High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays

Abstract

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.

Keeping in Touch with Type-III Secretion System Effectors: Mass Spectrometry-Based Proteomics to Study Effector–Host Protein–Protein Interactions

“…The availability of various interactomics approaches and their application have provided insights into the strengths and weaknesses with respect to studying host-pathogen interactions. The often complementary nature of the different interactomics methods currently at hand, stresses the need to use more than one approach to obtain a complete and less biased picture of EH-PPI repertoires ( Figure 3 ), as illustrated by several studies discussed above [ 99 , 101 , 102 ]. AP-MS and derived technologies thereof are usually not successful in detecting weak and dynamic PPIs, due to their highly dynamic nature, or membrane targets, due to their poor solubilization and thus extraction during native protein extraction.…”

“…Virotrap on the other hand benefits from its unique feature being the evasion of cell lysis by hijacking vesicle-forming properties of HIV-1 GAG protein. In addition, especially for poorly soluble proteins, such as membrane and cytoskeletal proteins, BioID and Virotrap have proven to clearly outperform AP-MS screenings [ 97 , 99 , 101 , 102 ]. Since the total amount of membrane-associated effectors is estimated to be around 30%, there is a clear necessity for techniques capable of targeting this protein category [ 95 ].…”

“…GFP fused to a PBL, for example, has frequently been used as a control for non-bait specific biotinylation [ 83 ]. In addition, Virotrap has proven to be complementary to BioID in identifying PPIs [ 99 ], and both techniques are based on a completely different principle, making the methods also intrinsically complementary. Although both techniques deliver only partially overlapping protein identifications, they also harbor a specific set of missing interactions, or false negatives.…”

“…Hence, the endogenous location of Virotrap bait-interactions should preferentially be the cytosol, as this is the most physiologically relevant location for interactions in Virotrap. Currently, studies on 33 Virotrap target hits [98,99] that reside in or at the plasma membrane or fulfill (one of) their function(s) in the cytosol have been published.…”

“…In this context, it is noteworthy that VLPs have successfully been isolated from plant tissue expressing full-length HIV-1 GAG [100], so application of Virotrap in plants could be an interesting subject of future investigation. Virotrap has fruitfully assisted in selecting candidate interactions for human ring finger protein 41 (RNF41) and proved to be orthogonal to BioID and AP-MS in that regard [97,99]. Furthermore, we recently obtained proof-of-concept data that Virotrap can be used for studying EH-PPIs by fusing the C-terminus of GAG to Salmonella T3Es, by the identification of known host interactors (unpublished data).…”