Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc
Structural bases for the interaction and stabilization of the human amino acid transporter LAT2 with its ancillary protein 4F2hc
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DOI：
10.1073/pnas.1323779111
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发表时间：
2014-02-25
影响因子：
11.1
通讯作者：
Fotiadis, Dimitrios
中科院分区：
文献类型：
--
作者：
Rosell, Albert;Meury, Marcel;Fotiadis, Dimitrios
异聚体氨基酸转运蛋白（HAT）是已知的唯一例子，在所有的生命领域，溶质转运蛋白由两个亚基连接的保守的二硫键。在后生动物中，重亚基负责将异源二聚体运输到质膜，轻亚基是转运蛋白。HAT参与人类病理学，如氨基酸尿症、肿瘤生长和侵袭、病毒感染和可卡因成瘾。然而，关于HAT的重亚基和轻亚基之间的相互作用的结构信息很少。在这项工作中，纯化的人4F 2 hc/L型氨基酸转运蛋白2（LAT 2）异源二聚体在酵母中过表达的透射电子显微镜和单颗粒分析，连同对接分析和交联实验，揭示了4F 2 hc的胞外结构域与LAT 2相互作用，几乎完全覆盖了转运蛋白的胞外面。4F 2 hc增加了轻亚基LAT 2在去污剂溶解的毕赤酵母属膜中的稳定性，允许异二聚体功能性重建成脂蛋白体。此外，4F 2 hc的胞外结构域足以稳定溶解的LAT 2。4F 2 hc与LAT 2的相互作用提供了对轻亚基识别的结构基础和辅助蛋白在HAT中的稳定作用的见解。
Heteromeric amino acid transporters (HATs) are the unique example, known in all kingdoms of life, of solute transporters composed of two subunits linked by a conserved disulfide bridge. In metazoans, the heavy subunit is responsible for the trafficking of the heterodimer to the plasma membrane, and the light subunit is the transporter. HATs are involved in human pathologies such as amino acidurias, tumor growth and invasion, viral infection and cocaine addiction. However structural information about interactions between the heavy and light subunits of HATs is scarce. In this work, transmission electron microscopy and single-particle analysis of purified human 4F2hc/L-type amino acid transporter 2 (LAT2) heterodimers overexpressed in the yeast Pichia pastoris, together with docking analysis and crosslinking experiments, reveal that the extracellular domain of 4F2hc interacts with LAT2, almost completely covering the extracellular face of the transporter. 4F2hc increases the stability of the light subunit LAT2 in detergent-solubilized Pichia membranes, allowing functional reconstitution of the heterodimer into proteoliposomes. Moreover, the extracellular domain of 4F2hc suffices to stabilize solubilized LAT2. The interaction of 4F2hc with LAT2 gives insights into the structural bases for light subunit recognition and the stabilizing role of the ancillary protein in HATs.