The Usher syndrome 1G protein SANS participates in the transport of ciliary cargo in photoreceptor cells

摘要

Human Usher syndrome (USH) is the most common formof combined deaf-blindness, characterized by profoundcongenital deafness, constant vestibular dysfunction andpre-pubertal onset of retinitis pigmentosa. The USH1Gprotein SANS (scaffold protein containing ankyrin repeatsand SAM domain) is associated with microtubules andmediates a transport related periciliary protein network inphotoreceptor cells. Here we aim to enlighten the involvementof SANS in ciliary transport of photoreceptor cellsby identifying proteins associated with SANS in transportcomplexes. In Y2H screen of retinal cDNA library weidentified the direct binding of SANS to dynactin-1(p150Glued), a subunit of the dynactin complex and cargolinker to the cytoplasmic dynein motor. This interactionwas validated in complementary interaction assays in vitroand in cell culture. In addition, we demonstrated the integrationof SANS into the cytoplasmic dynein transportcomplex by GST-pull down of SANS with cytoplasmicdynein intermediate chain (cDIC74). Correlative immunofluorescenceand -electron microscopy analyses of photoreceptorinner segments revealed co-localization of essentialdynein-dynactin components and SANS at microtubules,the known transport routes for opsin bearing vesicles.Co-immunoprecipitation revealed that SANS is part of anopsin containing protein complex together with transportregulating rab-GTPases. Finally, the analysis of SANS deficientmice revealed altered distribution of opsin in theinner segment, indicating a transport delay. In conclusionwe identified SANS as a component of the cytoplasmicdynein motor complex which is crucial for opsin transportto photoreceptor connecting cilia. Defects in these transportmechanisms lead to retinal degeneration as characteristicfor USH1G patients.