RETRIEVAL PATHWAY IN GOLGI STACK TARGETING

描述(由申请人提供)：蛋白质靶向机制建立和维持细胞内隔间。以高尔基体为目标是一个很好的案例研究，因为它发生时，尽管细胞器中的膜通量令人印象深刻，而且它涉及成熟的亚室。高尔基池的成熟要求从较晚的隔室快速和选择性地取回高尔基体居民，推测是通过取回复合体--通过直接或受体介导的与居民的相互作用，选择性地为位于囊芽部位的高尔基体居民提供丰富的细胞膜。那么，检索复合体是什么，它们又是在哪里形成的呢？我们正在使用两个高尔基体蛋白作为标记来探索这些问题，GPP130和GP73不同于任何其他已知的蛋白，它们存在于早期高尔基体中，但在管腔pH中断时重新分配到内体，并在pH恢复后进行内体到高尔基体的恢复。我们的目标是确定信号、信号受体和细胞质外壳在高尔基体局部和远端内体中作用，以介导这些蛋白质的恢复。我们的进展表明，信号位于盘绕线圈的管腔干区，并包含可分离的元件，使高尔基体和内体靶向。高尔基体决定簇调节高尔基体或内体的检索，而内体决定簇将一部分蛋白质池从高尔基体转移到内体，在内体中经历对pH敏感的分选，进入检索载体，在返回高尔基体的途中绕过晚期的内体。我们将进一步细化信号并分析它们在循环中的作用，我们的主要工作将是鉴定与这些信号功能相互作用的蛋白质，并使用完整、半完整和无细胞的分析方法来鉴定介导GPP130和GP73在高尔基体和内体中检索分选的细胞质外壳成分。对于同时在内质网/高尔基体和TGN/内体系统中循环的单个高尔基体蛋白，对局部和远距离循环的需求进行比较可能会对膜靶向的共同和独特特征产生重要的见解。

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.