Abstract
The human Usher syndrome (USH) is a retinal ciliopathy, characterized by profound congenital deafness, variable vestibular dysfunction and pre-pubertal onset of retinitis pigmentosa. In the effected sensory cells, USH protein networks are assumed to function in ciliary transport processes. The USH1G protein SANS is a scaffold of the ciliary/periciliary USH protein network of photoreceptor cells. Moreover, SANS is associated with microtubules, the transport routes for protein delivery toward the cilium. To enlighten the role of SANS in ciliary transport processes, we aimed to identify transport related proteins associated with SANS. The intraflagellar transport (IFT) system is a conserved mechanism for bi-directional transport toward and through primary cilia. Thus, we tested the direct binding of SANS to IFT molecules, namely IFT20, IFT57, and IFT74 in 1:1 yeast-two-hybrid assay. The identified SANS-IFT interactions were validated in vitro via independent complementary interaction assays and in cells by applying membrane targeting assays. Quantitative immunofluorescence microscopy revealed the co-localization of SANS with IFT20, IFT52, and IFT57 particularly at ciliary base of wild type mouse photoreceptor cells. Analysis of photoreceptor cells of SANS knock out mice revealed the decrease of IFTs in the ciliary compartment indicating a role of SANS in the proper positioning of IFT-B molecules in primary cilia. Our study demonstrated direct binding of IFT complex B proteins IFT52 and IFT57 to the N-terminal ankyrin repeats and the central domain of SANS. Our data also indicate that pathologic mutations in the N-terminus of SANS lead to the loos of SANS binding to IFT-B molecules. Our findings provide direct evidence for a molecular link between the ciliary USH protein network and the IFT transport module in primary cilia.
Highlights
The human Usher syndrome (USH) is a complex autosomal recessive disease, characterized by combined vision and hearing loss, accompanied by vestibular dysfunction (Davenport and Omenn, 1977; Wolfrum, 2011; Mathur and Yang, 2015)
We have previously demonstrated that USH1G protein SANS acts in transport modules of primary cilia of cultured cells and retinal photoreceptors (Papal et al, 2013; Bauss et al, 2014)
We transfected yeast cells with either SANS full length fused to the activation domain or full length intraflagellar transport (IFT) molecules fused to the DNA-binding domain of the GAL4 reporter gene
Summary
The human Usher syndrome (USH) is a complex autosomal recessive disease, characterized by combined vision and hearing loss, accompanied by vestibular dysfunction (Davenport and Omenn, 1977; Wolfrum, 2011; Mathur and Yang, 2015). Two other USH proteins, harmonin, whirlin and the genetic modifier of USH PDZD7 bind with their PDZ domains to the C-terminal PBM (Adato et al, 2005; Maerker et al, 2008; Schneider et al, 2009; Sorusch et al, 2017) interacts the MAGUK protein MAGI2 to an internal PBM in the SAM domain (Bauss et al, 2014) In summary this data qualifies SANS as one of the key organizers of the protein networks related to USH (Maerker et al, 2008; van Wijk et al, 2009; Yang et al, 2010; Zallocchi et al, 2010; Overlack et al, 2011; Bauss et al, 2014; Sorusch et al, 2017)
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