Membrane Fusion ATPases and the Golgi Apparatus

膜融合 ATP 酶和高尔基体

• 批准号: 6777897
• 负责人: GRAHAM B WARREN
• 金额: $ 31.94万
• 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 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6777897
• 关键词: Golgi apparatus; adenosinetriphosphatase; cell cycle; cell free system; enzyme mechanism; laboratory rabbit; laboratory rat; membrane fusion; membrane proteins; phosphorylation; protein structure function; syntaxin

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）：使用无细胞的系统，而不是模仿高尔基体的有丝分裂周期，我们能够识别出与该细胞器的碎片化和重新组装有关的许多蛋白质。最后一个授予期的重点是由两个ATPases NSF和P97催化的高尔基水库的重新组装。 NSF被证明具有其在揭开圈圈复合物中良好表征作用的活动。发现它可以将泛素样蛋白Gate-16添加到V-SNARE，GOS-28中，该过程阻止了非生产性的顺式SNARE SNARE COMPLISES的形成，并为其与Cognate T-SNARE的相互作用启动了SNARE，SNANARE-SUNDTAXIN-5。 p97 ATPase利用P47作为适配器分子，这被证明可以识别单次泛素化的蛋白作为高尔基体重新组装过程的一部分。鉴定了其他衔接子分子并表征了尤其是UFD1P/NP14复合物，这证明可以在从与ER相关的降解到重新组装的过程中介导P97作用。本提案继续对这两种ATPase驱动的途径进行分析，重点是以下目的：1：研究P115绑定蛋白，以精确地阐明它如何编排CIS指导的COPI转运囊泡的捕获和对接。 2：表征一种新的束缚复合物，该复合物可能介导其他Copi囊泡的捕获和对接到中间/跨cistemae。这些囊泡的组成和功能也将被表征，而其他囊泡通过不同的系群捕获而鉴定和隔离。 3：确定泛素在高尔基体重新组装的P97途径中所起的作用。将确定和表征泛素化的靶标。 4：测试p97脱离T-T SNARE复合物的想法就像NSF脱离V-T SNARE复合物一样。萌芽酵母中ER膜的融合将用作测定。尽管该应用程序的主要目的是研究基本膜交通过程，但仍有医疗意义。在Sjogren综合征患者中，首先将绑扎蛋白鉴定为自动抗原，并且在一种情况下，在一种伴侣的情况下，伴有PIP2磷酸酶，与眼球脑脑综合征有关。 UFD1P在一种先天性发育障碍的Digeorge综合征中突变，P97在解散蛋白质聚集体中所扮演的作用已将这种ATPase牵涉到从阿尔茨海默氏症到亨廷顿病的神经退行性疾病中。因此，对高尔基体重新组装的分子机制的见解可能会提供对这些医疗状况的见解。