RETRIEVAL PATHWAY IN GOLGI STACK TARGETING

描述（由申请人提供）：蛋白质靶向机制建立并维持细胞内区室。靶向高尔基体是一个很好的案例研究，因为它发生，尽管令人印象深刻的膜通量通过细胞器，它涉及成熟的亚室。高尔基体池的成熟任务快速和选择性检索高尔基居民从后来的车厢，大概是通过检索复合物-细胞质外套选择性丰富的高尔基居民在囊泡芽网站通过直接或受体介导的相互作用与居民。那么，什么是提取复合物，它们在哪里形成？我们正在追求这些问题，使用两个高尔基体蛋白作为标记，GPP 130和GP 73，不像任何其他已知的蛋白质驻留在早期高尔基体，但重新分配到内体破坏内腔pH值和经历内体到高尔基体回收pH值恢复后。我们的目标是确定信号，信号受体，和细胞质外套在高尔基体和远端的内体中发挥作用，以介导这些蛋白质的检索。我们的进展表明，信号定位在卷曲螺旋腔干区域，并包含可分离的元件，赋予高尔基体和内体靶向。高尔基体决定簇介导从高尔基体或核内体的检索，并且核内体决定簇将一部分蛋白质池从高尔基体转移到核内体，在核内体中，其经历pH敏感性分选进入检索载体，所述检索载体在返回高尔基体的途中绕过晚期核内体。我们将进行进一步完善的信号和分析其在循环中的作用，我们的主要工作将是识别功能上与这些信号相互作用的蛋白质，并使用完整的，半完整的，和无细胞的测定，以确定介导检索排序的GPP 130和GP 73在高尔基体和内体的细胞质外壳组件。对于在ER/高尔基体和TGN/内体系统中循环的单个高尔基体蛋白，对局部和长距离循环的要求的比较可能会产生对内膜靶向中的共享和不同特征的重要见解。

DESCRIPTION (provided by applicant): Protein targeting mechanisms establish and maintain intracellular compartments. Targeting to the Golgi is an excellent case study as it occurs despite impressive membrane flux through the organelle and it involves subcompartments that mature. Maturation of Golgi cisternae mandates rapid and selective retrieval of Golgi residents from later compartments, presumably by retrieval complexes- cytoplasmic coats selectively enriching for Golgi residents at vesicle bud sites through direct or receptor-mediated interactions with the residents. Thus, what are the retrieval complexes and where do they form? We are pursuing these questions using two Golgi proteins as markers, GPP130 and GP73 that unlike any other known proteins reside in the early Golgi yet redistribute to endosomes upon disruption of lumenal pH and undergo endosome-to-Golgi retrieval upon pH restoration. Our goal is to identify the signals, the signal receptors, and the cytoplasmic coats acting locally in the Golgi and distally in endosomes to mediate retrieval of these proteins. Our progress indicates that the signals are positioned in a coiled-coil lumenal stem region and contain separable elements that confer Golgi and endosomal targeting. The Golgi determinants mediate retrieval from either the Golgi or endosomes and the endosomal determinant diverts a fraction of the protein pool out of the Golgi to endosomes where it undergoes pH-sensitive sorting into retrieval carriers that bypass late endosomes en route back to the Golgi. We will carry out further refinement of the signals and analysis of their role in cycling, and our major effort will be on identification of proteins that functionally interact with these signals and the use of intact, semi-intact, and cell-free assays to identify the cytoplasmic coat components that mediate retrieval sorting of GPP130 and GP73 at the Golgi and in endosomes. For single Golgi proteins that cycle in both the ER/Golgi and TGN/endosome systems, a comparison of requirements for local and long distance cycling may yield significant insights into shared and distinct features in endomembrane targeting.