Inter-subunit interactions between Glutamate-Like Receptors in Arabidopsis

The plant Glutamate-Like Receptors (GLRs) are homologs of animal ionotropic glutamate receptors (iGluRs), and are hypothesized to be potential amino acid sensors in plants. Genetic studies of proteins from this family implicate individual GLRs in a diversity of physiological roles in plants. Recently, amino-acid gated channel activities have been proven for a few plant GLRs, suggesting that at least some of the functional mechanisms are conserved between plant GLRs and animal iGluRs. Animal iGluRs generally form heterotetramers, and the ligand-binding specificity and channel functionality is determined by interaction between the subunits. In order to investigate whether plant GLRs interact with each other, a modified yeast-2-hybrid system (mbSUS) approach was taken on 15 of the 20 Arabidopsis GLRs to identify potential interaction partners. Using this approach, we have successfully identified GLR subunits that are capable of interacting with multiple other GLRs. Unlike iGluRs, sequence similarity between the subunit was not correlated with the likelihood of interaction among 2 given subunits. Interactions between selected GLRs (GLR1.1, 2.9, 3.2, and 3.4) were further tested in another heterologous expression system, mammalian HEK293 cells, using Förster resonance energy transfer (FRET). Two separate approaches (sensitized FRET and acceptor photobleaching) indicated that GLRs 1.1 and 3.4 are capable of forming homomers, whereas other combinations did not result in detectable FRET between the subunits.     

Genetically encoded sensors for metabolites.

BACKGROUND: Metabolomics, i.e., the multiparallel analysis of metabolite changes occurring in a cell or an organism, has become feasible with the development of highly efficient mass spectroscopic technologies. Functional genomics as a standard tool helped to identify the function of many of the genes that encode important transporters and metabolic enzymes over the past few years. Advanced expression systems and analysis technologies made it possible to study the biochemical properties of the corresponding proteins in great detail. We begin to understand the biological functions of the gene products by systematic analysis of mutants using systematic PTGS/RNAi, knockout and TILLING approaches. However, one crucial set of data especially relevant in the case of multicellular organisms is lacking: the knowledge of the spatial and temporal profiles of metabolite levels at cellular and subcellular levels. METHODS: We therefore developed genetically encoded nanosensors for several metabolites to provide a basic set of tools for the determination of cytosolic and subcellular metabolite levels in real time by using fluorescence microscopy. RESULTS: Prototypes of these sensors were successfully used in vitro and also in vivo, i.e., to measure sugar levels in fungal and animal cells. CONCLUSIONS: One of the future goals will be to expand the set of sensors to a wider spectrum of substrates by using the natural spectrum of periplasmic binding proteins from bacteria and by computational design of proteins with altered binding pockets in conjunction with mutagenesis. This toolbox can then be applied for four-dimensional imaging of cells and tissues to elucidate the spatial and temporal distribution of metabolites as a discovery tool in functional genomics, as a tool for high-throughput, high-content screening for drugs, to test metabolic models, and to analyze the interplay of cells in a tissue or organ.