) and the RING domain (orange; 620–659). LCD, the truncation-experiment-mapped region (green; 227–527) required to form visible condensates in N. benthamiana. f In vitro ubiquitination assay to evaluate the self-ubiquitination capacity of E3IDP45. E1, E2, and ubiquitin (Ub) are components of the ubiquitin-transfer cascade with MBP-tagged E3IDP45 (MBP–E3IDP45) simultaneously as an E3 and substrate. g, h Immunoblot analysis (g) and microscopic observation (h) of E3IDP45–YFP in the absence (Mock) or presence of E1 inhibitor TAK-243, the proteasome inhibitor MG-132, and the autophagy inhibitor E64d. Ponceaus S staining (g) was used as a control for protein loading. Semi-quantitative RT-PCR was conducted against YFP with NbUBQ as the internal control (g). For microscopic observation, 35S::CFP serves as the control (c, d, h). Scale bar, 10 µm (c, d, h). Source data are provided as a Source Data file.

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