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区室。靶向高尔基体是一个很好的案例研究，因为尽管通过细胞器的膜通量令人印象深刻，但它仍然发生，并且涉及成熟的亚室。高尔基池的成熟要求高尔基居民从以后的隔室中快速和选择性地回收，可能是通过回收复合体-通过直接或受体介导的相互作用，在囊泡芽位选择性地富集高尔基居民的细胞质外壳。因此，什么是检索复合体，它们在哪里形成？我们正在使用两种高尔基蛋白作为标记物，GPP130和GP73，这两种蛋白与其他已知的高尔基蛋白不同，它们存在于早期高尔基体中，但在腔内pH破坏时重新分布到核内体中，并在pH恢复时进行核内体到高尔基体的恢复。我们的目标是识别信号，信号受体，以及在高尔基体局部和远端在核内体中作用的细胞质外壳，以介导这些蛋白质的检索。我们的研究进展表明，这些信号位于线圈状管茎区域，并包含赋予高尔基体和内体靶向的可分离元件。高尔基体决定因子介导从高尔基体或核内体的回收，核内体决定因子将一部分蛋白质池从高尔基体转移到核内体，在那里它经历ph敏感的分选，进入回收载体，绕过后期核内体返回高尔基体。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、半完整和无细胞测定来鉴定介导高尔基体和内体中GPP130和GP73检索分选的细胞质外壳成分。对于在内质网/高尔基体和TGN/内体系统中循环的单个高尔基蛋白，比较局部和长距离循环的需求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.