Molecular mechanism of electrogenic carrier- and pump-mediated vacuolar transport
Final Report Abstract
Within the DFG research group FOR1061, we elucidated the molecular mechanisms of solute transport across the vacuolar membrane mediated by the two proton pumps V-ATPase and V-PPases, and several electrogenic sugar transporters (AtSUC4, AtERDL6, AtTMTs, BvTSTs). With respect to the V-PPase and the proton-coupled sugar transporters, our patch-clamp studies were initially strongly hindered by limitations in the current resolution, the result of low transport rates and/or transporter densities. By attempting different strategies, we overcame these limitations and greatly improved the current resolution, primarily by increasing the size of the relevant transporter population, and where possible, also decreasing the background conductance. For this, the transporter in question was transiently or stably overexpressed in mesophyll cells, and eventually combined with loss-of-function mutants of the interfering background transporter activities and/or of the transporter of interest itself. By applying the whole-vacuole patch clamp configuration to mesophyll vacuoles under certain experimental conditions (electrochemical gradients, substrate concentrations, regulators), we revealed the following transport features: V-ATPase is characterized by a complex pH regulation where the proton concentration at both sides of the vacuolar membrane feeds back on proton pump capacity and ATP consumption. According to our mathematical model, efficient V-ATPase function requires greater dissociation of transported protons from the pump protein when the pH increases. This feature results in an optimization of proton pumping by the V-ATPase to the existing proton concentrations. We further demonstrated that Cys256 within the VHA-A subunit of the peripheral ATP-hydrolyzing V1 complex, but not Cys535, is involved in oxidative inhibition of the V-ATPase activity. V-PPase. Two pyrosphosphatases from Nicotiana benthamiana were identified: NbPPase1.1 and NbPPase1.2, and both show a high identity in their primary structure with AtAVP1 and other V-PPases. NbPPase1.1 and AtAVP1 exhibit a similar affinity for PPi binding, which was independent from luminal pH, and where PPi consumption is well in the range of cellular PPi levels. Under salinity, a rise in the V-PPase proton pump activity in N. benthamiana mesophyll vacuoles was determined. This increase compensated for the reduction in V-ATPase-dependent proton pumping. However, extensive overloading with V-PPases caused cell death under normal growth conditions, but it was rescued by salinity treatment. This effect was related to the rise in proton pumping rather than to any changes in the cellular PPi homoeostasis. Thus, V-PPase hyperactivity can have a negative feedback effect on cellular viability when there is no increased demand for a proton motif force to energize transport processes. Accordingly, the plant needs to adapt the V-PPase proton pump activity to the cellular demands under the prevailing environmental conditions. AtTMT1/2 represents the first proton-coupled sugar antiporter capable of high capacity glucose and sucrose loading into the vacuole. BvTST2.1 was identified as a sucrose-specific sugar beet transporter that mediates the loading of vacuoles with sucrose in a proton-driven antiport mechanism. With respect to its high expression in taproots, BvTST2.1 is responsible for sucrose accumulation in this storage organ. AtSUC4 operates as a proton-coupled sucrose symporter for sugar release from the vacuole. AtERDL6 mediates glucose release across the vacuolar membrane in symport with protons.
Publications
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(2010) Arabidopsis V-ATPase activity at the tonoplast is required for efficient nutrient storage but not for sodium accumulation. Proc. Natl. Acad. Sci. USA 107: 3251-3256
Krebs,M., Beyhl,D., Görlich,E., Al-Rasheid,K.A., Marten,I., Stierhof,Y.D., Hedrich,R., and Schumacher,K.
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(2010) Increased activity of the vacuolar monosaccharide transporter TMT1 alters cellular sugar partitioning, sugar signalling and seed yield in Arabidopsis. Plant Physiol. 154: 665-677
Wingenter,K., Schulz,A., Wormit,A., Wic,S., Trentmann,O., Hoermiller,I.I., Heyer,A.G., Marten,I., Hedrich,R. and Neuhaus,H.E.
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(2011) A member of the mitogen-activated proteinactivated protein 3-kinase is involved in the regulation of plant vacuolar glucose uptake. Plant J. 68: 890-900
Wingenter,K., Trentmann,O., Winschuh,I., Hörmiller,I.I., Heyer,A.G., Reinders,J., Schulz,A., Geiger,D., Hedrich,R., and Neuhaus,H.E.
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(2011) Proton-driven sucrose symport and antiport is provided by the vacuolar transporters SUC4 and TMT1/2. Plant J. 68: 129-136
Schulz,A., Beyhl,D., Marten,I., Wormit,A., Neuhaus,E., Poschet,G., Büttner,M., Schneider,S., Sauer,N. and Hedrich,R.
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(2012) Luminal and cytosolic pH feedback on proton pump activity and ATP affinity of the V-type ATPase from Arabidopsis. J. Biol. Chem. 287: 8986-8993
Rienmüller,F., Dreyer,I., Schönknecht,G., Schulz,A., Schumacher,K., Nagy,R., Martinoia,E., Marten,I., and Hedrich,R.
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(2012) Regulation of the V-type ATPase by redox modulation. Biochem. J. 448: 243-251
Seidel,T., Scholl,S., Krebs,M., Rienmüller,F., Marten,I., Hedrich,R., Hanitzsch,M., Janetzki,P., Dietz, K.J., and Schumacher K.
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(2012) Vacuoles release sucrose via tonoplast-localised SUC4-type transporters. Plant Biol. 14: 325-336
Schneider,S., Hulpke,S., Schulz,A., Yaron,I., Höll,J., Imlau,A., Schmitt,B., Batz,S., Wolf,S., Hedrich R., and Sauer,N.
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(2014) Overexpression of a proton-coupled vacuolar glucose exporter impairs freezing tolerance and seed germination. New Phytol. 202: 188-197
Klemens,P.A.W., Patzke,K., Trentmann,O., Poschet,G., Büttner,M., Schulz,A., Marten,I., Hedrich,R., and Neuhaus,H.E.
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(2015) Identification and molecular characterization of the sucrose loader in sugar beet taproots. Nature Plants 14001
Jung,B., Ludewig,F., Schulz,A., Meißner,G., Wöstefeld,N., Flügge,U.I., Pommerrenig,B., Wirsching,P., Sauer,N., Koch,W., Sommer,F., Mühlhaus,T., Schroda,M., Cuin,T.A., Graus,D., Marten,I., Hedrich,R., and Neuhaus,H.E.
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(2015) Sugar transport across the plant vacuolar membrane: nature and regulation of carrier proteins. Curr. Opin. Plant Biol. 25: 63-70
Hedrich,R., Sauer,N., and Neuhaus,H.E.
