Protein targeting in vertebrate photoreceptors

描述(申请人提供)：这项提案的总体目标是揭示脊椎动物光感受器利用的蛋白质运输机制，以建立和维持其独特的亚细胞区隔。光感受器的质膜由两个蛋白质组成和功能不同的主要隔室组成--外段质膜和内段质膜。这种分离对于光感受器对光产生可靠的电响应是必不可少的。先前的研究表明，某些外节段膜蛋白含有特定的靶向信号，以确保它们被正确分类。然而，对于蛋白质靶向其他感光细胞周围的质膜的分子和细胞机制还知之甚少。在本申请中提出的实验中，我们将研究三种不同蛋白质靶向内段质膜的机制：超极化激活的环核苷酸门控通道(HCN1)、Na/K-ATPase和突触素。我们认为，控制HCN1和Na/K-ATPase靶向的机制涉及Ankyrin-B，它是膜细胞骨架中的一种必不可少的接头蛋白。膜细胞骨架排列在所有细胞的质膜上；它提供机械稳定性，并调节膜亚结构域的蛋白质组成。在光感受器中，锚蛋白-B装饰着外节外的整个质膜，这使得它成为蛋白质靶向该区域的主要组织者的一个有吸引力的候选者。目的1验证一种假设，即甲型流感病毒与锚蛋白B结合，这种相互作用是靶向甲型流感病毒所必需的。目的2验证这样一种假说：在光感受器中，已知的锚蛋白结合蛋白Na/K-ATPase与锚蛋白B而不是其他锚蛋白的选择性相互作用是光感受器中表达的Na/K-ATPase的一个特征，并且这种选择性相互作用是靶向Na/K-ATPase所必需的。值得注意的是，与Ankyrin-B的相互作用非常稳定，而一些蛋白质在整合到细胞器(如突触小泡)之前，会短暂地定位于质膜。在目标3中，我们将重点研究一种这样的突触小泡驻留蛋白--突触素。我们在突触素中发现了一个新的靶向序列，并将通过鉴定与该序列结合的蛋白(S)来继续这一发现，并调节突触素的靶向。总之，这些实验将提供对两种靶向途径的机械洞察，这些靶向途径用于将膜蛋白特异性地驱动到内段质膜。 与公共卫生相关：这项工作与理解视网膜失明退行性疾病的分子基础有关，这些疾病的特征通常是光感受器蛋白运输或维持细胞区隔的障碍。阐明控制这些过程的机制对于开发治疗干预措施至关重要。

DESCRIPTION (provided by applicant): The overall goal of this proposal is to uncover the mechanisms of protein trafficking utilized by the vertebrate photoreceptor to establish and maintain its unique subcellular compartmentalization. The plasma membrane of the photoreceptor consists of two major compartments with distinct protein composition and function - the outer segment plasma membrane and the inner segment plasma membrane. This separation is essential for photoreceptors to produce a reliable electrical response to light. Previous studies revealed that certain outer segment membrane proteins contain specific targeting signals to ensure they are properly sorted. However, nothing is known about the molecular and cellular mechanisms responsible for protein targeting to the plasma membrane surrounding the rest of the photoreceptor cell. In the experiments proposed in this application we will investigate the mechanisms controlling the targeting of three different proteins to the inner segment plasma membrane: the hyperpolarization-activated cyclic nucleotide-gated channel (HCN1), Na/K-ATPase and synaptophysin. We propose that the mechanism controlling the targeting of both HCN1 and Na/K-ATPase involves ankyrin-B, an essential adaptor protein within the membrane cytoskeleton. The membrane cytoskeleton lines the plasma membrane of all cells; it provides mechanical stability and regulates the protein composition of membrane subdomains. In photoreceptors, ankyrin-B decorates the entire plasma membrane outside the outer segment, which makes it an attractive candidate for serving as a master organizer of protein targeting into this domain. Aim 1 tests the hypothesis that HCN1 binds to ankyrin-B and that this interaction is required for the targeting of HCN1. Aim 2 tests the hypothesis that in photoreceptors the selective interaction of Na/K-ATPase, a known ankyrin binding protein, with ankyrin-B but not other ankyrins is a feature of the specific alpha subunit of Na/K-ATPase expressed in photoreceptors and that this selective interaction is necessary for the targeting of Na/K-ATPase. Notably, interactions with ankyrin-B are very stable, whereas some proteins are targeted to the plasma membrane transiently prior to their incorporation into organelles such as synaptic vesicles. In Aim 3 we will focus on one such synaptic vesicle resident protein, synaptophysin. We have found a novel targeting sequence within synaptophysin and will pursue this finding by identifying the protein(s) that bind to this sequence and regulate the targeting of synaptophysin. Together, these experiments will provide mechanistic insight into two targeting pathways used to drive membrane proteins specifically to the inner segment plasma membrane. PUBLIC HEALTH RELEVANCE: This work is relevant to understanding the molecular bases of blinding degenerative diseases of the retina, often characterized by disorders in photoreceptor protein trafficking or the maintenance of cellular compartmentalization. Elucidating the mechanisms controlling these processes is essential for developing therapeutic interventions.