Department of Neurobiology, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Traffic. 2010 Mar;11(3):394-404. doi: 10.1111/j.1600-0854.2009.01020.x. Epub 2009 Nov 19.
SNAREs are clustered membrane proteins essential for intracellular fusion steps. During fusion, three to four SNAREs with a Q(a)-, Q(b)-, Q(c)- and R-SNARE-motif form a complex. The core complex represents a Q(a)Q(b)Q(c)R-SNARE-motif bundle, most certainly assembling in steps. However, to date it is unknown which intermediate SNARE complex observed in vitro also exists in vivo. Here we have applied comparative fluorescence recovery after photobleaching (FRAP)-studies as a novel approach for studying in intact cells a SNARE interaction involved in synaptic vesicle fusion [catalyzed by syntaxin 1A (Q(a)), SNAP25 (Q(b)/Q(c)) and synaptobrevin 2 (R)]. We find that the Q(b)-SNARE-motif of SNAP25 interacts reversibly with clustered syntaxin. The interaction requires most of the alpha helical Q(b)-SNARE-motif and depends on its position within the molecule. We conclude that a zippered Q(a)Q(b)-SNARE complex represents a short-lived SNARE intermediate in intact cells, most likely providing an initial molecular platform toward membrane fusion.
SNAP 受体是细胞内融合步骤所必需的聚集膜蛋白。在融合过程中，具有 Q(a)-、Q(b)-、Q(c)-和 R-SNARE 基序的三到四个 SNAP 受体形成复合物。核心复合物代表一个 Q(a)Q(b)Q(c)R-SNARE 基序束，很可能以逐步的方式组装。然而，迄今为止，尚不清楚体外观察到的哪种中间 SNAP 复合物也存在于体内。在这里，我们应用比较荧光恢复后光漂白（FRAP）研究作为一种新的方法来研究参与突触囊泡融合的完整细胞中的 SNAP 相互作用[由突触融合蛋白 1A（Q(a)）、SNAP25（Q(b)/Q(c)）和突触融合蛋白 2（R）催化]。我们发现 SNAP25 的 Q(b)-SNARE 基序与聚集的突触融合蛋白可逆地相互作用。这种相互作用需要 Q(b)-SNARE 基序的大部分α螺旋结构，并取决于其在分子中的位置。我们得出结论，拉链式 Q(a)Q(b)-SNARE 复合物代表完整细胞中短暂存在的 SNARE 中间产物，很可能为膜融合提供了初始的分子平台。
