Lobingier Braden T, Nickerson Daniel P, Lo Sheng-Ying, Merz Alexey J
Department of Biochemistry, University of Washington School of Medicine, Seattle, United States.
Department of Biochemistry, University of Washington School of Medicine, Seattle, United States Department of Physiology and Biophysics, University of Washington School of Medicine, Seattle, United States
Elife. 2014 May 16;3:e02272. doi: 10.7554/eLife.02272.
Secretory and endolysosomal fusion events are driven by SNAREs and cofactors, including Sec17/α-SNAP, Sec18/NSF, and Sec1/Munc18 (SM) proteins. SMs are essential for fusion in vivo, but the basis of this requirement is enigmatic. We now report that, in addition to their established roles as fusion accelerators, SM proteins Sly1 and Vps33 directly shield SNARE complexes from Sec17- and Sec18-mediated disassembly. In vivo, wild-type Sly1 and Vps33 function are required to withstand overproduction of Sec17. In vitro, Sly1 and Vps33 impede SNARE complex disassembly by Sec18 and ATP. Unexpectedly, Sec17 directly promotes selective loading of Sly1 and Vps33 onto cognate SNARE complexes. A large thermodynamic barrier limits SM binding, implying that significant conformational rearrangements are involved. In a working model, Sec17 and SMs accelerate fusion mediated by cognate SNARE complexes and protect them from NSF-mediated disassembly, while mis-assembled or non-cognate SNARE complexes are eliminated through kinetic proofreading by Sec18.DOI: http://dx.doi.org/10.7554/eLife.02272.001.
分泌性囊泡与内溶酶体的融合事件由SNARE蛋白和辅助因子驱动，包括Sec17/α-SNAP、Sec18/NSF和Sec1/Munc18（SM）蛋白。SM蛋白在体内融合过程中至关重要，但其发挥这一作用的机制尚不清楚。我们现在报告，除了作为融合促进因子的既定作用外，SM蛋白Sly1和Vps33还能直接保护SNARE复合体免受Sec17和Sec18介导的拆解。在体内，需要野生型Sly1和Vps33的功能来抵抗Sec17的过量产生。在体外，Sly1和Vps33会阻碍Sec18和ATP介导的SNARE复合体拆解。出乎意料的是，Sec17能直接促进Sly1和Vps33选择性地加载到同源SNARE复合体上。一个较大的热力学屏障限制了SM蛋白的结合，这意味着其中涉及显著的构象重排。在一个工作模型中，Sec17和SM蛋白加速同源SNARE复合体介导的融合，并保护它们免受NSF介导的拆解，而错误组装或非同源的SNARE复合体则通过Sec18的动力学校对被清除。DOI: http://dx.doi.org/10.7554/eLife.02272.001 。
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