Tarafdar Pradip K, Chakraborty Hirak, Bruno Michael J, Lentz Barry R
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Molecular and Cellular Biophysics Program, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina; Molecular and Cellular Biophysics Program, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina.
Biophys J. 2015 Nov 3;109(9):1863-72. doi: 10.1016/j.bpj.2015.08.051.
Although the importance of a SNARE complex in neurotransmitter release is widely accepted, there exist different views on how the complex promotes fusion. One hypothesis is that the SNARE complex's ability to bring membranes into contact is sufficient for fusion, another points to possible roles of juxtamembrane regions (JMRs) and transmembrane domains (TMDs) in catalyzing lipid rearrangement, and another notes the complex's presumed ability to bend membranes near the point of contact. Here, we performed experiments with highly curved vesicles brought into contact using low concentrations of polyethylene glycol (PEG) to investigate the influence of the synaptobrevin (SB) TMD with an attached JMR (SB-JMR-TMD) on the rates of stalk and pore formation during vesicle fusion. SB-JMR-TMD enhanced the rates of stalk and fusion pore (FP) formation in a sharply sigmoidal fashion. We observed an optimal influence at an average of three peptides per vesicle, but only with phosphatidylserine (PS)-containing vesicles. Approximately three SB-JMR-TMDs per vesicle optimally ordered the bilayer interior and excluded water in a similar sigmoidal fashion. The catalytic influences of hexadecane and SB-JMR-TMD on fusion kinetics showed little in common, suggesting different mechanisms. Both kinetic and membrane structure measurements support the hypotheses that SB-JMR-TMD 1) catalyzes initial intermediate formation as a result of its basic JMR disrupting ordered interbilayer water and permitting closer interbilayer approach, and 2) catalyzes pore formation by forming a membrane-spanning complex that increases curvature stress at the circumference of the hemifused diaphragm of the prepore intermediate state.
尽管SNARE复合体在神经递质释放中的重要性已被广泛接受，但对于该复合体如何促进融合存在不同观点。一种假说是，SNARE复合体使膜接触的能力足以实现融合；另一种观点指出，近膜区域（JMRs）和跨膜结构域（TMDs）在催化脂质重排中可能发挥作用；还有一种观点提到，该复合体可能具有在接触点附近使膜弯曲的能力。在此，我们使用低浓度聚乙二醇（PEG）使高度弯曲的囊泡相互接触，进行了实验，以研究带有附着JMR的突触小泡蛋白（SB）跨膜结构域（SB-JMR-TMD）对囊泡融合过程中柄和孔形成速率的影响。SB-JMR-TMD以急剧的S形方式提高了柄和融合孔（FP）的形成速率。我们观察到，平均每个囊泡有三个肽时具有最佳影响，但仅对含磷脂酰丝氨酸（PS）的囊泡有此效果。平均每个囊泡约有三个SB-JMR-TMD以类似的S形方式最佳地排列双层内部并排除水分。十六烷和SB-JMR-TMD对融合动力学的催化影响几乎没有共同之处，表明存在不同机制。动力学和膜结构测量均支持以下假说：1）SB-JMR-TMD通过其碱性JMR破坏有序的双层间水并允许更紧密的双层间接近，从而催化初始中间体形成；2）SB-JMR-TMD通过形成跨膜复合体来催化孔形成，该复合体增加了预孔中间体状态的半融合隔膜圆周处的曲率应力。
