Membrane Protein Transport in Photoreceptors

描述（由申请人提供）：光感受器外节膜，初始光子捕获启动光转导的位点，在整个生命周期中以10天的间隔进行更新和完全替换。对细胞完整性的一个挑战是 在光感受器的寿命期间，替代蛋白从内节到外节的生物合成递送。复杂的运输途径需要多种组分，如酰基结合蛋白、Rab GTP酶和中心蛋白。该应用程序将识别：i）参与膜蛋白的囊泡转运的关键Rab GTP酶，和ii）参与纤毛转运的中心蛋白。目的1主要研究Rab GTPases在光感受器囊泡转运中的作用。Rab（Ras analog in brain）蛋白是Ras超基因家族的成员，主要参与膜蛋白运输。已经鉴定了超过70种不同的Rab多肽。目的1a关注Rab 8和Rab 11亚型，已知它们在非洲爪蟾和果蝇模型中对光感受器运输很重要。令人惊讶的是，小鼠Rab 8a/Rab 11 a双敲除不产生有缺陷的运输表型。因此，我们将专注于密切相关的亚型，以确定负责膜蛋白组织和视紫红质运输的关键Rab蛋白。目的1b研究Rab 28，最近显示与隐性人类视锥/视杆细胞营养不良相关。目的2将研究中心体蛋白的功能，称为“中心蛋白”，这是钙调素超家族的20 kD EF-手型钙结合蛋白（四个EF-手型基序）。中心蛋白首先在单细胞绿色藻类中被描述，在那里它们与鞭毛基部装置相关联。光感受器中心蛋白（亚型1-4）位于睫状体腔和基底体，在那里它们通过其T？亚单位。一种假设是，中心蛋白可能参与调节转导蛋白从外节到内节的易位，反之亦然。作为初步结果，我们发现，centrin 1是所需的核-基体连接在小鼠精子发生，和centrin 2调节交通的ACIII和通道亚基的嗅觉感觉神经元。这一目的旨在确定感光中心蛋白3和中心蛋白4的作用，与基体和/或连接纤毛轴丝密切相关。我们期望产生具有视网膜变性的综合征疾病的小鼠模型，例如，Joubert或Senior-Loken综合征。

DESCRIPTION (provided by applicant): Photoreceptor outer segment membranes, the site of initial photon capture initiating phototransduction, undergo renewal with total replacement occurring at ten day intervals throughout life. A challenge to cellular integrity concerns the post biosynthetic delivery of replacement proteins from the inner, to the outer, segment over the lifetime of the photoreceptor. Complex trafficking pathways require multiple components, such as acyl-binding proteins, Rab GTPases and centrins. This application will identify: i) key Rab GTPases involved in vesicular transport of membrane protein, and ii) centrins participating in ciliary transport. Aim 1 will focus on Rab GTPases in photoreceptor vesicular transport. Rab (Ras analog in brain) proteins are members of the Ras supergene family involved mainly in membrane protein trafficking. More than 70 distinct Rab polypeptides have been identified. Aim 1a focuses on Rab8 and Rab11 isoforms which are known to be important for photoreceptor trafficking in Xenopus and Drosophila models. Surprisingly, a mouse Rab8a/Rab11a double knockout does not produce a defective trafficking phenotype. Therefore, we will focus on closely-related isoforms to identify key Rab proteins responsible for membrane protein organization and rhodopsin trafficking. Aim 1b investigates Rab28, shown recently associated with recessive human cone/rod dystrophy. Aim 2 will examine the function of centrosomal proteins, called "centrins," which are 20kD EF- hand calcium binding proteins (four EF-hand motifs) of the calmodulin superfamily. Centrins were first described in unicellular green algae where they are associated with the flagellar basal apparatus. Photoreceptor centrins (isoforms 1-4) are located in the ciliary lumen and basal body where they associate with transducin via its T¿?-subunits. One hypothesis is that centrins may be involved in regulation of transducin translocation from the outer, to the inner, segment or vice-versa. As preliminary results, we found that centrin1 is required for the nucleus-basal body connection during mouse spermiogenesis, and centrin2 regulates trafficking of ACIII and channel subunits in olfactory sensory neurons. This aim seeks to identify the roles of photoreceptor centrin3 and centrin4 which associate strongly with the basal body and/or connecting cilium axoneme. We expect to generate mouse models of syndromic disease with retina degeneration, e.g., Joubert or Senior-Loken syndromes.