β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function
β-Subunit of the Ostα-Ostβ Organic Solute Transporter Is Required Not Only for Heterodimerization and Trafficking but Also for Function
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DOI：
10.1074/jbc.m112.352245
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发表时间：
2012-06-15
影响因子：
4.8
通讯作者：
Ballatori, Nazzareno
中科院分区：
文献类型：
--
作者：
Christian, Whitney V.;Li, Na;Ballatori, Nazzareno
有机溶质转运体Ost/Slc51由两种不同的蛋白质组成，它们必须异二聚化才能产生运输活性，但单个亚基在介导运输活性中的作用尚不清楚。本研究确定了Ost β中与Ost α异二聚化、Ost α -Ost β复合物运输到质膜以及HEK293细胞胆汁酸运输活性所需的区域。双分子荧光互补分析显示，含有Ost β跨膜（TM）结构域的25个氨基酸肽与Ost α异二聚体，尽管所产生的复合物未能到达质膜并产生细胞[H-3]牛磺胆酸运输活性。Ost β的单个TM结构域的缺失消除了与Ost α的相互作用，这表明TM片段对于与Ost α形成异质复合物是必要和充分的。在Ost β的TM结构域中高度保守的色氨酸-天冬酰胺序列突变为丙氨酸并不能阻止细胞表面运输，但会破坏运输活性。去除Ost β的n端27个氨基酸导致转运蛋白复合物到达质膜，并在30℃时表现出运输活性。完全删除Ost β的C端会取消[H-3]牛胆酸盐的运输活性，但在TM结构域的C端重新插入两个天然精氨酸可以修复这一缺陷。这些带正电荷的残基根据正电荷内规则建立了肽的正确N-exo/C-cyt拓扑结构。总之，研究结果表明，Ost β对于Ost α的适当运输和功能运输单元的形成都是必需的，并确定了对这些过程至关重要的Ost β的特定残基。
The organic solute transporter, Ost/Slc51, is composed of two distinct proteins that must heterodimerize to generate transport activity, but the role of the individual subunits in mediating transport activity is unknown. The present study identified regions in Ost beta required for heterodimerization with Ost alpha, trafficking of the Ost alpha-Ost beta complex to the plasma membrane, and bile acid transport activity in HEK293 cells. Bimolecular fluorescence complementation analysis revealed that a 25-amino acid peptide containing the Ost beta transmembrane (TM) domain heterodimerized with Ost alpha, although the resulting complex failed to reach the plasma membrane and generate cellular [H-3]taurocholate transport activity. Deletion of the single TM domain of Ost beta abolished interaction with Ost alpha, demonstrating that the TM segment is necessary and sufficient for formation of a heteromeric complex with Ost alpha. Mutation of the highly conserved tryptophan-asparagine sequence within the TM domain of Ost beta to alanines did not prevent cell surface trafficking, but abolished transport activity. Removal of the N-terminal 27 amino acids of Ost beta resulted in a transporter complex that reached the plasma membrane and exhibited transport activity at 30 degrees C. Complete deletion of the C terminus of Ost beta abolished [H-3]taurocholate transport activity, but reinsertion of two native arginines immediately C-terminal to the TM domain rescued this defect. These positively charged residues establish the correct N-exo/C-cyt topology of the peptide, in accordance with the positive inside rule. Together, the results demonstrate that Ost beta is required for both proper trafficking of Ost alpha and formation of the functional transport unit, and identify specific residues of Ost beta critical for these processes.