Rab8b GTPase, a protein transport regulator, is an interacting partner of otoferlin, defective in a human autosomal recessive deafness form

Abstract

Mutations within OTOF encoding otoferlin lead to a recessive disorder called DFNB9. Several studies have indicated otoferlin's association with ribbon synapses of cochlear sensory hair cells, as well as data showing the protein's presence in neurons, nerve fibers and hair cells, suggesting a more ubiquitous function. Otoferlin's co-localization not only with ribbon synaptic proteins, but also with additional endosomal (EEA1) or Golgi proteins (GM130) were motivation for a search for further binding partners of otoferlin by a yeast two-hybrid screen in a rodent cochlear cDNA library (P3-P15). This screen identified Rab8b GTPase as a novel interacting partner, substantiated by transient co-expression and co-localization in HEK 293 cells and co-immunoprecipitation of the complex using tagged proteins in vitro and native proteins from cochlea. This finding implies that otoferlin could be a part of components contributing to trans-Golgi trafficking.

Lack of Brain-Derived Neurotrophic Factor Hampers Inner Hair Cell Synapse Physiology, But Protects against Noise-Induced Hearing Loss

“…Proteins were extracted using the NucleoSpin RNA/protein kit (Macherey-Nagel) following the manufacturer's instructions. SDS-PAGE and Western blotting were performed using the "XCell II SureLock Mini-Cell and XCell II Blot Module" (Invitrogen), as previously described (Heidrych et al, 2008). The blotted proteins were incubated with either rabbit polyclonal or mouse monoclonal antibodies: BDNF (Santa Cruz Biotechnology); GAPDH (Abcam).…”

Otoferlin is a calcium sensor that directly regulates SNARE-mediated membrane fusion

“…However, subcellular localization studies have led to conflicting conclusions. An immunogold staining study reports localization to synaptic vesicles (Roux et al, 2006), whereas immunofluorescence indicated that otoferlin is localized predominantly in the Golgi and possibly in early endosomes (Schug et al, 2006;Heidrych et al, 2008). Thus, otoferlin may have alternative or additional functions, such as vesicle recycling or membrane trafficking in the Golgi.…”

“…Thus, otoferlin may have alternative or additional functions, such as vesicle recycling or membrane trafficking in the Golgi. Recently, it was reported that point mutations within otoferlin resulted in plasma membrane infolding (Heidrych et al, 2008). This suggests a role for otoferlin in the endocytosis and vesicle recycling and could explain why otoferlin IHC knockouts exhibit defects in exocytosis.…”

The otoferlin interactome in neurosensory hair cells: Significance for synaptic vesicle release and trans-Golgi network (Review)

“…Its subcellular distribution beyond regions of synaptic vesicle fusion and its association with Golgi markers suggested a more ubiquitous role. Protein-protein association studies demonstrated that otoferlin can not only interact with syntaxin 1 and Snap25, both ribbon synaptic components (27) but with Rab8 and Myo6, two proteins involved in the transGolgi network and in cargo sorting (31,32). Screening for otoferlin's partners using the yeast-2-hybrid system suggested further candidates (33) ( Table I).…”