Transmembrane topology of vesicular glutamate transporter 2
Transmembrane topology of vesicular glutamate transporter 2
复制标题
DOI：
10.1248/bpb.29.547
复制
发表时间：
2006-03-01
影响因子：
2
通讯作者：
Omote, H
中科院分区：
文献类型：
--
作者：
Jung, SK;Morimoto, R;Omote, H
囊泡谷氨酸转运蛋白（VGLUT）通过分泌囊泡中L-谷氨酸的囊泡储存，在神经元和一些外周组织的L-谷氨酸信号传导中发挥着重要作用。为了研究膜中VGLUT的拓扑结构，我们制备了针对氨基末端（抗N）、第一假定环（抗L）和羧基末端（抗C）区域的定点抗体。没有抗体与COS细胞中表达的VGLUT 2反应，因为它们不能接近抗原。然而，当细胞用毛地黄皂苷透化时，抗N和抗C抗体都识别VGLUT 2，而抗L抗体不识别。当细胞用Triton X-100透化时，出现对抗L-抗体的免疫反应性。这些结果表明，膜中VGLUT 2的氨基末端和羧基末端区域都面向细胞质，而第一环面向管腔。
Vesicular glutamate transporter (VGLUT) plays an essential role in L-glutamate signaling in neurons and some peripheral tissues through vesicular storage of L-glutamate in secretory vesicles. To investigate the topology of VGLUT in membranes, we prepared site-directed antibodies against the amino-terminal (anti-N), 1st putative loop (anti-L), and carboxyl terminal (anti-C) regions. None of the antibodies reacted with VGLUT2 expressed in COS cells because they could not gain access to the antigen. However, both the anti-N and anti-C antibodies recognized VGLUT2 when the cells were permeabilized with digitonin, while the anti-L antibodies did not. Immunological reactivity to anti-L-antibodies appeared when the cells were permeabilized with Triton X-100. These results suggest that both the amino-terminal and carboxyl-terminal regions of VGLUT2 in membranes face the cytoplasm while the 1st loop faces the lumen.