title: Tryptophan-rich basic protein (WRB) mediates insertion of the tail-anchored protein otoferlin and is required for hair cell exocytosis and hearing.
abstract: The transmembrane recognition complex (TRC40) pathway mediates the insertion of tail-anchored (TA) proteins into membranes. Here, we demonstrate that otoferlin, a TA protein essential for hair cell exocytosis, is inserted into the endoplasmic reticulum (ER) via the TRC40 pathway. We mutated the TRC40 receptor tryptophan-rich basic protein (Wrb) in hair cells of zebrafish and mice and studied the impact of defective TA protein insertion. Wrb disruption reduced otoferlin levels in hair cells and impaired hearing, which could be restored in zebrafish by transgenic Wrb rescue and otoferlin overexpression. Wrb-deficient mouse inner hair cells (IHCs) displayed normal numbers of afferent synapses, Ca2+channels, and membrane-proximal vesicles, but contained fewer ribbon-associated vesicles. Patch-clamp of IHCs revealed impaired synaptic vesicle replenishment. In vivo recordings from postsynaptic spiral ganglion neurons showed a use-dependent reduction in sound-evoked spiking, corroborating the notion of impaired IHC vesicle replenishment. A human mutation affecting the transmembrane domain of otoferlin impaired its ER targeting and caused an auditory synaptopathy. We conclude that the TRC40 pathway is critical for hearing and propose that otoferlin is an essential substrate of this pathway in hair cells.
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.|Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Vogl
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.|Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Göttingen Graduate School for Neurosciences, Biophysics and Molecular Biosciences, University of Göttingen, Göttingen, Germany.
authors.forename: Iliana
authors.initials: I
authors.lastname: Panou
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Auditory Systems Physiology Group and InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany.
authors.forename: Gulnara
authors.initials: G
authors.lastname: Yamanbaeva
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Molecular Architecture of Synapses Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Carolin
authors.initials: C
authors.lastname: Wichmann
authors.affiliation: Otolaryngology Division, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA.
authors.forename: Sara J
authors.initials: SJ
authors.lastname: Mangosing
authors.affiliation: Institute of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Fabio
authors.initials: F
authors.lastname: Vilardi
authors.affiliation: Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
authors.forename: Artur A
authors.initials: AA
authors.lastname: Indzhykulian
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Synaptic Physiology of Mammalian Vestibular Hair Cells Junior Research Group, Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Tina
authors.initials: T
authors.lastname: Pangršič
authors.affiliation: Department of Neurosciences, University of Padova, Padova, Italy.|Audiology and Phoniatrics Service, Treviso Regional Hospital, Treviso, Italy.
authors.forename: Rosamaria
authors.initials: R
authors.lastname: Santarelli
authors.affiliation: Servicio de Genetica, Hospital Universitario Ramon y Cajal, IRYCIS, Madrid, Spain.
authors.forename: Montserrat
authors.initials: M
authors.lastname: Rodriguez-Ballesteros
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Weber
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany.|Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.
authors.forename: Sangyong
authors.initials: S
authors.lastname: Jung
authors.affiliation: Otolaryngology Division, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA.
authors.forename: Elena
authors.initials: E
authors.lastname: Cardenas
authors.affiliation: Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, MA, USA.
authors.forename: Xudong
authors.initials: X
authors.lastname: Wu
authors.affiliation: Department of Molecular Neurobiology, Max-Planck-Institute for Experimental Medicine, Göttingen, Germany.
authors.forename: Sonja M
authors.initials: SM
authors.lastname: Wojcik
authors.affiliation: W. M. Keck Center for Collaborative Neuroscience, Nelson Lab-D250, Rutgers University, Piscataway, NJ, USA.
authors.forename: Kelvin Y
authors.initials: KY
authors.lastname: Kwan
authors.affiliation: Servicio de Genetica, Hospital Universitario Ramon y Cajal, IRYCIS, Madrid, Spain.|Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER), Madrid, Spain.
authors.forename: Ignacio
authors.initials: I
authors.lastname: Del Castillo
authors.affiliation: Institute of Molecular Biology, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Blanche
authors.initials: B
authors.lastname: Schwappach
authors.affiliation: Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Auditory Systems Physiology Group and InnerEarLab, Department of Otolaryngology, University of Göttingen Medical Center, Göttingen, Germany.
authors.forename: Nicola
authors.initials: N
authors.lastname: Strenzke
authors.affiliation: Howard Hughes Medical Institute and Department of Neurobiology, Harvard Medical School, Boston, MA, USA david_corey@hms.harvard.edu shuh-yow@ucsd.edu tmoser@gwdg.de.
authors.forename: David P
authors.initials: DP
authors.lastname: Corey
authors.affiliation: Otolaryngology Division, Department of Surgery, School of Medicine, University of California San Diego, La Jolla, CA, USA david_corey@hms.harvard.edu shuh-yow@ucsd.edu tmoser@gwdg.de.
authors.forename: Shuh-Yow
authors.initials: SY
authors.lastname: Lin
authors.affiliation: Institute for Auditory Neuroscience and InnerEarLab, University Medical Center Göttingen, Göttingen, Germany david_corey@hms.harvard.edu shuh-yow@ucsd.edu tmoser@gwdg.de.|Collaborative Research Center 889, University of Göttingen, Göttingen, Germany.|Synaptic Nanophysiology Group, Max Planck Institute for Biophysical Chemistry, Göttingen, Germany.|Center for Nanoscale Microscopy and Molecular Physiology of the Brain, University Medical Center Göttingen, Göttingen, Germany.
authors.forename: Tobias
authors.identifier: 0000-0001-7145-0533
authors.initials: T
authors.lastname: Moser
mesh_terms: D000818:Animals; D053501:Arsenite Transporting ATPases / Q000378:metabolism*; D005089:Exocytosis; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006198:Hair Cells, Auditory / Q000378:metabolism*; D006309:Hearing; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism*; D015027:Zebrafish; D029961:Zebrafish Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: C111546:GET1 protein, human; D008565:Membrane Proteins; D009687:Nuclear Proteins; C118457:OTOF protein, human; D029961:Zebrafish Proteins; C516233:otoferlin protein, mouse; C000620532:wrb protein, zebrafish; D053501:Arsenite Transporting ATPases
keywords: deafness; endoplasmic reticulum; protein targeting; synapse; tail‐anchored protein