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

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、神经传递以及所有胞吐和 内吞交通。其机制从酵母到人类都是保守的。当前的范例 表明称为 SNARE 的膜蛋白在锚定于 并置的膜作为反式圈套复合物。虽然需要 SNARE，但基因研究 从酵母到人类的研究表明 Rab GTP 酶、其效应子、SM 蛋白和 SNARE 陪伴者也必不可少。我们对酵母液泡融合的研究提供了新的方法 范例，阐明了这些其他必需蛋白质的整合机制并显示 他们的行为超出了跨网罗复杂水平的监管范围。液泡融合 研究已经从最初的遗传学发展到我们对体外细胞器的广泛研究 与蛋白脂质体融合体的融合，我们已用所有纯化和定义的物质重新构建了融合蛋白 蛋白质和脂质：SNARE、SNARE 拆卸分子伴侣 Sec18p/Sec17p、Rab GTPase Ypt7p、六聚 Rab 效应复合物 HOPS 和脂质（包括酸性和脂质） 双层厌恶的头基和特定的脂肪酰基链。通过严格的融合测定 受保护的管腔内容物混合，我们的研究表明融合是由几个合作驱动的 因素：反式 SNARE 复合体组装产生的双层应力、膜不稳定 非双层脂质，以及通过多亚基系链的作用而弯曲的双层。融合是 通过省略 SNARE、非双层倾向的脂质或束缚因子，甚至 尽管在后两种情况下 SNARE 仍然以反式配对。我们的化学成分重构 融合允许 SNARE 浓度、非双层脂质浓度的独立变化， 以及 HOPS 和 Rab 束缚蛋白，同时分析物理关联和 融合功能。测试和扩展这个模型系统正在改变我们对融合的看法。在光下 融合在整个人类生理学中的基本作用以及人类的核心作用 HOPS 用于马尔堡病毒和埃博拉病毒以及病原体等细菌的细胞感染 伯内特立克克斯体，这些研究也将具有医学意义。