Membrane Fusion ATPases and the Golgi Apparatus

膜融合 ATP 酶和高尔基体

• 批准号: 7219534
• 负责人: Ayano Satoh
• 金额: $ 31.25万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-03-01 至 2008-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7219534
• 关键词: ATP phosphohydrolase; Alzheimer' s Disease; Animals; Antigens; Binding; Biochemical; Biological Assay; CDC48 protein; Cell-Free System; Cells; Coat Protein Complex I; Complex; Condition; Coupled; Cytosol; DiGeorge Syndrome; Docking; Event; Golgi Apparatus; Grant; Guanosine Triphosphate Phosphohydrolases; Homologous Gene; Huntington Disease; In Vitro; Incubated; Interphase; Link; Liver; Medial; Mediating; Medical; Membrane; Membrane Fusion; Membrane Protein Traffic; Mitosis; Mitotic; Molecular; Mono-S; Mutate; Nature; Neurodegenerative Disorders; Nuclear Envelope; Oculocerebrorenal Syndrome; Organelles; Pathway interactions; Patients; Phosphatidylinositol 4,5-Diphosphate; Phosphoric Monoester Hydrolases; Phosphorylation; Population; Process; Proteins; Range; Rattus; Regulation; Relative (related person); Role; Role playing therapy; SNAP receptor; Saccharomycetales; Sjogren' s Syndrome; Testing; Transport Vesicles; Tube; Ubiquitin; Ubiquitin Like Proteins; Vesicle; Work; Yeasts; developmental disease; human AKAP13 protein; in vivo; insight; interest; macrogolgin; p97 ATPase; prevent; protein aggregate; protein misfolding; rab GTP-Binding Proteins; syntaxin 5; target SNARE proteins; vesicular SNARE proteins

DESCRIPTION (provided by applicant): Using a cell-free system than mimics the mitotic cycle of the Golgi apparatus, we have been able to identify many of the proteins involved in the fragmentation and reassembly of this organelle. The last granting period focused on the reassembly of Golgi cisternae that is catalyzed by two ATPases, NSF and p97. NSF was shown to have an activity additional to its well-characterized role in unraveling SNARE complexes. It was found to add the ubiquitin-like protein, GATE-16, to the v-SNARE, GOS-28, a process that prevented the formation of non-productive cis SNARE complexes, and primed the SNARE for its interaction with the cognate t-SNARE, syntaxin-5. The p97 ATPase utilizes p47 as an adaptor molecule and this was shown to recognize mono-ubiquitinated proteins as part of the Golgi reassembly process. Other adaptor molecules were identified and characterized, notably the Ufd1p/Np14 complex, and this was shown to mediate p97 action in processes ranging from ER-associated degradation through to nuclear envelope reassembly. The present proposal continues the analysis of these two ATPase-driven pathways focusing on the following aims: 1: Studying the p115 tethering protein to work out precisely how it choreographs the capture and docking of cis-directed COPI transport vesicles. 2: Characterizing a new tethering complex that likely mediates the capture and docking of other COPI vesicles to medial/trans cistemae. The composition and function of these vesicles will also be characterized as will others identified and isolated through capture by different tethers. 3: Determining the role played by ubiquitin in the p97 pathway of Golgi reassembly. Ubiquitinated targets will be identified and characterized. 4: Testing the idea that p97 unravels t-t SNARE complexes just as NSF unravels v-t SNARE complexes. The fusion of ER membranes in budding yeast will be used as the assay. Though the main thrust of this application is the study of fundamental membrane traffic processes, there are medical implications. The tethering proteins were first identified as auto-antigens in patients with Sjogren's syndrome, and in one case as a partner for OCRL1, a PIP2 phosphatase, implicated in oculocerebrorenal syndrome. Ufd1p is mutated in DiGeorge syndrome, a congenital developmental disorder, and the role played by p97 in unraveling protein aggregates has implicated this ATPase in neurodegenerative diseases ranging from Alzheimer's to Huntington disease. Insights into the molecular mechanism of Golgi reassembly may therefore provide insight into these medical conditions.

描述（由申请人提供）：使用模拟高尔基体有丝分裂周期的无细胞系统，我们已经能够鉴定出参与该细胞器的断裂和重组的许多蛋白质。最后一个资助期的重点是由两种 ATP 酶 NSF 和 p97 催化的高尔基池的重组。 NSF 被证明除了其在解开 SNARE 复合体方面的明确作用之外，还具有其他活性。研究发现将泛素样蛋白 GATE-16 添加到 v-SNARE、GOS-28 中，这一过程可防止非生产性顺式 SNARE 复合物的形成，并为 SNARE 与同源 t 的相互作用做好准备。 -SNARE，syntaxin-5。 p97 ATP 酶利用 p47 作为接头分子，这表明它可以识别单泛素化蛋白，作为高尔基体重组过程的一部分。其他接头分子也被鉴定和表征，特别是 Ufd1p/Np14 复合物，并且这被证明可以介导 p97 在从 ER 相关降解到核膜重新组装等过程中的作用。目前的提案继续分析这两个 ATP 酶驱动的途径，重点关注以下目标： 1：研究 p115 束缚蛋白，以精确弄清楚它如何编排顺式定向 COPI 转运囊泡的捕获和对接。图 2：表征一种新的束缚复合物，该复合物可能介导其他 COPI 囊泡与内侧/反式池的捕获和对接。这些囊泡的组成和功能也将被表征，其他囊泡也将通过不同系链的捕获来识别和分离。图3：确定泛素在高尔基体重组的p97途径中所起的作用。泛素化目标将被识别和表征。图 4：测试 p97 解开 t-t SNARE 复合体的想法，就像 NSF 解开 v-t SNARE 复合体一样。出芽酵母中内质网膜的融合将被用作检测方法。尽管该应用的主要目的是研究基本的膜交通过程，但也具有医学意义。这些束缚蛋白首先被鉴定为干燥综合征患者的自身抗原，在一个病例中，它是 OCRL1（一种 PIP2 磷酸酶）的伴侣，与眼脑肾综合征有关。 Ufd1p 在 DiGeorge 综合征（一种先天性发育障碍）中发生突变，p97 在分解蛋白质聚集体中所发挥的作用表明这种 ATP 酶与从阿尔茨海默病到亨廷顿病等神经退行性疾病有关。因此，深入了解高尔基体重组的分子机制可能有助于了解这些医疗状况。

项目成果

mTrs130 is a component of a mammalian TRAPPII complex, a Rab1 GEF that binds to COPI-coated vesicles.

• DOI: 10.1091/mbc.e09-05-0387
• 发表时间: 2009-10
• 期刊: Molecular biology of the cell
• 影响因子: 3.3
• 作者: A. Yamasaki;Shekar Menon;Sidney Yu;Jemima Barrowman;T. Meerloo;V. Oorschot;J. Klumperman;Ayano Satoh;S. Ferro-Novick