Identifying molecular triggers for ammonium transporter trafficking to target membranes in root cells.

多细胞生物的进化增加了代谢物和底物在特化细胞及其细胞膜之间的转移和交换的需要，从而增加了载体运输系统的需要。尽管在真核生物中膜运输的许多方面可能是共享的，但高等植物的膜系统显示出不同的组织特征，可能需要适应性的膜运输专门化。根据营养供给和营养需求水平，营养转运蛋白在翻译后有效地控制蛋白质的数量和定位。尽管已经阐明了转运途径和参与外吞和内吞作用的几种蛋白质，但对于哪些细胞内信号触发这些过程，从而确定靶膜中存在的转运蛋白的实际数量，人们仍然知之甚少。该项目的总体目标是发现植物细胞中氮(N)营养状态信号级联中的新成分。我们的概念首先建立在氨转运蛋白（AMT）中肽结构域或氨基酸序列的存在上，这些结构域或氨基酸序列负责随着植物氮营养状况或外部氮有效性的变化而改变细胞膜内定位。随后，这些肽结构域将被用于寻找细胞内信号，这些信号改变了AMT蛋白在细胞内的定位，依赖于植物细胞的氮营养状况。为了实现这一目标，我们将在第一个工作包（WP）中可视化gfp标记的拟南芥AMTs （AMT1;1/AMT1；3和AMT1；2）响应N状态的膜重新定位，并在交换差异靶向AMTs的肽结构域以评估其N响应膜定位（WP 3）之前，通过独立的生化方法（WP 2）确认其膜定位。通过在酿酒酵母（Saccharomyces cerevisiae, delta mep1,2,3）和拟南芥四重AMT突变体qko （WP 4）中的异源表达，对修饰肽结构域的AMT进行功能分析。然后，我们将使用n反应性gfp标记的AMT来研究代谢触发因素对AMT运输的影响，最后将体内药物应用于转基因拟南芥系的根，以鉴定活细胞成像方法中对抑制剂敏感的n反应性膜运输过程（WP 5）。最后，将对所有工作包中获得的结果进行汇总，以描绘出AMT结构域如何控制蛋白质运输并影响底物运输特性，以响应氮缺乏或氮充足生长条件下的代谢线索。

The evolution of multicellular organisms increased the need for the transfer and exchange of metabolites and substrates across specialized cells and their cell membranes and thus for vectorial transport systems. Although many aspects of membrane trafficking are likely to be shared among eukaryotes, the endomembrane system of higher plants displays distinct organizational features that may entail adaptive specializations in membrane trafficking. Nutrient transporters underlie an effective posttranslational control of protein amount and localization in response to the level of nutrient supply and nutrient demand. Despite the elucidation of trafficking pathways and of several of the proteins being involved in exo- and endocytosis, it is still poorly understood which intracellular signals trigger these processes and thereby determine the actual amount of transport proteins present in a target membrane. The overall goal of the proposed project is to uncover new components in the signaling cascade of the nitrogen (N) nutritional status in a plant cell. Our concept builds first on the existence of peptide domains or amino acid sequences in ammonium transporter (AMT) proteins that are responsible for a change of the intracellular membrane localization upon changes in the N nutritional status of the plant or external N availability. Subsequently, these peptide domains will be employed to search for intracellular signals that alter the intracellular localization of AMT proteins in dependence of the N nutritional status of the plant cell. To reach this aim we will visualize in a first work package (WP) the membrane relocation of GFP-tagged Arabidopsis AMTs (AMT1;1/AMT1;3 and AMT1;2) in response to the N status and confirm their membrane localization in an independent biochemical approach (WP 2) before swapping peptide domains in differentially targeted AMTs to assess their N-responsive membrane localization (WP 3). AMTs with modified peptide domains will be functionally analyzed by heterologous expression in an ammonium transport-deficient strain of Saccharomyces cerevisiae (delta mep1,2,3) and the Arabidopsis quadruple AMT mutant qko (WP 4). We will then use N-responsive GFP-tagged AMTs to investigate the effect of metabolic triggers on AMT trafficking and finally apply pharmacological agents in vivo to roots of transgenic Arabidopsis lines to identify inhibitor-sensitive, N-responsive membrane trafficking processes in live cell imaging approaches (WP 5). Finally, results obtained in all work packages will be assembled to draw a picture on how AMT domains control protein trafficking and affect substrate transport properties in response to metabolic cues under N-deficient or N-replete growth conditions.