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

点击翻译按钮获取中文摘要

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.