Characterization of mammalian COG complex-interacting Golgi trafficking machinery

描述（由申请人提供）：在所有真核细胞中，细胞内膜运输对于一系列重要的细胞功能至关重要，包括蛋白质分泌、pot-translational修饰、细胞信号传导、细胞极化和细胞维持。膜运输的缺陷可加重或甚至加剧许多人类疾病，包括癌症、糖尿病、阿尔茨海默氏病、囊性纤维化、Hermansky-Pudlak综合征和先天性糖基化障碍。我们开创了保守寡聚高尔基体（COG）的功能分析，这是一种进化上保守的8种基因产物的复合体，每种基因产物对高尔基体中的膜运输至关重要。COG复合物与SNARE、SM蛋白、Rab、卷曲螺旋系链和COPI涂层相互作用，以组织转运中间体与其受体膜的特异性对接和融合。至少有一半，但可能是所有的COG亚基主要由螺旋束串联堆叠，形成长的灵活杆。这种结构特征也在三种相关的拴系复合物Dsl/ZW 10、GARP和外囊中发现，其作用是将囊泡拴系到ER、TGN和质膜，表明共同的潜在拴系机制。这项提议旨在更详细地探讨COG复合体协调高尔基复合体贩运的机制。1.我们将检验以下假设：膜结合COG亚基以几种组合排列，包括在空间上分离并可能参与不同膜运输步骤的叶A、叶B和叶A/B复合物。2.我们将测试的假设，COG 4和COG 8蛋白可以启动两个不同的拴系平台，吸引两个人口的高尔基体贩运中间形成。3.我们将测试的假设，COG亚基和它们的蛋白质伴侣之间的相互作用是必要的，以维持COG复合物的拴系逆行囊泡回收高尔基体酶和逆行货物的功能。4.我们将使用生物化学和显微镜方法来研究COG复合物和其他CATALYSTS复合物之间的串扰，特别关注COG复合物的叶B和GARP复合物之间的串扰，这两者都是trans-Golgi SNARE复合物组装所需的。

DESCRIPTION (provided by applicant): In all eukaryotic cells intracellular membrane trafficking is critical for a range of important cellular functions including protein secretion, pot-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.