Energy transport in cells. Physiological functions and structural aspects of nucleotide transport proteins
Plant Biochemistry and Biophysics
Final Report Abstract
During the founding period we were able to identify and to uncover the biochemical and physiologic function of diverse plant transport proteins belonging to the mitochondrial carrier family: the plastid and mitochondrial NAD+ transporters AtNDT1 and AtNDT2, the first plasma membrane located ATP transporter PM-ANT1 and a mitochondrial Ca2+ regulated ATP/phosphate transporter. Additionally, we have very valuable hints that a close homolog to the Brittle1 protein (catalyzes export on adenylates out of plastids), namely AttZT1 is responsible for intracellular transport of the phytohormone t-Zeatin. This will represent a breakthrough in (plant) cell biology. Impaired provision of ATP, respectively energy, to the plants ER results in a dramatic phenotype of er-ant1 mutant plants. A detailed investigation of these plant lines showed for the first time a close connection between proper ER function and photorespiration. Most likely, disturbed cellular redox biology in er-ant1 lines is causative for impaired activity of the mitochondrial enzyme GDC. Several independent EMS bases er-ant1 suppressor lines (with low or (still) high glycine levels, but no inhibition of grown under low CO2) are very promising candidates to decipher novel components connecting ER metabolism to photorespiration. . Novel EMS based er-ant1 suppressor mutants provide an excellent basis for further work clarifying the connection between ER metabolism and photorespiration. We see no other way to analyze this interaction than exploiting the power of a forward-genetic approach. Further insights into the physiological relevance of nocturnal ATP provision to chloroplasts plastids were obtained. Limited provision with energy not only impairs primary nocturnal metabolism, also hormonal crosstalk, prime defense and disease resistance is affected. We moreover, substantially expended the current knowledge about the distribution and physiological role of NTT-type nucleotide carries in the plant kingdom by analyzing corresponding transporters from algae, including those with complex plastids. At the beginning of our project we speculated that PHT4,6 might be a Golgi located nucleotide carrier. However, detailed analyses showed that PHT4,6 acts as a Pi exporter involved in shuttling inorganic phosphate back into the cytosol. Impaired subcellular phosphate homeostasis provoked by the absence of PHT4,6 in the Golgi apparatus severely affects the plant overall vitality. Novel EMS based pht4,6 suppressor mutants provide an excellent basis for further work clarifying the connection between a Golgi located Pi carrier and vacuolar Pi levels, which are affected in pht4,6 plants. Last but not least I have to mention that we established a “cornucopia” of new methods in the lab like e.g. a FPLC based protein purification and “polishing”, a protein crystallization platform (including a pipetting robot), EMS based mutagenesis of Arabidopsis mutants, primary metabolite/ion profiling, up to date cloning methodology etc. This won’t have been possible without the Reinhart Koselleck grant. Especially due to starting the crystallization platform we made significant progress in our attempts to crystallize PamNTT1 protein. However, the final aim to solve the X-ray structure of these proteins is ongoing work. Nevertheless, we are very confident to finish this work in a conceivable time period. I have to admit that I was unable to reach two of my aims: Firstly, I was not successful in crystallizing PamNTT1 at a quality sufficient to solve the transporter structure with help of X-ray analysis. Secondly, my efforts to identify the “real” ER located ATP carrier failed. Although I identified with ER-ANT1 and tZT1 two ER located ATP/ADP transporters, none of these carriers represent the “real” ER located ATP importer. This conclusion is based on the observations that er-ant1 mutants develop nicely under high CO2 (absence of photorespiration) and tZT1 mutants do not show a dramatic phenotypic appearance under ambient growth. Maybe this failure is also a consequence of the high risk nature of Reinhart Koselleck grants.
Publications
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2009. Molecular identification and functional characterization of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins. J. Biol. Chem. 284: 31249-31259
Palmieri F, Rieder B, Ventrella A, Blanco E, Do PT, Nunes-Nesi A, Trauth AU, Fiermonte G, Tjaden J, Agrimi G, Kirchberger S, Paradies E, Fernie AR, Neuhaus HE
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2010. Limitation of nocturnal ATP import into chloroplasts seems to affect hormonal crosstalk, prime defense, and enhance disease resistance in Arabidopsis thaliana. Mol.Plant Microbe Interact. 23: 1584-1591
Schmitz G, Reinhold T, Gobel C, Feussner I, Neuhaus HE, Conrath U
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2011. Adenine nucleotide transport in plants: much more than a mitochondrial issue. Trends Plant Sci. 16: 507-515
Haferkamp I, Fernie AR, Neuhaus HE
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2011. Identification of an Arabidopsis plasma membrane located ATP transporter important for anther development. Plant Cell 23: 1932-1944
Rieder B, Neuhaus HE
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2012. Lack of the Golgi phosphate transporter PHT4;6 causes strong developmental defects, constitutively activated disease resistance mechanisms and altered intracellular phosphate compartmentation in Arabidopsis. Plant J. 72: 732-744
Hassler S, Lemke L, Jung B, Möhlmann T, Krüger F, Schumacher K, Espen L, Martinoia E, Neuhaus HE
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2013. From endoplasmic reticulum to mitochondria: absence of the Arabidopsis ATP antiporter Endoplasmic Reticulum Adenylate Transporter1 perturbs photorespiration. Plant Cell 25: 2647-2660
Hoffmann C, Plocharski B, Haferkamp I, Leroch M, Ewald R, Bauwe H, Riemer J, Herrmann JM, Neuhaus HE
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2014. Nucleotides and nucleosides: transport, metabolism and signaling function of extracellular ATP. In: Lüttge U, Beyschlag W, Cushman JC, eds. Prog. Bot. 75. Heidelberg, Germany: Springer, 119-144
Möhlmann T, Steinebrunner I, Neuhaus HE
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2015. In vitro analyses of mitochondrial ATP/phosphate carriers from Arabidopsis thaliana revealed unexpected Ca2+-effects. BMC Plant Biol. 15: 238
Lorenz A, Lorenz M, Vothknecht UC, Niopek-Witz S, Neuhaus HE, Haferkamp I
