Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis.
Membrane fusion catalyzed by a Rab, SNAREs, and SNARE chaperones is accompanied by enhanced permeability to small molecules and by lysis.
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DOI：
10.1091/mbc.e11-08-0680
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发表时间：
2011-12
影响因子：
3.3
通讯作者：
Zick M
中科院分区：
文献类型：
--
作者：
Zucchi PC;Zick M
生物膜的融合需要脂双层的剧烈重排。开发了区分融合与裂解的新测定以研究酵母液泡融合机器的体外重建。这些测定揭示，真正的融合伴随着对小分子的强烈增强的膜渗透性和裂解。密封的生物膜的融合在混合其膜双层的同时连接其封闭的水性隔室。重构的融合反应通常通过脂质混合来测定，其可以由真融合或由裂解及其伴随的膜再退火产生。融合也经常使用低分子量的探针通过混合内腔水室来测定。利用几种探针（生物素、甲基伞形酮基-N-乙酰基-α-d-神经氨酸和连二亚硫酸盐），我们发现酵母液泡SNARE（SNAP [可溶性NSF附着蛋白]受体）增加了膜对小分子的渗透性，并且这种渗透性被同型融合和液泡蛋白分选复合物（HOPS）和Sec 17 p/Sec 18 p（液泡系留和SNARE伴侣蛋白）增强。我们现在报告一种新的检测方法的发展，允许平行评估脂质混合，完整的内腔室的混合，发生的任何裂解，和小分子的膜渗透。将该测定应用于由空泡脂质、4个空泡SNARE、SNARE拆解分子伴侣Sec 17 p和Sec 18 p、Rab Ypt 7 p和Rab效应子/SM蛋白复合物HOPS组成的全纯化重建系统，我们表明真正的融合伴随着对小分子的膜渗透性的强烈增强和可测量的裂解率。
The fusion of biological membranes entails a drastic rearrangement of the lipid bilayer. New assays that distinguish fusion from lysis were developed to study an in vitro reconstitution of the yeast vacuolar fusion machinery. These assays revealed that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and by lysis. The fusion of sealed biological membranes joins their enclosed aqueous compartments while mixing their membrane bilayers. Reconstituted fusion reactions are commonly assayed by lipid mixing, which can result from either true fusion or from lysis and its attendant reannealing of membranes. Fusion is also frequently assayed by the mixing of lumenal aqueous compartments, using probes of low molecular weight. With several probes (biotin, methylumbelliferyl-N-acetyl-α-d-neuraminic acid, and dithionite), we find that yeast vacuolar SNAREs (SNAP [Soluble NSF attachment protein] Receptors) increase the permeability of membranes to small molecules and that this permeabilization is enhanced by homotypic fusion and vacuole protein sorting complex (HOPS) and Sec17p/Sec18p, the vacuolar tethering and SNARE chaperone proteins. We now report the development of a novel assay that allows the parallel assessment of lipid mixing, the mixing of intact lumenal compartments, any lysis that occurs, and the membrane permeation of small molecules. Applying this assay to an all-purified reconstituted system consisting of vacuolar lipids, the four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, the Rab Ypt7p, and the Rab effector/SM protein complex HOPS, we show that true fusion is accompanied by strongly enhanced membrane permeability to small molecules and a measurable rate of lysis.