Morgan J R, Zhao X, Womack M, Prasad K, Augustine G J, Lafer E M
Department of Neurobiology, Duke University Medical Center, Durham, North Carolina 27710, USA.
J Neurosci. 1999 Dec 1;19(23):10201-12. doi: 10.1523/JNEUROSCI.19-23-10201.1999.
We have used the squid giant synapse to determine whether clathrin assembly by AP180 is important for synaptic vesicle endocytosis. The squid homolog of AP180 encodes a 751 amino acid protein with 40% sequence identity to mouse AP180. Alignment of squid AP180 with other AP180 homologs shows that amino acid identity was highest in the N-terminal inositide-binding domain of the protein and weakest in the C-terminal clathrin assembly domain. Recombinant squid AP180 was able to assemble clathrin in vitro, suggesting a conserved three-dimensional structure that mediates clathrin assembly despite the divergent primary sequence of the C-terminal domain. Microinjection of the C-terminal domains of either mouse or squid AP180 into the giant presynaptic terminal of squid enhanced synaptic transmission. Conversely, a peptide from the C-terminal domain of squid AP180 that inhibited clathrin assembly in vitro completely blocked synaptic transmission when it was injected into the giant presynaptic terminal. This inhibitory effect occurred over a time scale of minutes when the synapse was stimulated at low (0.03 Hz), physiological rates. Electron microscopic analysis revealed several structural changes consistent with the inhibition of synaptic vesicle endocytosis; peptide-injected terminals had far fewer synaptic vesicles, were depleted of coated vesicles, and had a larger plasma membrane perimeter than terminals injected with control solutions. In addition, the remaining synaptic vesicles were significantly larger in diameter. We conclude that the clathrin assembly domain of AP180 is important for synaptic vesicle recycling at physiological rates of activity and that assembly of clathrin by AP180 is necessary for maintaining a pool of releasable synaptic vesicles.
我们利用枪乌贼巨大突触来确定AP180介导的网格蛋白组装对于突触小泡内吞作用是否重要。枪乌贼AP180的同源物编码一种751个氨基酸的蛋白质，与小鼠AP180的序列同一性为40%。枪乌贼AP180与其他AP180同源物的比对显示，该蛋白质在N端肌醇结合结构域的氨基酸同一性最高，而在C端网格蛋白组装结构域的氨基酸同一性最弱。重组枪乌贼AP180能够在体外组装网格蛋白，这表明尽管C端结构域的一级序列不同，但存在介导网格蛋白组装的保守三维结构。将小鼠或枪乌贼AP180的C端结构域显微注射到枪乌贼的巨大突触前终末可增强突触传递。相反，一种来自枪乌贼AP180 C端结构域的在体外抑制网格蛋白组装的肽，当注射到巨大突触前终末时，完全阻断了突触传递。当在低（0.03Hz）生理频率刺激突触时，这种抑制作用在几分钟的时间尺度上发生。电子显微镜分析揭示了与突触小泡内吞作用抑制一致的几种结构变化；注射肽的终末突触小泡数量少得多，包被小泡耗尽，并且质膜周长比注射对照溶液的终末更大。此外，剩余的突触小泡直径明显更大。我们得出结论，AP180的网格蛋白组装结构域对于生理活动频率下的突触小泡循环很重要，并且AP180介导的网格蛋白组装对于维持可释放突触小泡池是必要的。
