Department of Pharmacology, University of Washington, Seattle, Washington 98195, USA.
J Neurosci. 2010 Apr 21;30(16):5569-78. doi: 10.1523/JNEUROSCI.4781-09.2010.
Synaptic vesicles are specialized cycling endosomes that contain a unique constellation of membrane proteins. Proteins are sorted to vesicles by short amino acid sequences that serve as binding sites for clathrin adaptor proteins. Here we show that a tyrosine-based endocytosis motif in the vesicle protein SV2 is required for trafficking to synaptic vesicles of both SV2 and the calcium sensor protein synaptotagmin. Aberrant neurotransmission in cultured hippocampal neurons lacking SV2 was rescued by expression of wild-type SV2A, but not by SV2A-Y46A, a mutant containing a disrupted endocytosis motif in SV2A's cytoplasmic N terminus. Neurons expressing SV2A-Y46A had significantly more SV2 on the plasma membrane, indicating reduced internalization. A screen for proteins that preferentially bound wild-type SV2A identified multiple endocytosis-related proteins, and in vitro binding studies confirmed binding to the clathrin adaptors AP2, EPS15, and amphiphysin 2/Bin1. Neurons lacking SV2 contained less synaptotagmin and had a higher proportion of synaptotagmin on the plasma membrane. Expression of either wild-type SV2A or SV2A-Y46A restored synaptotagmin expression levels; however, only wild-type SV2A restored a normal proportion of synaptotagmin on the plasma membrane. These findings indicate that SV2 influences the expression and trafficking of synaptotagmin via separate mechanisms. Synaptic vesicles immunoisolated from SV2A/B double knock-out mice had significantly less synaptotagmin than vesicles isolated from wild-type mice. Our results indicate that SV2 plays a major role in regulating the amount of synaptotagmin in synaptic vesicles and provide an explanation for the observation that synapses lacking SV2 have fewer vesicles competent for calcium-induced fusion.
突触小泡是专门的循环内体，包含独特的膜蛋白组合。蛋白质通过作为笼形蛋白衔接蛋白结合位点的短氨基酸序列被分拣到小泡中。在这里，我们表明，囊泡蛋白 SV2 中的一个基于酪氨酸的内吞作用基序对于 SV2 和钙传感器蛋白突触融合蛋白 synaptotagmin 向突触小泡的运输都是必需的。在缺乏 SV2 的培养海马神经元中，异常的神经传递可以通过表达野生型 SV2A 来挽救，但不能通过 SV2A-Y46A 来挽救，SV2A-Y46A 是一种在 SV2A 的细胞质 N 末端含有破坏的内吞作用基序的突变体。表达 SV2A-Y46A 的神经元质膜上的 SV2 明显更多，表明内化减少。对优先与野生型 SV2A 结合的蛋白质进行筛选，鉴定出多种内吞作用相关蛋白，体外结合研究证实与网格蛋白衔接蛋白 AP2、EPS15 和 amphiphysin 2/Bin1 结合。缺乏 SV2 的神经元含有较少的突触融合蛋白 synaptotagmin，并且突触融合蛋白 synaptotagmin 在质膜上的比例更高。表达野生型 SV2A 或 SV2A-Y46A 均可恢复突触融合蛋白 synaptotagmin 的表达水平；然而，只有野生型 SV2A 可恢复突触融合蛋白 synaptotagmin 在质膜上的正常比例。这些发现表明 SV2 通过独立的机制影响突触融合蛋白 synaptotagmin 的表达和运输。从 SV2A/B 双敲除小鼠中免疫分离的突触小泡中的突触融合蛋白 synaptotagmin 明显少于从野生型小鼠中分离的突触小泡。我们的结果表明 SV2 在调节突触小泡中突触融合蛋白 synaptotagmin 的数量方面起着主要作用，并为缺乏 SV2 的突触中融合能力较弱的囊泡较少的观察结果提供了解释。
