| Design and Functional Analysis of Fluorescent Nitrate and Peptide Transporter Activity Sensors in Yeast Cultures
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Author:
Date:
2016-02-05
[Abstract] This protocol describes the methods used to engineer and deploy genetically encoded fluorescence activity reporters for nitrate and peptide transporter activity in yeast cells. Fusion of the dual-affinity nitrate transceptor CHL1/AtNRT1.1/AtNPF6.3 or four different peptide transporters (AtPTR1, 2, 4, and 5) from Arabidopsis to a pair of fluorescent proteins with different spectral properties, enabled us to engineer the NiTracs (nitrate transporter activity tracking sensors) and the PepTracs (peptide transporter activity tracking sensors), ratiometric fluorescence activity sensors that monitor the activity of the plasma membrane nitrate transceptor or the peptide transporters in vivo (Ho et al., 2014). The NiTrac1 sensor responds specifically and reversibly to ...
[摘要] 该协议描述了用于在酵母细胞中设计和部署遗传编码的荧光活性报告人硝酸盐和肽转运蛋白活性的方法。将双亲硝酸盐转运体CHL1 / AtNRT1.1 / AtNPF6.3或四种不同的肽转运蛋白（AtPTR1,2,4和5）从拟南芥融合成具有不同光谱性质的一对荧光蛋白，使我们能够设计NiTracs（硝酸盐转运蛋白活性跟踪传感器）和PepTracs（肽转运蛋白活性跟踪传感器），比例式荧光活性传感器，监测体外硝酸盐转运体或肽转运蛋白的活性（Ho et al。，2014）。 NiTrac1传感器特异性和可逆地响应于硝酸盐的添加，而PepTracs通过减少供体和受体发射来响应添加二肽，而受体激发的发射保持不变，或荧光团发射比例的变化。所有传感器都适用于比例成像。 NiTrac1传感器响应[从μM到mM（Liu和Tsay，2003）]和硝酸盐转运动力学的两相动力学的相似性暗示了NiTrac1在运输循环期间提供了关于构象重排的信息，从而报告在广泛的外部硝酸盐浓度范围内的转运蛋白活性。 NiTrac的几种变体已被设计，它们对硝酸盐（NiTrac1：CHL1; NiTracT101A：CHL1T101A）的亲和力不同。 NiTrac还能识别氯酸盐。在这里，我们描述了使用分光荧光计设计，实施和检测酵母细胞中硝酸盐受体活性的简单方法。
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| Quantification of Extracellular Ammonium Concentrations and Transporter Activity in Yeast Using AmTrac Fluorescent Sensors
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Author:
Date:
2015-01-05
[Abstract] AmTracs are the first example of “activity sensors”, since they report the activity of ammonium transporters by means of fluorescence readout in vivo (De Michele et al., 2013). AmTracs are based on a single fluorescent protein, a circularly permuted GFP (cpGFP), inserted into the cytosolic loop connecting the two pseudosymmetrical halves of the Arabidopsis and yeast plasma membrane ammonium transporters AtAMTs and ScMEP (Figure 1). Recently, FRET-based activity sensors for nitrate and peptide transporters have also been developed (Ho et al., 2014). Since transporter activity directly depends on the availability of substrate, AmTracs measure extracellular ammonium concentrations. Several versions of AmTrac exist, with different fluorescence intensity ...
[摘要] AmTracs是“活性传感器”的第一个例子，因为它们通过体内荧光读数来报告铵转运蛋白的活性（De Michele et al。，2013）。 AmTracs基于单个荧光蛋白，循环置换的GFP（cpGFP），插入连接拟南芥和酵母质膜铵转运蛋白AtAMTs和ScMEP的两个伪对称半部的细胞溶质环（图1）。最近，还开发了基于FRET的硝酸盐和肽转运蛋白活性传感器（Ho et al。，2014）。由于转运蛋白活性直接取决于底物的可用性，因此AmTracs测量细胞外铵浓度。存在几种版本的AmTrac，具有不同的荧光强度（FI）响应和铵的亲和力，并且基于不同的铵转运蛋白（AmTrac：AtAMT1; 3; AmTrac1; 2：At AMT1; 2; MepTrac：ScMEP2）。目前，最有用的AmTrac版本可能是AmTrac-GS（明亮，Km为50μM）和AmTrac-100（高容量版本，Km为100μM）。用于测量荧光计中酵母细胞中铵浓度的方案对于所有版本都是相同的。
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