Mechanisms of Membrane Fusion

膜融合机制

• 批准号: 9069290
• 负责人: WILLIAM Tobey WICKNER
• 金额: $ 74.7万
• 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/9069290
• 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; 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; public health relevance; rab GTP-Binding Proteins; reconstitution; trafficking

 DESCRIPTION (provided by applicant): 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.

 描述(申请人提供)：膜融合是激素分泌、神经传递以及所有胞外和胞内运输的基础。它的机制从酵母到人类都是保守的。目前的一个范例表明，当被称为SNARS的膜蛋白作为反式SNARE复合体锚定在相对的膜上时，它将无情地推动融合。虽然圈套是必需的，但从酵母到人类的遗传学研究表明，Rab GTP酶、它们的效应器、SM蛋白和圈套伴侣也是必不可少的。我们对酵母液泡融合的研究提供了一个新的范式，阐明了这些其他基本蛋白的整合机制，并表明它们的作用超出了对反式SNARE复合体水平的调节。液泡融合的研究已经从最初的遗传学通过我们对体外细胞器融合的广泛研究发展到蛋白质脂质体融合，我们已经用所有纯化和定义的蛋白质和脂重组：SNARs，SNAR分解伴侣Sec18p/Sec17p，Rab GTPase Ypt7p，一个六聚体Rab效应复合Hop，以及包括酸性和双层厌恶头基和特定脂肪酰链的脂类。通过对受保护的管腔内容物混合进行严格的融合分析，我们的研究表明融合是由几个协同因素驱动的：跨SNARE复合体组装造成的双层应力，非双层脂类造成的膜不稳定，以及通过多亚单位系绳作用的双层弯曲。尽管在后两种情况下，陷阱仍然以反式方式配对，但融合会被陷阱、非双层倾向脂类或系留因子的省略所阻止。 我们化学定义的融合重组允许SNARE浓度、非双层脂质浓度以及Hop和Rab拴系蛋白的独立变化，所有这些都是在分析物理关联和融合功能的同时进行的。测试和扩展这一模型系统正在改变我们对融合的看法。鉴于融合在整个人类生理学中的基础作用，以及人类啤酒花在马尔堡和埃博拉病毒以及伯氏柯克斯体等细菌的细胞感染中的核心作用，这些研究也将具有医学意义。