Synaptic Vesicle Recycling at CNS Synapses without AP-2
Synaptic Vesicle Recycling at CNS Synapses without AP-2
复制标题
没有 AP-2 的中枢神经系统突触的突触小泡再循环
DOI：
10.1523/jneurosci.5639-08.2009
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发表时间：
2009-03-25
影响因子：
5.3
通讯作者：
Ryan, Timothy A.
中科院分区：
文献类型：
--
作者：
Kim, Sung Hyun;Ryan, Timothy A.
突触囊泡（SV）是由10种跨膜蛋白组成，在神经递质胞吐后必须回收。胞吐后这些蛋白质一旦并入质膜，就重新进入突触囊泡的机制尚不清楚。适配器复合物AP-2是用于质膜上货物识别的主要网格蛋白相关适配器。在这里，我们研究了它在突触囊泡内吞作用中的作用。shRNA介导的AP-2复合物的敲低导致原代神经元中该蛋白复合物的类似于96%的减少。我们同时表达shRNA和pHluorin标记的囊泡组分，表明AP-2的缺失显著减缓但不能阻止四种主要突触囊泡跨膜蛋白的内吞作用。我们发现，在AP-2的情况下，AP-1适配器复合物似乎在功能上替代AP-2，但复杂的内化动力学，现在是敏感的鸟嘌呤核苷酸交换因子ADP-核糖基化因子GTdR（ARF-GEF）抑制剂布雷菲德菌素-A（BFA）。同时去除AP-2和AP-1阻止了这种补偿性取代，并导致缓慢但功能性的内吞作用。这些结果表明，在AP-2的情况下，SV蛋白仍然成为内吞，和突触囊泡循环仍然运作。
Synaptic vesicles (SVs) are composed of similar to 10 types of transmembrane proteins that must be recycled after exocytosis of neurotransmitter. The mechanisms for resorting these proteins into synaptic vesicles once incorporated into the plasma membrane after exocytosis are poorly understood. The adaptor complex AP-2 is the major clathrin-associated adaptor for cargo recognition at the plasma membrane. Here, we have investigated its role in synaptic vesicle endocytosis. shRNA-mediated knockdown of the AP-2 complex results in an similar to 96% reduction of this protein complex in primary neurons. We used simultaneous expression of shRNA and pHluorin-tagged vesicle components to show that the absence of AP-2 significantly slows but does not prevent the endocytosis of four of the major synaptic vesicle transmembrane proteins. We show that in the absence of AP-2, the AP-1 adaptor complex appears to functionally substitute for AP-2 but results in complex internalization kinetics that are now sensitive to the guanine-nucleotide exchange factor for ADP-ribosylation factor GTPase (ARF-GEF) inhibitor brefeldin-A (BFA). Simultaneous removal of both AP-2 and AP-1 prevents this compensatory substitution and results in slowed but functional endocytosis. These results demonstrate that in the absence of AP-2, SV proteins still become endocytosed, and synaptic vesicle recycling remains operational.