Characterization of mammalian COG complex-interacting Golgi trafficking machinery

描述(申请人提供)：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、POT翻译修饰、细胞信号、细胞极化和细胞维持。膜转运的缺陷可加重或加重许多人类疾病，包括癌症、糖尿病、阿尔茨海默氏症、囊性纤维化、Hermansky-Pudlak综合征和先天性糖基化紊乱。我们率先对保守的寡聚体高尔基体(COG)进行了功能分析，COG是一个进化上保守的由八个基因产物组成的复合体，每个基因产物都对高尔基体的膜运输至关重要。COG复合体与SNARS、SM蛋白、RABS、卷曲圈套和COPI涂层相互作用，组织转运中间体与受体膜的特异性对接和融合。至少有一半，但很可能是所有COG亚基主要由串联堆叠的螺旋束组成，形成长的柔性棒。这一结构特征也在三个相关的拴系复合体DSL/ZW10、GARP和外囊中发现，它们将小泡拴在内质网、TGN和质膜上，这表明了一种共同的潜在拴系机制。这项提案旨在更详细地探讨COG建筑群策划高尔基建筑群贩运的机制。1.我们将检验这一假设，即膜结合的COG亚基排列在几个组合中，包括叶A、叶B和叶A/B复合体，这些复合体在空间上是分开的，可能参与不同的膜转运步骤。2.我们将检验这一假设，即COG4和COG8蛋白可以启动两个不同的系留平台的形成，从而吸引两个高尔基体运输中间体。3.我们将检验这一假设，即COG亚基与其蛋白质伙伴之间的相互作用对于维持COG复合体的功能是必要的，该复合体连接逆行小泡、回收高尔基酶和逆行货物。4.我们将使用生化和显微镜方法研究COG复合体和其他CATCHR复合体之间的串扰，特别是COG复合体的叶B和GARP复合体之间的串扰，这两个叶都是组装跨高尔基体SNARE复合体所必需的。

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.