Ferreira Lucimar T, Santos Magda S, Kolmakova Natalia G, Koenen Janaina, Barbosa Jose, Gomez Marcus V, Guatimosim Cristina, Zhang Xiaodong, Parsons Stanley M, Prado Vania F, Prado Marco A M
Program in Molecular Pharmacology, Departamento de Farmacologia, Belo Horizonte, Brazil.
J Neurochem. 2005 Aug;94(4):957-69. doi: 10.1111/j.1471-4159.2005.03244.x.
The vesicular acetylcholine transporter (VAChT) regulates the amount of acetylcholine stored in synaptic vesicles. However, the mechanisms that control the targeting of VAChT and other synaptic vesicle proteins are still poorly comprehended. These processes are likely to depend, at least partially, on structural determinants present in the primary sequence of the protein. Here, we use site-directed mutagenesis to evaluate the contribution of the C-terminal tail of VAChT to the targeting of this transporter to synaptic-like microvesicles in cholinergic SN56 cells. We found that residues 481-490 contain the trafficking information necessary for VAChT localization and that within this region L485 and L486 are strictly necessary. Deletion and alanine-scanning mutants lacking most of the carboxyl tail of VAChT, but containing residues 481-490, were still targeted to microvesicles. Moreover, we found that clathrin-mediated endocytosis of VAChT is required for targeting to microvesicles in SN56 and PC12 cells. The data provide novel information on the mechanisms and structural determinants necessary for VAChT localization to synaptic vesicles.
囊泡乙酰胆碱转运体（VAChT）调节储存在突触囊泡中的乙酰胆碱量。然而，控制VAChT和其他突触囊泡蛋白靶向的机制仍知之甚少。这些过程可能至少部分取决于蛋白质一级序列中存在的结构决定因素。在这里，我们使用定点诱变来评估VAChT的C末端尾巴对该转运体在胆碱能SN56细胞中靶向突触样微囊泡的作用。我们发现481-490位残基包含VAChT定位所需的运输信息，并且在该区域内L485和L486是绝对必需的。缺少VAChT大部分羧基末端尾巴但包含481-490位残基的缺失和丙氨酸扫描突变体仍靶向微囊泡。此外，我们发现SN56和PC12细胞中VAChT的网格蛋白介导的内吞作用是靶向微囊泡所必需的。这些数据提供了关于VAChT定位于突触囊泡所需机制和结构决定因素的新信息。
