Mechanisms of Membrane Fusion

 描述（由申请人提供）：膜融合是激素分泌、神经传递和所有胞吐和胞吞运输的基础。其机制是保守的从酵母到人类。目前的范例表明，膜蛋白称为SNARE无情地驱动融合时，锚定在并列膜作为反式SNARE复合物。虽然SNARE是必需的，但从酵母到人类的遗传研究表明，Rab GTP酶、其效应子、SM蛋白和SNARE伴侣也是必需的。我们对酵母液泡融合的研究提供了一个新的范例，阐明了这些其他必需蛋白质的整合机制，并表明它们的作用超出了trans-SNARE复合物水平的调节。通过我们对体外细胞器融合的广泛研究，蛋白脂质体融合研究已经从最初的遗传学进展到蛋白脂质体融合，我们已经用所有纯化和确定的蛋白质和脂质重建了蛋白脂质体融合：SNARE，SNARE拆卸分子伴侣Sec 18 p/Sec 17 p，Rab GTp 7 p，六聚体Rab效应复合物HOPS，以及包括酸性和双层厌恶头基和特定脂肪酰基链的脂质。与严格的融合试验的保护内腔内容物混合，我们的研究表明，融合是由几个合作因素驱动：双层应力trans-SNARE复合物组装，膜不稳定的非双层脂质，和双层弯曲通过多亚基系链的作用。融合被SNARE、非双层倾向脂质或系留因子的缺失所阻断，即使SNARE在后两种条件下仍然以反式配对。 我们的化学定义的融合重建允许SNARE浓度，非双层脂质浓度，HOPS和Rab拴系蛋白的独立变化，同时测定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。鉴于融合在整个人类生理学中的基本作用，以及人类HOPS在马尔堡和埃博拉病毒以及病原体贝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。

 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.