Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes
Surface-localized glycine transporters 1 and 2 function as monomeric proteins in Xenopus oocytes
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DOI：
10.1073/pnas.041329498
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发表时间：
2001-02-13
影响因子：
11.1
通讯作者：
Betz, H
中科院分区：
文献类型：
--
作者：
Horiuchi, M;Nicke, A;Betz, H
依赖于Na+/Cl-的神经递质转运体形成了一个跨膜蛋白超家族，它们共享12个跨膜区域。为了获得这些转运蛋白的四级结构的信息，我们在非洲爪哇卵母细胞中异源表达了胶质甘氨酸转运蛋白GlyT1及其神经元同源物GlyT2。通过用[S-35]蛋氨酸代谢标记或质膜不通透性试剂表面标记，然后进行亲和纯化，我们分别分析了新合成的GlyT及其功能质膜结合组分的细胞池。在蓝色天然凝胶电泳中，发现表面定位的转运蛋白仅以复杂糖基化的单体形式存在，而胞内GlyT1和GlyT2的很大一部分是核心糖基化和寡聚体。相反，即使用交联剂戊二醛处理后，表面甘氨酸也不能作为寡聚蛋白迁移。这些结果表明，质膜结合的GlyT1和GlyT2是单体蛋白，因此，钠/氯依赖的神经递质转运体不需要寡聚来进行底物转位。
Na+/Cl--dependent neurotransmitter transporters form a superfamily of transmembrane proteins that share 12 membrane-spanning regions. To gain information about the quaternary structure of these transporter proteins, we heterologously expressed the glial glycine transporter GlyT1 and its neuronal homolog GlyT2 in Xenopus oocytes. By using metabolic labeling with [S-35]methionine or surface labeling with a plasma membrane impermeable reagent followed by affinity purification, we separately analyzed the total cellular pools of newly synthesized GlyTs and its functional plasma membrane-bound fractions. Upon blue native gel electrophoresis, the surface-localized transporter proteins were found to exist exclusively in complex-glycosylated monomeric form, whereas a significant fraction of the intracellular GlyT1 and GlyT2 was core-glycosylated and oligomeric. In contrast, even after treatment with the crosslinker glutaraldehyde, surface GlyTs failed to migrate as oligomeric proteins. These results indicate that plasma membrane-bound GlyT1 and GlyT2 are monomeric proteins, Thus, Na+/Cl--dependent neurotransmitter transporters do not require oligomerization for substrate translocation.