RNF41 interacts with the VPS52 subunit of the GARP and EARP complexes

RNF41 (Ring Finger Protein 41) is an E3 ubiquitin ligase involved in the intracellular sorting and function of a diverse set of substrates. Next to BRUCE and Parkin, RNF41 can directly ubiquitinate ErbB3, IL-3, EPO and RARa receptors or downstream signaling molecules such as Myd88, TBK1 and USP8. In this way it can regulate receptor signaling and routing. To further elucidate the molecular mechanism behind the role of RNF41 in intracellular transport we performed an Array MAPPIT (Mammalian Protein-Protein Interaction Trap) screen using an extensive set of proteins derived from the human ORFeome collection. This paper describes the identification of VPS52, a subunit of the GARP (Golgi-Associated Retrograde Protein) and the EARP (Endosome-Associated Recycling Protein) complexes, as a novel interaction partner of RNF41. Through interaction via their coiled coil domains, RNF41 ubiquitinates and relocates VPS52 away from VPS53, a common subunit of the GARP and EARP complexes, towards RNF41 bodies

High-confidence interactome for RNF41 built on multiple orthogonal assays

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

L'assurance perte de bénéfices et frais généraux ou profits insurance : thèse pour le doctorat... / présentée... par P. R. de Magnin... ; Université de Paris, Faculté de droit

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High-Confidence Interactome for RNF41 Built on Multiple Orthogonal Assays

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

The Prader-Willi syndrome proteins MAGEL2 and necdin regulate leptin receptor cell surface abundance through ubiquitination pathways

In Prader-Willi syndrome (PWS), obesity is caused by the disruption of appetite-controlling pathways in the brain. Two PWS candidate genes encode MAGEL2 and necdin, related melanoma antigen proteins that assemble into ubiquitination complexes. Mice lacking Magel2 are obese and lack leptin sensitivity in hypothalamic pro-opiomelanocortin neurons, suggesting dysregulation of leptin receptor (LepR) activity. Hypothalamus from Magel2-null mice had less LepR and altered levels of ubiquitin pathway proteins that regulate LepR processing (Rnf41, Usp8, and Stam1). MAGEL2 increased the cell surface abundance of LepR and decreased their degradation. LepR interacts with necdin, which interacts with MAGEL2, which complexes with RNF41 and USP8. Mutations in the MAGE homology domain of MAGEL2 suppress RNF41 stabilization and prevent the MAGEL2-mediated increase of cell surface LepR. Thus, MAGEL2 and necdin together control LepR sorting and degradation through a dynamic ubiquitin-dependent pathway. Loss of MAGEL2 and necdin may uncouple LepR from ubiquitination pathways, providing a cellular mechanism for obesity in PWS

RNF41 ubiquitinates and relocates VPS52.

<p><b>(A)</b> RNF41 enhances VPS52 ubiquitination. HEK293T cells co-transfected with a plasmid encoding Flag-tagged VPS52 and untagged RNF41, L163Q or soluble IL5Rα (mock) were incubated overnight with 5mM MG132 to inhibit proteasomal degradation. Flag-VPS52 was immunoprecipitated and ubiquitination was determined by Western Blotting with an anti-ubiquitin antibody (upper panels). Expression and loading controls were visualized using anti-Flag, RNF41 and anti-β-actin antibodies (lower panels). Statistical analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178132#pone.0178132.s008" target="_blank">S5A Fig</a>. <b>(B)</b> RNF41 co-resides with VPS52 in the RIPA insoluble pellet fraction. HEK293T cells transiently co-transfected with a plasmid encoding untagged VPS52 and untagged RNF41, L163Q, DN RNF41 or soluble IL5Rα (mock) were either sonicated in 2x Laemmli buffer (left) or lysed with RIPA buffer (middle). Insoluble RIPA pellets were sonicated in 2x Laemmli buffer (right). Protein levels were detected by Western blotting using anti-VPS52, anti-RNF41 and anti-β-actin (loading control). Statistical analysis is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178132#pone.0178132.s008" target="_blank">S5B Fig</a>. <b>(C)</b> RNF41 relocates VPS52 and not VPS53. (I) Confocal microscopy of HeLa cells transiently transfected with a vector encoding E-tagged VPS52 together with soluble IL5Rα (mock, upper panel), untagged RNF41 (middle panel) or L163Q (lower panel) (right side shows merged images) were fixed and stained with antibodies against Etag (secondary Alexa Fluor 488, green), RNF41 (secondary Alexa Fluor 568, magenta) and Golgi marker p230 (secondary Alexa Fluor 647, red). (II) Same setup as in I, with co-transfection of VPS53 instead of E-tagged VPS52, and the use of anti-VPS53 (secondary Alexa Fluor 488, green) and co-staining with Golgi marker GM130 (secondary Alexa Fluor 647, red). n = 3 or more independent experiments. <b>(D)</b> Confocal microscopy and <b>(E)</b> AlphaScreen analysis show that Ectopic RNF41 expression disrupts the VPS52-VPS53 interaction. For confocal microscopy, HeLa cells were transiently transfected with a plasmid encoding E-tagged VPS52 and untagged VPS53 together with soluble IL5Rα (mock, upper panel), untagged RNF41 (middle panel) or L163Q (lower panel) (right side shows merged images), fixed and stained with antibodies against Etag (secondary Alexa Fluor 488, green), VPS53 (secondary Alexa Fluor 568, red) and RNF41 (secondary Alexa Fluor 647, magenta). The inset shows a magnification of the boxed area. The white overlay represents the intersect between the golgi marker and E-tagged VPS52 or VPS53 (C) or between E-tagged VPS52 and VPS53 (D) with a threshold set on standard deviation using Volocity 6.3 software. n = 3 or more independent experiments. Scale bar, 10μm. For AlphaScreen analysis, HEK293T cells were transiently co-transfected with a plasmid encoding E-tagged VPS52 and Flag-tagged VPS53 together with WT RNF41, L163Q or soluble IL5Rα (mock). Values are means ± s.d from triplicate samples from one of three representative experiments. Data and statistical analysis of biological replicates are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0178132#pone.0178132.s006" target="_blank">S3 Fig</a>.</p