Graham M E, Washbourne P, Wilson M C, Burgoyne R D
The Physiological Laboratory, University of Liverpool, Crown Street, Liverpool, L69 3BX, UK.
J Cell Sci. 2001 Dec;114(Pt 24):4397-405. doi: 10.1242/jcs.114.24.4397.
Considerable data support the idea that intracellular membrane fusion involves a conserved machinery containing the SNARE proteins. SNAREs assembled in vitro form a stable 4-helix bundle and it has been suggested that formation of this complex provides the driving force for bilayer fusion. We have tested this possibility in assays of exocytosis in cells expressing a botulinum neurotoxin E (BoNT/E)-resistant mutant of SNAP-25 in which additional disruptive mutations have been introduced. Single or double mutations of glutamine to glutamate or to arginine in the central zero layer residues of SNAP-25 did not impair the extent, time course or Ca2+-dependency of exocytosis in PC12 cells. Using adrenal chromaffin cells, we found that exocytosis could be reconstituted in cells transfected to express BoNT/E. A double Q-->E mutation did not prevent reconstitution and the kinetics of single granule release events were indistinguishable from control cells. This shows a high level of tolerance of changes in the zero layer indicating that the conservation of these residues is not due to an essential requirement in vesicle docking or fusion and suggests that formation of a fully stable SNARE complex may not be required to drive membrane fusion.
大量数据支持这样一种观点，即细胞内膜融合涉及一种包含SNARE蛋白的保守机制。在体外组装的SNARE蛋白形成一个稳定的四螺旋束，有人提出这种复合物的形成提供了双层膜融合的驱动力。我们在表达对肉毒杆菌神经毒素E（BoNT/E）具有抗性的SNAP-25突变体（其中引入了额外的破坏性突变）的细胞的胞吐作用测定中测试了这种可能性。在SNAP-25中央零层残基中，谷氨酰胺单突变或双突变为谷氨酸或精氨酸，并不损害PC12细胞中胞吐作用的程度、时间进程或对Ca2+的依赖性。利用肾上腺嗜铬细胞，我们发现转染以表达BoNT/E的细胞中可以重建胞吐作用。双Q→E突变并不妨碍重建，单个颗粒释放事件的动力学与对照细胞没有区别。这表明对零层变化具有高度耐受性，表明这些残基的保守性并非由于囊泡对接或融合中的基本要求，这表明驱动膜融合可能不需要形成完全稳定的SNARE复合物。
