title: Ergic2, a brain specific interacting partner of Otoferlin.
abstract: BACKGROUND
  Otoferlin, a postulated calcium sensor of 230 kDa, was proposed to trigger calcium dependent fusion of vesicles with plasma membrane in the ribbon synapses of cochlear IHCs. Otoferlin's interaction with Rab8b and Myo6, proteins involved in the intracellular membrane trafficking, extended the previous hypothesis assigning Otoferlin an additional role in trans-Golgi trafficking. Here, we present another Otoferlin binding partner, Ergic2, a protein with a still unknown function but presenting sequence homology to other proteins involved in ER/Golgi vesicle trafficking.
  
  
  METHODS
  Novel binding partners of Otoferlin were searched by yeast two-hybrid screening in a rodent cochlear cDNA library (P3-P15). RT-PCR, western blot, immunohistochemistry staining and co-immunoprecipitation were applied to analyze and confirm an interaction between Ergic2 and Otoferlin.
  
  
  RESULTS
  The Y-2-H screening, using baits covering parts of Otoferlin's C2D domain, identified Ergic2 as an interacting protein for Otoferlin. Both are co-expressed (mRNA and protein level) in rodent cochlea and brain before- and after-onset of hearing. By RT-PCR Ergic2 was detected in cochlear IHCs and OHCs and in brain regions where Otoferlin is known to be present. Co-localization studies revealed an overlap of Ergic2 and Otoferlin signals in IHCs and neurons of cerebral cortical layer I making Ergic2 the promising binding candidate. However, while Ergic2 was co-precipitated by an anti-Otoferlin antibody in protein lysates from murine brain, this specific protein interaction was not detected in cochlea.
  
  
  CONCLUSIONS
  Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.
authors.affiliation: University of Tübingen, Div. of Moleculer Genetics, Inst. of Human Genetics, Tübingen, Germany.
authors.forename: Magdalena
authors.initials: M
authors.lastname: Żak
authors.forename: Andreas
authors.initials: A
authors.lastname: Bress
authors.forename: Niels
authors.initials: N
authors.lastname: Brandt
authors.forename: Christoph
authors.initials: C
authors.lastname: Franz
authors.forename: Peter
authors.initials: P
authors.lastname: Ruth
authors.forename: Markus
authors.initials: M
authors.lastname: Pfister
authors.forename: Marlies
authors.initials: M
authors.lastname: Knipper
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Blin
mesh_terms: D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D003051:Cochlea / Q000378:metabolism; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000378:metabolism; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D008565:Membrane Proteins / Q000378:metabolism*; D051379:Mice; D008817:Mice, Mutant Strains; D011485:Protein Binding; D021381:Protein Transport; D060888:Real-Time Polymerase Chain Reaction; D020798:Two-Hybrid System Techniques; D033921:Vesicular Transport Proteins
chemical_list: D002352:Carrier Proteins; D017931:DNA Primers; C576154:Ergic2 protein, mouse; D008565:Membrane Proteins; D033921:Vesicular Transport Proteins; C516233:otoferlin protein, mouse