Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology · 2012
Ergic2, a brain specific interacting partner of Otoferlin.
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Żak Magdalena, Bress Andreas, Brandt Niels, Franz Christoph, Ruth Peter, Pfister Markus, Knipper Marlies, Blin Nikolaus
Аннотация
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.
CONCLUSION: Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.
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.
CONCLUSION: Our new data on Otoferlin's interactome suggest that Otoferlin can form different, tissue-specific protein complexes.