The transport efficiency (TE) describes the performance of a transport protein for a specific substrate. To compare the TE of different transporters, the number of active transporters in the plasma membrane must be monitored, as it may vary for each transporter and experiment. Available methods, like LC–MS quantification of tryptic peptides, fail to discriminate inactive intracellular transporters or, like cell-surface biotinylation followed by affinity chromatography and Western blotting, are imprecise and very laborious. We wanted to normalize active transporters by the activity of a second transporter. A transporter tandem, generated by joining two transporter cDNAs into a single open reading frame, should guarantee a 1 : 1 stoichiometry. Here we created a series of tandems with different linkers between the human ergothioneine (ET) transporter ETT (gene symbol SLC22A4) and organic cation transporter OCT2 (SLC22A2). The linker sequence strongly affected the expression strength. The stoichiometry was validated by absolute peptide quantification and untargeted peptide analysis. Compared with wild-type ETT, the normalized ET clearance of the natural variant L503F was higher (f = 1.34); G462E was completely inactive. The general usefulness of the tandem strategy was demonstrated by linking several transporters with ETT; every construct was active in both parts. Transporter tandems can be used - without membrane isolation or protein quantification — as precise tools for transporter number normalization, to identify, for example, relevant transporters for a drug. It is necessary, however, to find suitable linkers, to check the order of transporters, and to verify the absence of functional interference by saturation kinetics.

中文翻译：

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转运蛋白双链膜：用于标准化质膜中主动转运蛋白的精确工具

转运效率（TE）描述了转运蛋白对特定底物的性能。为了比较不同转运蛋白的TE，必须监测质膜中活性转运蛋白的数量，因为每种转运蛋白和实验的活跃转运蛋白数量可能会有所不同。可用的方法，例如LC-MS定量分析胰蛋白酶肽，不能区分无活性的细胞内转运蛋白，或者像细胞表面生物素化然后进行亲和色谱和Western印迹一样，方法不精确且费力。我们希望通过第二个转运蛋白的活动来规范活跃转运蛋白。通过将两个转运蛋白cDNA连接到一个开放阅读框中而产生的转运蛋白串联，应保证化学计量比为1：1。在这里，我们创建了一系列在人类麦角硫氨酸（ET）转运蛋白ETT（基因符号SLC22A4）和有机阳离子转运蛋白OCT2（SLC22A2）之间具有不同接头的双链烷。接头序列强烈影响表达强度。通过绝对肽定量和非靶向肽分析验证了化学计量。与野生型ETT相比，自然变体L503F的归一化ET清除率更高（f = 1.34）；G462E完全不活动。通过将多个转运蛋白与ETT相连，证明了串联策略的一般用途。每个构造在这两个部分都活跃。转运蛋白的膜可以用作转运蛋白数量归一化的精确工具，而无需进行膜分离或蛋白质定量分析，例如识别药物的相关转运蛋白。有必要，