Hundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We... Show moreHundreds of E3 ligases play a critical role in recognizing specific substrates for modification by ubiquitin (Ub). Separating genuine targets of E3s from E3-interactors remains a challenge. We present BioE3, a powerful approach for matching substrates to Ub E3 ligases of interest. Using BirA-E3 ligase fusions and bioUb, site-specific biotinylation of Ub-modified substrates of particular E3s facilitates proteomic identification. We show that BioE3 identifies both known and new targets of two RING-type E3 ligases: RNF4 (DNA damage response, PML bodies), and MIB1 (endocytosis, autophagy, centrosome dynamics). Versatile BioE3 identifies targets of an organelle-specific E3 (MARCH5) and a relatively uncharacterized E3 (RNF214). Furthermore, BioE3 works with NEDD4, a HECT-type E3, identifying new targets linked to vesicular trafficking. BioE3 detects altered specificity in response to chemicals, opening avenues for targeted protein degradation, and may be applicable for other Ub-likes (UbLs, e.g., SUMO) and E3 types. BioE3 applications shed light on cellular regulation by the complex UbL network. Show less
The fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity... Show moreThe fast dynamics and reversibility of posttranslational modifications by the ubiquitin family pose significant challenges for research. Here we present SUMO-ID, a technology that merges proximity biotinylation by TurboID and protein-fragment complementation to find SUMO-dependent interactors of proteins of interest. We develop an optimized split-TurboID version and show SUMO interaction-dependent labelling of proteins proximal to PML and RANGAP1. SUMO-dependent interactors of PML are involved in transcription, DNA damage, stress response and SUMO modification and are highly enriched in SUMO Interacting Motifs, but may only represent a subset of the total PML proximal proteome. Likewise, SUMO-ID also allow us to identify interactors of SUMOylated SALL1, a less characterized SUMO substrate. Furthermore, using TP53 as a substrate, we identify SUMO1, SUMO2 and Ubiquitin preferential interactors. Thus, SUMO-ID is a powerful tool that allows to study the consequences of SUMO-dependent interactions, and may further unravel the complexity of the ubiquitin code. Show less
