Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae.
Functional characterization of a Na+-dependent dicarboxylate transporter from Vibrio cholerae.
复制标题
DOI：
10.1085/jgp.201311141
复制
发表时间：
2014-06
期刊：
影响因子：
--
通讯作者：
Mindell JA
中科院分区：
文献类型：
--
作者：
Mulligan C;Fitzgerald GA;Wang DN;Mindell JA
VcINDY是一种与果蝇寿命和哺乳动物胰岛素抵抗有关的转运蛋白的细菌同系物，是一种高亲和力、产电性、Na+依赖性二羧酸转运蛋白。SLC 13转运蛋白家族，其成员在调节脂肪酸合成、肥胖、胰岛素抵抗和其他过程中发挥关键的生理作用，催化克雷布斯循环中间体和硫酸盐穿过哺乳动物细胞的质膜的转运。SLC 13转运蛋白是二价阴离子：Na+同向转运蛋白（DASS）家族的一部分，该家族包括几个充分表征的细菌成员。尽管共享显着的序列相似性，DASS家族成员的功能特性不同，就其底物和耦合离子的依赖性。细菌DASS家族成员二聚体VcINDY的高分辨率结构的发表为该转运蛋白家族提供了重要的结构见解。然而，将这种结构的见解与目前对该家族的功能理解相结合还需要对转运蛋白的功能特性进行全面分析。为此，我们纯化了VcINDY，将其重组为脂质体，并确定其基本功能特性。我们的数据表明，VcINDY是一种高亲和力的Na+依赖性转运蛋白，偏好C4-和C5-二羧酸。运输的模型基板，琥珀酸盐，是高度pH值依赖性，一致的VcINDY强烈偏好基板的双阴离子形式。VcINDY转运是产电的，琥珀酸与三种或更多种Na+离子的转运偶联。相反，琥珀酸，柠檬酸，结合在VcINDY晶体结构（在一个面向内的构象），似乎只有微弱的相互作用与转运蛋白在体外。这些传输特性一起提供了一个功能框架，为未来的实验和计算检查的VcINDY传输机制。
VcINDY, a bacterial homolog of transporters implicated in lifespan in fruit flies and insulin resistance in mammals, is a high affinity, electrogenic, Na+-dependent dicarboxylate transporter. The SLC13 transporter family, whose members play key physiological roles in the regulation of fatty acid synthesis, adiposity, insulin resistance, and other processes, catalyzes the transport of Krebs cycle intermediates and sulfate across the plasma membrane of mammalian cells. SLC13 transporters are part of the divalent anion:Na+ symporter (DASS) family that includes several well-characterized bacterial members. Despite sharing significant sequence similarity, the functional characteristics of DASS family members differ with regard to their substrate and coupling ion dependence. The publication of a high resolution structure of dimer VcINDY, a bacterial DASS family member, provides crucial structural insight into this transporter family. However, marrying this structural insight to the current functional understanding of this family also demands a comprehensive analysis of the transporter’s functional properties. To this end, we purified VcINDY, reconstituted it into liposomes, and determined its basic functional characteristics. Our data demonstrate that VcINDY is a high affinity, Na+-dependent transporter with a preference for C4- and C5-dicarboxylates. Transport of the model substrate, succinate, is highly pH dependent, consistent with VcINDY strongly preferring the substrate’s dianionic form. VcINDY transport is electrogenic with succinate coupled to the transport of three or more Na+ ions. In contrast to succinate, citrate, bound in the VcINDY crystal structure (in an inward-facing conformation), seems to interact only weakly with the transporter in vitro. These transport properties together provide a functional framework for future experimental and computational examinations of the VcINDY transport mechanism.
登录
查看更多内容
影响因子：
20.6
作者：
Alderighi, L;Gans, P;Vacca, A
通讯作者：
Vacca, A
影响因子：
16.8
作者：
Hanelt, Inga;Wunnicke, Dorith;Slotboom, Dirk Jan
通讯作者：
Slotboom, Dirk Jan
影响因子：
4.1
作者：
Inoue, K;Fei, YJ;Ganapathy, V
通讯作者：
Ganapathy, V
影响因子：
4.8
作者：
Inoue, K;Zhuang, L;Ganapathy, V
通讯作者：
Ganapathy, V
影响因子：
4.8
作者：
Chen, XZ;Shayakul, C;Hediger, MA
通讯作者：
Hediger, MA