FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter
FUN26 (Function Unknown Now 26) Protein from Saccharomyces cerevisiae Is a Broad Selectivity, High Affinity, Nucleoside and Nucleobase Transporter
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DOI：
10.1074/jbc.m114.553503
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发表时间：
2014-08-29
影响因子：
4.8
通讯作者：
Hays, Franklin A.
中科院分区：
文献类型：
--
作者：
Boswell-Casteel, Rebba C.;Johnson, Jennifer M.;Hays, Franklin A.
背景：FUN26是一种在酵母空泡中表达的核苷转运蛋白。结果：纯化的FUN26蛋白脂质体研究表明，对C(2)-核糖修饰敏感的核苷和碱基摄取广泛。结论：FUN26是一种高亲和力、宽选择性的核苷和碱基转运蛋白。意义：相对于其他ENTs，FUN26具有独特的底物转运谱，并在洗涤剂溶解和纯化后保持活性。平衡核苷转运体(Ents)是一种多面体完整的膜蛋白，它能跨细胞膜运输核苷，并在较小程度上运输核碱基。ENTS以扩散控制的双向方式调节一系列人类疗法的疗效和功能。在分子水平上对ENT功能的详细了解仍然是难以捉摸的。FUN26(功能未知26)是酿酒酵母的ENT同源物，在液泡膜中表达。在目前的系统中，对纯化的FUN26的蛋白质脂质体研究表明，对于广泛的底物，核苷和核碱基摄取到管腔体积中的能力很强。这种转运活性对核糖上C(2)-和C(5)-位的核苷修饰很敏感，并且不受膜pH差的刺激。与未观察到[H-3]腺苷或[H-3]UTP转运的核苷相比，[H-3]腺嘌呤核苷酸碱基转运效率提高了近4倍。FUN26突变研究确定了破坏(G463A或G216A)或调节(F249I或L390A)转运蛋白功能的残基。这些结果表明，相对于已知的ENT家族成员，FUN26具有独特的底物转运谱，并且纯化的ENT可以在蛋白脂质体中重组，以在特定的系统中进行功能表征。
Background: FUN26 is a nucleoside transporter expressed in yeast vacuoles. Results: Proteoliposome studies of purified FUN26 reveal broad nucleoside and nucleobase uptake that is sensitive to C(2)-ribose modifications. Conclusion: FUN26 is a high affinity and broad selectivity nucleoside and nucleobase transporter. Significance: FUN26 has a unique substrate transport profile relative to other ENTs and retains activity following detergent solubilization and purification.Equilibrative nucleoside transporters (ENTs) are polytopic integral membrane proteins that transport nucleosides and, to a lesser extent, nucleobases across cell membranes. ENTs modulate efficacy for a range of human therapeutics and function in a diffusion-controlled bidirectional manner. A detailed understanding of ENT function at the molecular level has remained elusive. FUN26 (function unknown now 26) is a putative ENT homolog from S. cerevisiae that is expressed in vacuole membranes. In the present system, proteoliposome studies of purified FUN26 demonstrate robust nucleoside and nucleobase uptake into the luminal volume for a broad range of substrates. This transport activity is sensitive to nucleoside modifications in the C(2)- and C(5)-positions on the ribose sugar and is not stimulated by a membrane pH differential. [H-3]Adenine nucleobase transport efficiency is increased approximate to 4-fold relative to nucleosides tested with no observed [H-3]adenosine or [H-3]UTP transport. FUN26 mutational studies identified residues that disrupt (G463A or G216A) or modulate (F249I or L390A) transporter function. These results demonstrate that FUN26 has a unique substrate transport profile relative to known ENT family members and that a purified ENT can be reconstituted in proteoliposomes for functional characterization in a defined system.