75 Integrating Membrane Transport with Male Gametophyte ... - TAIR

323 Fluorescent amino acid sensors report amino acid dynamics in living cells
Martin Bogner, Uwe Ludewig
Zentrum fuer Molekularbiologie der Pflanzen (ZMBP), Pflanzenphysiologie, Universitaet Tuebingen
All organisms require sufficient cytosolic amino acid levels to maintain protein synthesis at adequate rate. In addition
to their function as building blocks of proteins, amino acids also serve other functions in plants, e.g as nitrogen storage
compounds or osmotic protectants. The cytosolic amino acid concentrations differ between organs and are subject
to metabolic changes and compartmentation. In order to measure amino acid changes with subcellular specificity in
plants, fluorescent proteins have been designed and constructed that respond specifically to changes in the amino acid
concentrations. These sensors are based on the bacterial protein QBP from E.coli, which is known to commit large
conformational changes upon the high-affinity binding of its substrate, glutamine. We attached two different green
fluorescent protein variants to QBP, in a way each is conceted to a different hemisphere. Fluorescence resonance energy
transfer (FRET) between the attached chromophores was observed. In the purified recombinant protein a change in
fluorescence was observed after addition of argenine. Affinity for glutamine could be partially restored by mutations in
the binding pocket. This goes well with the computer model which indicates changes in the morphology of the binding
pocket, because of the insertion of one GFP varinat into the linear sequence of QBP. Measurements with all proteinogenic
amino acids indicated selective FRET changes for different constructs. The sensors were expressed in E.coli, in yeast and
in plants. Fluorescence changes were observed upon addition of amino acids, in accordance with amino acid changes in
the cytoplasm. Fluorescent amino acid sensors appear to be a versatile tool to study the in vivo dynamics of metabolism
and compartmentation, especially in large-scale genomic approaches and upon environmental changes.
324 Identification and Characterization of a Novel Plastid Envelope Protein
Andrea Braeutigam 1 , Susanne Hoffmann-Benning 2 , Andreas Weber 3
1
Michigan State University, Genetics Program, 2 Michigan State University, Department of Biochemistry and
Molecular Biology, 3 Michigan State University, Department of Plant Biology
C4 plants such as maize use a carbon concentration mechanism which renders their carbon fixation more efficient
compared to C3 plants. This carbon concentration mechanism requires spatial separation and compartimentation of
several enzymatic reactions and thus requires immense metabolite fluxes across the plastid envelope. We hypothesized
that we can identify candidates for the transport proteins that catalyze those fluxes by analyzing the proteome of the
plastid envelope of maize plastids. Specifically we hypothesize that we can identify candidate genes for the transport of
oxaloacetate, malate and pyruvate.
We analyzed the mesophyll plastid envelope by proteomics and identified more than 30 proteins with high confidence.
Within the highly hydrophobic protein fraction a band is visible in SDS gels which is absent from the envelopes of C3
plants and we identified a protein with unknown function from this band. This protein is present throughout the plant
kingdom including red algae, green algae and land plants but notably absent from cyanobacteria.
To understand the in planta function of this novel protein we identified a null allele in Arabidopsis thaliana. A detailed
analysis of the phenotype will be presented.