title: Retromer binding to FAM21 and the WASH complex is perturbed by the Parkinson disease-linked VPS35(D620N) mutation.
abstract: Retromer is a protein assembly that plays a central role in orchestrating export of transmembrane-spanning cargo proteins from endosomes into retrieval pathways destined for the Golgi apparatus and the plasma membrane [1]. Recently, a specific mutation in the retromer component VPS35, VPS35(D620N), has linked retromer dysfunction to familial autosomal dominant and sporadic Parkinson disease [2, 3]. However, the effect of this mutation on retromer function remains poorly characterized. Here we established that in cells expressing VPS35(D620N) there is a perturbation in endosome-to-TGN transport but not endosome-to-plasma membrane recycling, which we confirm in patient cells harboring the VPS35(D620N) mutation. Through comparative stable isotope labeling by amino acids in cell culture (SILAC)-based analysis of wild-type VPS35 versus the VPS35(D620N) mutant interactomes, we establish that the major defect of the D620N mutation lies in the association to the actin-nucleating Wiskott-Aldrich syndrome and SCAR homolog (WASH) complex. Moreover, using isothermal calorimetry, we establish that the primary defect of the VPS35(D620N) mutant is a 2.2 ± 0.5-fold decrease in affinity for the WASH complex component FAM21. These data define the primary molecular defect in retromer assembly that arises from the VPS35(D620N) mutation and, by revealing functional effects on retromer-mediated endosome-to-TGN transport, provide new insight into retromer deregulation in Parkinson disease.
authors: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Ian J
authors: IJ
authors: McGough
authors: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Florian
authors: F
authors: Steinberg
authors: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors: Da
authors: D
authors: Jia
authors: Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK.
authors: Peter A
authors: PA
authors: Barbuti
authors: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Kirsty J
authors: KJ
authors: McMillan
authors: Proteomics Facility, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK.
authors: Kate J
authors: KJ
authors: Heesom
authors: Institute of Clinical Neurosciences, University of Bristol, Frenchay Hospital, Bristol BS16 1LE, UK.
authors: Alan L
authors: AL
authors: Whone
authors: Henry Wellcome Laboratory for Integrative Neuroscience and Endocrinology, School of Clinical Sciences, University of Bristol, Dorothy Hodgkin Building, Whitson Street, Bristol BS1 3NY, UK.
authors: Maeve A
authors: MA
authors: Caldwell
authors: Departments of Biochemistry and Molecular Biology and Immunology, Mayo Clinic, Rochester, MN 55905, USA.
authors: Daniel D
authors: DD
authors: Billadeau
authors: Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA; Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA.
authors: Michael K
authors: MK
authors: Rosen
authors: The Henry Wellcome Integrated Signaling Laboratories, School of Biochemistry, Medical Sciences Building, University of Bristol, Bristol BS8 1TD, UK. Electronic address: pete.cullen@bristol.ac.uk.
authors: Peter J
authors: PJ
authors: Cullen
mesh_terms: D017089:Ankyrin Repeat / Q000235:genetics; D000951:Antigens, Neoplasm / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D011992:Endosomes / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins / Q000378:metabolism*; D018832:Molecular Chaperones / Q000378:metabolism; D009154:Mutation; D010300:Parkinson Disease / Q000235:genetics*; D011485:Protein Binding / Q000235:genetics; D021381:Protein Transport; D033921:Vesicular Transport Proteins / Q000235:genetics* / Q000378:metabolism*
chemical_list: D000951:Antigens, Neoplasm; C507525:DNAJC13 protein, human; C510453:ENTR1 protein, human; D047908:Intracellular Signaling Peptides and Proteins; D008840:Microfilament Proteins; D018832:Molecular Chaperones; C419573:VPS35 protein, human; D033921:Vesicular Transport Proteins; C549919:WASH protein, human