Characterization of mammalian COG complex-interacting Golgi trafficking machinery

摘要 在所有真核细胞中，细胞内膜运输对一系列重要的细胞功能至关重要 包括蛋白质分泌、翻译后修饰、细胞信号、细胞极化和细胞 维修。膜贩运的缺陷可能会突显，甚至加剧一些人 疾病包括癌症、糖尿病、阿尔茨海默氏症、囊性纤维化、赫曼斯基-普德拉克综合征和 先天性糖基化障碍。我们率先对保守的寡聚体进行了功能分析 高尔基体(COG)，由八种基因产物组成的进化保守的复合体，每一种都对 高尔基体中的膜运输。COG复合体与SNARS、SM蛋白、RABS、 盘绕绳索和COPI涂层，用于组织运输中间体与其 受体膜。至少一半，但很可能所有COG亚基都主要由堆积在一起的螺旋束组成 系列，以形成长的柔性杆。这一建筑特征也出现在三个相关的系链建筑群中 DSL/ZW10、GARP和外囊将囊泡拴在内质网、TGN和质膜上，提示 共同的底层系留机制。这项提案旨在通过以下方式更详细地探讨这一机制 COG建筑群策划了高尔基建筑群的走私活动。 1.我们将检验膜结合COG亚基以几种组合排列的假设 包括在空间上分离的叶A、叶B和叶A/B复合体 不同的膜转运步骤。 2.我们将检验COG4和COG8蛋白可以启动两个不同的 吸引两个高尔基人贩运中间人的系链平台。 3.我们将检验COG亚单位与其蛋白质伙伴之间的相互作用是 维持COG复合体功能是维持逆行囊泡循环高尔基酶所必需的 和逆行货物。 4.我们将使用生化和显微镜方法来研究COG复合体之间的串扰 和其他CATCHR复合体，特别关注COG复合体的叶B之间的串扰 和GARP复合体，这两个都是组装反高尔基圈套复合体所必需的。

ABSTRACT In all eukaryotic cells intracellular membrane trafficking is critical for a range of important cellular functions including protein secretion, post-translational modifications, cell signaling, cell polarization, and cell maintenance. Defects in membrane trafficking can underline, or even exacerbate, a number of human diseases including cancer, diabetes mellitus, Alzheimer’s, cystic fibrosis, Hermansky-Pudlak syndrome and Congenital Disorders of Glycosylation. We have pioneered the functional analysis of the Conserved Oligomeric Golgi (COG), an evolutionarily conserved complex of eight gene products, each of which is critical for the membrane trafficking in the Golgi apparatus. The COG complex interacts with SNAREs, SM proteins, Rabs, coiled-coil tethers and COPI coat to organize specific docking and fusion of transport intermediates with their acceptor membrane. At least half, but probably all COG subunits consist largely of helical bundles stacked in series to form long flexible rods. This architectural feature is also found in three related tethering complexes Dsl/ZW10, GARP and exocyst that act to tether vesicles to ER, TGN and plasma membrane, suggesting a common underlying tethering mechanism. This proposal seeks to probe in greater detail the mechanism by which the COG complex orchestrates trafficking in the Golgi complex. 1. We will test the hypothesis that membrane bound COG subunits are arranged in several combinations including Lobe A, Lobe B and Lobe A/B complexes that are spatially separated and likely to participate in different membrane trafficking steps. 2. We will test the hypothesis that the COG4 and COG8 proteins can initiate the formation of two different tethering platforms which attract two populations of Golgi trafficking intermediates. 3. We will test the hypothesis that the interactions between COG subunits and their protein partners are necessary to maintain the COG complexes function of tethering retrograde vesicles recycling Golgi enzymes and retrograde cargo. 4. We will use biochemical and microscopy approaches to investigate cross-talk between the COG complex and other CATCHR complexes specifically focusing on the cross-talk between Lobe B of the COG complex and the GARP complex, both of which are required for the assembly of trans-Golgi SNARE complex.