title: Endosomal recruitment of the WASH complex: active sequences and mutations impairing interaction with the retromer.
abstract: BACKGROUND
  The Wiskott-Aldrich syndrome protein and scar homolog (WASH) complex is the major Arp2/3 activator at the surface of endosomes. The branched actin network, that the WASH complex induces, contributes to cargo sorting and scission of transport intermediates destined for most endosomal routes. A major challenge is to understand how the WASH molecular machine is recruited to the surface of endosomes. The retromer endosomal machinery has been proposed by us and others to play a role in this process.
  
  
  RESULTS
  In this work, we used an unbiased approach to identify the endosomal receptor of the WASH complex. We have delineated a short fragment of the FAM21 subunit that is able to displace the endogenous WASH complex from endosomes. Using a proteomic approach, we have identified the retromer cargo selective complex (CSC) as a partner of the active FAM21 sequence displacing the endogenous WASH complex. A point mutation in FAM21 that abolishes CSC interaction also impairs WASH complex displacement activity. The CSC is composed of three subunits, VPS35, VPS29 and VPS26. FAM21 directly binds the VPS35 subunit of the retromer CSC. Additionally, we show that a point mutant of VPS35 that blocks binding to VPS29 also prevents association with FAM21 and the WASH complex revealing a novel role for the VPS35-VPS29 interaction in regulating retromer association with the WASH complex.
  
  
  CONCLUSIONS
  This novel approach of endogenous WASH displacement confirms previous suggestions that the retromer is the receptor of the WASH complex at the surface of endosomes and identify key residues that mediate this interaction. The interaction between these two endosomal machineries, the WASH complex and the retromer, is likely to play a critical role in forming platforms at the surface of endosomes for efficient sorting of cargoes.
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Emmanuèle
authors: E
authors: Helfer
authors: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0XY, UK.
authors: Michael E
authors: ME
authors: Harbour
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Véronique
authors: V
authors: Henriot
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Goran
authors: G
authors: Lakisic
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Carla
authors: C
authors: Sousa-Blin
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Larisa
authors: L
authors: Volceanov
authors: Department of Clinical Biochemistry, Cambridge Institute for Medical Research, University of Cambridge, Addenbrooke's Hospital, Cambridge CB2 0XY, UK.
authors: Matthew N J
authors: MNJ
authors: Seaman
authors: Centre de Recherche de Gif, Laboratoire d'Enzymologie et Biochimie Structurales, CNRS UPR3082, Avenue de la Terrasse, Gif-sur-Yvette Cedex 91198, France.
authors: Alexis
authors: A
authors: Gautreau
mesh_terms: D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002352:Carrier Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011992:Endosomes / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D051379:Mice; D008840:Microfilament Proteins / Q000378:metabolism*; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D041681:NIH 3T3 Cells; D028044:Phosphate-Binding Proteins; D017354:Point Mutation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D033921:Vesicular Transport Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; C584240:FAM21 protein, mouse; D008840:Microfilament Proteins; D046912:Multiprotein Complexes; D028044:Phosphate-Binding Proteins; D021122:Protein Subunits; C503111:VPS29 protein, mouse; D033921:Vesicular Transport Proteins; C089989:Vps26 protein, mouse; C101274:Vps35 protein, mouse; C577014:WASH protein, mouse