High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays

• Published in: Journal of proteome research Vol. 17; no. 4; pp. 1348 - 1360
• Main Authors: Masschaele, Delphine, Wauman, Joris, Vandemoortele, Giel, De Sutter, Delphine, De Ceuninck, Leentje, Eyckerman, Sven, Tavernier, Jan
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 06.04.2018
• Subjects: BioID; AP−MS; interactomics; cytokine receptor signaling; AP2S1; virotrap; microarray MAPPIT; high-confidence interactome; Y2H; RNF41
• ISSN: 1535-3893; 1535-3907
• DOI: 10.1021/acs.jproteome.7b00704

Ring finger protein 41 (RNF41) is an E3 ubiquitin ligase involved in the ubiquitination and degradation of many proteins including ErbB3 receptors, BIRC6, and parkin. Next to this, RNF41 regulates the intracellular trafficking of certain JAK2-associated cytokine receptors by ubiquitinating and suppressing USP8, which, in turn, destabilizes the ESCRT-0 complex. To further elucidate the function of RNF41 we used different orthogonal approaches to reveal the RNF41 protein complex: affinity purification–mass spectrometry, BioID, and Virotrap. We combined these results with known data sets for RNF41 obtained with microarray MAPPIT and Y2H screens. This way, we establish a comprehensive high-resolution interactome network comprising 175 candidate protein partners. To remove potential methodological artifacts from this network, we distilled the data into a high-confidence interactome map by retaining a total of 19 protein hits identified in two or more of the orthogonal methods. AP2S1, a novel RNF41 interaction partner, was selected from this high-confidence interactome for further functional validation. We reveal a role for AP2S1 in leptin and LIF receptor signaling and show that RNF41 stabilizes and relocates AP2S1.