Shi G, Faúndez V, Roos J, Dell'Angelica E C, Kelly R B
Department of Biochemistry and Biophysics and the Hormone Research Institute, University of California, San Francisco, California 94143-0534, USA.
J Cell Biol. 1998 Nov 16;143(4):947-55. doi: 10.1083/jcb.143.4.947.
In the neuroendocrine cell line, PC12, synaptic vesicles can be generated from endosomes by a sorting and vesiculation process that requires the heterotetrameric adaptor protein AP3 and a small molecular weight GTPase of the ADP ribosylation factor (ARF) family. We have now discovered a second pathway that sorts the synaptic vesicle-associated membrane protein (VAMP) into similarly sized vesicles. For this pathway the plasma membrane is the precursor rather than endosomes. Both pathways require cytosol and ATP and are inhibited by GTPgammaS. The second pathway, however, uses AP2 instead of AP3 and is brefeldin A insensitive. The AP2-dependent pathway is inhibited by depletion of clathrin or by inhibitors of clathrin binding, whereas the AP3 pathway is not. The VAMP-containing, plasma membrane-derived vesicles can be readily separated on sucrose gradients from transferrin (Tf)-containing vesicles generated by incubating Tf-labeled plasma membrane preparations at 37 degreesC. Dynamin- interacting proteins are required for the AP2-mediated vesiculation from the plasma membrane, but not from endosomes. Thus, VAMP is sorted into small vesicles by AP3 and ARF1 at endosomes and by AP2 and clathrin at the plasma membrane.
在神经内分泌细胞系PC12中，突触小泡可通过一种分选和小泡形成过程从内体产生，该过程需要异源四聚体衔接蛋白AP3和ADP核糖基化因子（ARF）家族的一种小分子量GTP酶。我们现在发现了第二条将突触小泡相关膜蛋白（VAMP）分选到大小相似的小泡中的途径。对于这条途径，质膜是前体而非内体。两条途径都需要胞质溶胶和ATP，并被GTPγS抑制。然而，第二条途径使用AP2而非AP3，且对布雷菲德菌素A不敏感。依赖AP2的途径可通过网格蛋白耗竭或网格蛋白结合抑制剂来抑制，而AP3途径则不然。含VAMP的、源自质膜的小泡可通过蔗糖梯度很容易地与在37℃孵育转铁蛋白（Tf）标记的质膜制剂所产生的含转铁蛋白（Tf）的小泡分离。动力蛋白相互作用蛋白是AP2介导的从质膜形成小泡所必需的，但不是从内体形成小泡所必需的。因此，VAMP在内体由AP3和ARF1分选到小泡中，在质膜由AP2和网格蛋白分选到小泡中。
