Kim Myung-Hee, Hersh Louis B
Department of Molecular and Cellular Biochemistry, University of Kentucky, 800 Rose Street, Lexington, KY 40536, USA.
J Biol Chem. 2004 Mar 26;279(13):12580-7. doi: 10.1074/jbc.M310681200. Epub 2004 Jan 14.
In neuronal cells the neurotransmitter acetylcholine is transferred from the cytoplasm into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). The cytoplasmic tail of VAChT has been shown to contain signals that direct its sorting and trafficking. The role of clathrin-associated protein complexes in VAChT sorting to synaptic vesicles has been examined. A fusion protein between the VAChT cytoplasmic tail and glutathione S-transferase was used to identify VAChT-clathrin-associated protein adaptor protein 1, adaptor protein 2 and adaptor protein 180 complexes from a rat brain extract. In vivo coimmunoprecipitation confirmed adaptin alpha and adaptin gamma complexes, but adaptor protein 180 complexes were not detected by this technique. Deletion and site directed mutagenesis show that the VAChT cytoplasmic tail contains multiple trafficking signals. These include a non-classical tyrosine motif that serves as the signal for adaptin alpha and a dileucine motif that serves as the signal for adaptin gamma. A classical tyrosine motif is also involved in VAChT trafficking, but does not interact with any known adaptor proteins. There appear to be two endocytosis motifs, one involving the adaptor protein 1 binding site and the other involving the adaptor protein 2 binding site. These results suggest a complex trafficking pathway for VAChT.
在神经元细胞中，神经递质乙酰胆碱通过囊泡乙酰胆碱转运体（VAChT）从细胞质转移到突触小泡中。已证明VAChT的细胞质尾部含有指导其分选和运输的信号。研究了网格蛋白相关蛋白复合物在VAChT分选至突触小泡中的作用。使用VAChT细胞质尾部与谷胱甘肽S-转移酶之间的融合蛋白，从大鼠脑提取物中鉴定VAChT-网格蛋白相关蛋白衔接蛋白1、衔接蛋白2和衔接蛋白180复合物。体内共免疫沉淀证实了衔接蛋白α和衔接蛋白γ复合物，但该技术未检测到衔接蛋白180复合物。缺失和定点诱变表明，VAChT细胞质尾部含有多个运输信号。这些信号包括作为衔接蛋白α信号的非经典酪氨酸基序和作为衔接蛋白γ信号的双亮氨酸基序。一个经典的酪氨酸基序也参与VAChT的运输，但不与任何已知的衔接蛋白相互作用。似乎有两个内吞基序，一个涉及衔接蛋白1结合位点，另一个涉及衔接蛋白2结合位点。这些结果表明VAChT存在复杂的运输途径。
