MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion.
MARCKS Effector Domain, a reversible lipid ligand, illuminates late stages of membrane fusion.
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MARCKS 效应器结构域是一种可逆脂质配体，阐明了膜融合的后期阶段。
DOI：
10.1091/mbc.e23-06-0228
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发表时间：
2023
影响因子：
3.3
通讯作者：
Wickner,William
中科院分区：
文献类型：
--
作者：
Orr,Amy;Wickner,William
酵母空泡HOPS系膜，催化R-和Q-SNARE之间的transans-SNARE组装，Shepherds SNARE通过Sec 17的早期抑制。在部分SNARE拉链后，通过完成SNARE拉链或Sec 17/Sec 18缓慢驱动融合，但快速融合需要拉链和Sec 17/Sec 18。使用重组空泡融合，我们发现MARCKS效应域（MED）肽，脂质配体，在后期反应阶段可逆地阻断融合。MED融合阻断可通过盐提取、MED配体钙调蛋白灭活或添加Sec 17/Sec 18来克服。在与MED一起孵育期间，SNARE在体内组装稳定的复合物，并且融合体变得对Qa SNARE的抗体具有抗性。当Q-SNARE被预组装时，合成系链可以取代HOPS进行融合。使用合成系链，融合需要完整的SNARE拉链和Sec 17/Sec 18来克服MED阻滞。相反，当SNARE结构域只有三分之二的拉链时，只有HOPS将支持Sec 17/Sec 18驱动的融合，而不需要完全拉链。因此，HOPS在拉炼期间保持与SNARE接合。MED有助于研究不同的融合阶段：栓系、初始反式SNARE组装及其对Sec 17的敏感性、SNARE拉链、Sec 17/Sec 18接合以及脂质和管腔混合。
Yeast vacuolar HOPS tethers membranes, catalyzestrans-SNARE assembly between R- and Q-SNAREs, and shepherds SNAREs past early inhibition by Sec17. After partial SNARE zippering, fusion is driven slowly by either completion of SNARE zippering or by Sec17/Sec18, but rapid fusion needs zippering and Sec17/Sec18. Using reconstituted-vacuolar fusion, we find that MARCKS Effector Domain (MED) peptide, a lipid ligand, blocks fusion reversibly at a late reaction stage. The MED fusion blockade is overcome by either salt extraction, inactivation with the MED ligand calmodulin, or addition of Sec17/Sec18. During incubation with MED, SNAREs assemble stable complexes intransand fusion becomes resistant to antibody to the Qa SNARE. When Q-SNAREs are preassembled, a synthetic tether can replace HOPS for fusion. With a synthetic tether, fusion needs both complete SNARE zippering and Sec17/Sec18 to overcome a MED block. In contrast, when SNARE domains are only two-third zippered, only HOPS will support Sec17/Sec18 driven fusion without needing complete zippering. HOPS thus remains engaged with SNAREs during zippering. MED facilitates the study of distinct fusion stages: tethering, initialtrans-SNARE assembly and its sensitivity to Sec17, SNARE zippering, Sec17/Sec18 engagement, and lipid and lumenal mixing.