Vesicular glutamate transporter contains two independent transport machineries
Vesicular glutamate transporter contains two independent transport machineries
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DOI：
10.1074/jbc.m607670200
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发表时间：
2006-12-22
影响因子：
4.8
通讯作者：
Moriyama, Yoshinori
中科院分区：
文献类型：
--
作者：
Juge, Narinobu;Yoshida, Yumi;Moriyama, Yoshinori
囊泡谷氨酸转运蛋白（VGLUT）负责L-谷氨酸的囊泡储存，并在中枢神经系统的谷氨酸信号传递中发挥重要作用。运输的分子机制仍不清楚。在这里，我们建立了一种新颖的体外测定程序，其中包括野生和突变 VGLUT2 的纯化以及用纯化的细菌 F0F1-ATPase (F-ATPase) 将其重建到脂质体中。添加 ATP 后，蛋白脂质体以膜电位 (Delta psi) 依赖性方式促进 L-谷氨酸的摄取。 ATP依赖性L-谷氨酸摄取表现出对类似于4mM Cl-的绝对需求，对伊文思蓝敏感，但对D,L-天冬氨酸不敏感。跨膜残基 Arg(184)、His(128) 和 Glu(191) 发生突变的 VGLUT2 显示 L-谷氨酸转运活性显着丧失，而 Na+ 依赖性无机磷酸盐 (Pi) 摄取仍与野生型相当。此外，Pi 运输不需要 Cl-，并且不会被伊文思蓝抑制。因此，VGLUT2 似乎拥有两种相互独立的内在转运机制：依赖于 Delta psi 的 L-谷氨酸摄取和依赖于 Na+ 的 Pi 摄取。
Vesicular glutamate transporters (VGLUTs) are responsible for the vesicular storage of L-glutamate and play an essential role in glutamatergic signal transmission in the central nervous system. The molecular mechanism of the transport remains unknown. Here, we established a novel in vitro assay procedure, which includes purification of wild and mutant VGLUT2 and their reconstitution with purified bacterial F0F1-ATPase (F-ATPase) into liposomes. Upon the addition of ATP, the proteoliposomes facilitated L-glutamate uptake in a membrane potential (Delta psi)-dependent fashion. The ATP-dependent L-glutamate uptake exhibited an absolute requirement for similar to 4 mM Cl-, was sensitive to Evans blue, but was insensitive to D, L-aspartate. VGLUT2s with mutations in the transmembrane-located residues Arg(184), His(128), and Glu(191) showed a dramatic loss in L-glutamate transport activity, whereas Na+-dependent inorganic phosphate (Pi) uptake remained comparable to that of the wild type. Furthermore, Pi transport did not require Cl- and was not inhibited by Evans blue. Thus, VGLUT2 appears to possess two intrinsic transport machineries that are independent of each other: a Delta psi-dependent L-glutamate uptake and a Na+-dependent Pi uptake.