Quality control of the primary cilium proteome

项目总结 初级纤毛组织信号通路，如视觉、嗅觉和刺猬信号。这个 信号受体进入纤毛、内外纤毛的运动对于这些的正确调节至关重要。 途径，但我们对通过纤毛运输信号受体的基本机制的理解 仍然是支离破碎的。该实验室过去的工作确定并表征了BBSome，这是一种蛋白质复合体， 从纤毛中摆渡出信号受体。BBSome与人类健康和疾病的相关性就是证据 由于BBS某些功能障碍会导致Bardet-Biedl综合征(BBS)，这是一种遗传性疾病，其特征是 肥胖、视网膜变性、多指畸形和肾脏畸形。 这项建议的主要目标是确定BBSome如何选择要移除的信号受体 从cilia那里。这一资助阶段的重点将放在研究泛素在膜标记中的作用。 用于从纤毛中去除的蛋白质。初步数据表明，存在一种睫状结构，能够识别 激活的GPCRs，泛素化它们，并从纤毛中分类泛素化的GPCRs。我们将描述 在这些步骤中的每一步都作用于分子，使用信号依赖的GPCR退出的定量分析。这个 需要退出的蛋白质上附着和阅读泛素的分子齿轮和杠杆的分级排序 Cilia承诺揭示一种多步骤途径，使人想起ESCRT负责 降级排序。最后，退出纤毛的GPCR的命运将通过单分子成像进行追踪 确定内吞作用是否耦合到纤毛出口，或者GPCRs是否扩散到血浆中 出纤毛后的膜。 拟议的研究将为纤毛蛋白丰度的调节提供新的线索，并打开 对纤毛质量控制进行了机械性的调查。

PROJECT SUMMARY Primary cilia organize signaling pathways such as vision, olfaction and Hedgehog signaling. The movements of signaling receptors into, inside and out of cilium are critical for the correct regulation of these pathways, yet our understanding of the basic mechanisms governing signaling receptor trafficking through cilia remains fragmentary. Past work from the lab identified and characterized the BBSome, a protein complex that ferries signaling receptors out of cilia. The relevance of the BBSome to human health and disease is evidence by the fact that BBSome dysfunction causes Bardet-Biedl Syndrome (BBS), a hereditary disease characterized by obesity, retinal degeneration, polydactyly and kidney malformations. The major goal of this proposal is to determine how the BBSome selects signaling receptors for removal from cilia. The emphasis in this funding period will be on investigating the role of ubiquitin in tagging membrane proteins for removal from cilia. Preliminary data indicate the existence of a ciliary machinery that recognizes activated GPCRs, ubiquitinates them and sorts ubiquitinated GPCRs out of cilia. We will characterize the molecules acting at each of these steps using quantitative assays for signal-dependent GPCR exit. The hierarchical ordering of the molecular cogs and levers that affix and read ubiquitin on proteins that need to exit cilia promises to uncover a multi-step pathway reminiscent of the ESCRT machinery responsible for degradative sorting. Finally, the fate of GPCRs that exit cilia will be tracked by single-molecule imaging to determine whether endocytosis is coupled to ciliary exit or whether GPCRs instead diffuse into the plasma membrane after exiting cilia. The proposed studies will cast new light on how the ciliary abundance of proteins is regulated and open the door to a mechanistic investigation of ciliary quality control.