Mechanisms of Membrane Fusion

膜融合机制

• 批准号: 9353911
• 负责人: WILLIAM Tobey WICKNER
• 金额: $ 10.94万
• 依托单位: DARTMOUTH COLLEGE
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-07-01 至 2021-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9353911
• 关键词: Bacteria; Biological Assay; Biological Models; Cells; Complex; Controlled Study; Coxiella burnetii; Ebola virus; Frankfurt-Marburg Syndrome Virus; Genetic; Genetic study; Hormones; Human; Humulus; In Vitro; Infection; Light; Lipid Bilayers; Lipids; Medical; Membrane; Membrane Fusion; Membrane Proteins; Molecular Chaperones; Organelles; Physiology; Proteins; Regulation; Role; SNAP receptor; Somatotropin; Stress; System; Testing; Vacuole; Variant; Yeasts; cell growth; membrane assembly; neurotransmission; pathogen; pathogenic bacteria; proteoliposomes; rab GTP-Binding Proteins; reconstitution; trafficking; Bacteria; Biological Assay; Biological Models; Cells; Complex; Controlled Study; Coxiella burnetii; Ebola virus; Frankfurt-Marburg Syndrome Virus; Genetic; Genetic study; Hormones; Human; Humulus; In Vitro; Infection; Light; Lipid Bilayers; Lipids; Medical; Membrane; Membrane Fusion; Membrane Proteins; Molecular Chaperones; Organelles; Physiology; Proteins; Regulation; Role; SNAP receptor; Somatotropin; Stress; System; Testing; Vacuole; Variant; Yeasts; cell growth; membrane assembly; neurotransmission; pathogen; pathogenic bacteria; proteoliposomes; rab GTP-Binding Proteins; reconstitution; trafficking

Membrane fusion underlies hormone secretion, neurotransmission, and all exocytic and endocytic traffic. Its mechanism is conserved from yeast to humans. A current paradigm suggests that membrane proteins termed SNAREs inexorably drive fusion when anchored in apposed membranes as a trans-SNARE complex. While SNAREs are required, genetic studies from yeast to humans show that Rab GTPases, their effectors, SM proteins, and SNARE chaperones are also essential. Our studies of yeast vacuole fusion are providing a new paradigm, illuminating the integrated mechanisms of these other essential proteins and showing that their actions extend beyond regulation of trans-SNARE complex levels. Vacuole fusion studies have progressed from initial genetics through our extensive study of in vitro organelle fusion to proteoliposome fusion, which we have reconstituted with all purified and defined proteins and lipids: SNAREs, SNARE disassembly chaperones Sec18p/Sec17p, the Rab GTPase Ypt7p, a hexameric Rab effector complex HOPS, and lipids which include acidic and bilayer-averse headgroups and specific fatty acyl chains. With a rigorous fusion assay of protected lumenal content mixing, our studies show that fusion is driven by several cooperating factors: bilayer stress from trans-SNARE complex assembly, membrane destabilization by nonbilayer lipids, and bilayer bending through the action of a multisubunit tether. Fusion is blocked by the omission of SNAREs, of nonbilayer-prone lipids, or of tethering factors, even though SNAREs still pair in trans in the latter 2 conditions. Our chemically-defined reconstitution of fusion allows independent variation of SNARE concentration, nonbilayer lipid concentration, and the HOPS and Rab tethering proteins, all while assaying both physical associations and fusion function. Testing and extending this model system is changing our view of fusion. In light of the fundamental role of fusion throughout human physiology, and the central role of human HOPS for cellular infection by Marburg and Ebola viruses and by bacteria such as the pathogen Coxiella burnetii, these studies will be of medical significance as well.

膜融合是激素分泌，神经传递以及所有外囊肿和 内吞交通。它的机制是从酵母到人类的。当前的范式 表明，锚定在 将膜作为跨弹药络合物。虽然需要咆哮，但遗传研究 从酵母到人类表明，Rab GTPases，它们的效应子，SM蛋白和军鼓 伴侣也是必不可少的。我们对酵母液泡融合的研究正在提供新的 范式，照亮这些其他必要蛋白的综合机制并显示 他们的行为超出了跨弹复合物水平的调节。液泡融合 研究已经从初始遗传学到我们对体外细胞器的广泛研究发展 融合到蛋白质体融合，我们已经用所有纯化和定义的重组 蛋白质和脂质：圈套，圈套拆卸伴侣SEC18P/SEC17P，RAB GTPase YPT7P，一种六聚体Rab效应复合物啤酒花和脂质，包括酸性和脂质 避开双层的头组和特定的脂肪酰基链。用严格的融合测定法 受保护的腔内含量混合，我们的研究表明，融合是由多个合作驱动的 因素：反式弹头复合体组装中的双层应力，膜不稳定 非叶脂脂质和双层弯曲的弯曲。融合是 由于省略贪食片，非叶叶式脂质或束缚因子的遗漏，甚至被阻止 尽管在后2条条件下，小偷在反式中仍然配对。我们的化学定义的重构 融合允许单独浓度，非质体脂质浓度独立变化， 以及啤酒花和rab绑扎蛋白，同时均测定了物理关联和 融合功能。测试和扩展此模型系统正在改变我们对融合的看法。在光线下 融合整个人类生理学的基本作用以及人类的核心作用 马堡和埃博拉病毒和细菌（例如病原体）感染细胞感染的啤酒花 Coxiella burnetii，这些研究也将具有医学意义。