Structure and transport cycle of H+/sucrose symporter ZmSUT1
Zusammenfassung der Projektergebnisse
Proton-coupled sucrose transporters play a pivotal role for cell to cell and long distance transport in plants. Utilizing the hyperpolarized membrane potential and the transmembrane proton gradient, they enable phloem cells to accumulate sucrose to concentrations up to ~1 M in the cytosol, which is about 1000-fold higher than in the apoplast. This fact suggests that plants developed transporters with unique structural and functional properties. The carrier protein can accomplish this task only because proton and sucrose transport are tightly coupled. Molecular insights into the process of sugar translocation via H+-coupled sugar carriers in plants are, however, still fragmentary. During the funding period our knowledge about the transport mechanism of plant sucrose transporters very well advanced. We used the maize sucrose transporter ZmSUT1 as a model transporter and Xenopus oocytes as a versatile heterologous expression system. The two-electrode voltage clamp technique combined with sophisticated fluorescence-based applications allowed us to dissect the transport cycle of ZmSUT1 which in turn deepened our understanding about the transport mechanism of plant sucrose transporters. Moreover, sitedirected mutant analysis based on a 3D model of ZmSUT1 elucidated the molecular grounds of sucrose binding to the ZmSUT1 protein. Detailed studies on the transport cycle and structure-function research resulted in the following major outcomes of this project: • Binding of protons to the transporter were characterized by pre-steady state currents • Binding of protons and sucrose to the outward-directed transporter appeared random • Sucralose represents a competitive inhibitor of ZmSUT1 • Voltage clamp fluorometry measurements visualized the conformational changes of ZmSUT1 • Sucralose inhibits the conformational change locking the transporter in its outwardfacing conformation • The movement between the outward and inward conformation represents the ratelimiting step in the reaction cycle of ZmSUT1 • ZmSUT1 changes its access from the cytosolic to the extracellular site with a rate of ~500 s^-1 • Two amino acids on TMD I and two on TMD IV were identified as putative sucrose binding residues. Mutations markedly altered the affinity of ZmSUT1 to its substrate • The position of the putative sucrose binding sites very well coincides with the developed 3D-model ofZmSUT1 • Comparison of the major sucrose transporting proteins (ZmSUT1 and UmSrt1) within the Ustilago maydis/Zea mays patho-system revealed major differences of the electrical properties between these competitors for apoplastic sucrose • The knowledge and techniques gained with the characterization of ZmSUT1 was successfully transferred to a new class of NRT/PTR transporter and was key for the identification and in detail characterization of glucosinulate carriers in Arabidopsis. Within the funding period of this project we developed techniques for studying cation-coupled sugar carriers and acquired new knowledge about the reaction cycle of plant sucrose transporters. Especially the dissection of the reaction cycle into its individual steps which can now be studied separately will help us in the future to elucidate structure-function relations of plant sugar transporters. Thus, we are now well equipped to elucidate the impact of mutantions on sugar-binding, conformational changes, H+ translocation and the coupling of proton and sugar transport. These topics will be major objectives of our subsequent work. Brokkoli & Co.: Senföle als chemische Keule. 2012 (http://www.uniwuerzburg.de/sonstiges/meldungen/single/artikel/brokkoli/) Pflanzen: Nano-Maschinen lassen Zuckersaft fließen. 2010 (http://www.uniwuerzburg.de/sonstiges/meldungen/single/artikel/pflanzen/)
Projektbezogene Publikationen (Auswahl)
- (2010). Sucrose- and H+-Dependent charge movements associated with the gating of sucrose transporter ZmSUT1. PLoS ONE 5, 1-10
Carpaneto, A., Koepsell, H., Bamberg, E., Hedrich, R., and Geiger, D.
- (2011). A member of the mitogen-activated protein 3-kinase family is involved in the regulation of plant vacuolar glucose uptake. Plant Journal 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.
- (2011). Plant sucrose transporters from a biophysical point of view. Molecular Plant 4, 395-406
Geiger, D.
- (2012). A substrate binding hinge domain is critical for transportrelated structural changes of organic cation transporter 1. Journal of Biological Chemistry 287, 31561-31573
Egenberger, B., Gorboulev, V., Keller, T., Gorbunov, D., Gottlieb, N., Geiger, D., Mueller, T.D., and Koepsell, H.
(Siehe online unter https://doi.org/10.1074/jbc.M112.388793) - (2012). NRT/PTR transporters are essential for translocation of glucosinolate defence compounds to seeds. Nature 488, 531-534
Nour-Eldin, H.H., Andersen, T.G., Burow, M., Madsen, S.R., Jørgensen, M.E., Olsen, C.E., Dreyer, I., Hedrich, R., Geiger, D., and Halkier, B.A.
(Siehe online unter https://doi.org/10.1038/nature11285) - (2013). Conformational changes represent the rate-limiting step in the transport cycle of maize sucrose transporter1. Plant Cell 25, 3010-3021
Derrer, C., Wittek, A., Bamberg, E., Carpaneto, A., Dreyer, I., and Geiger, D.
(Siehe online unter https://doi.org/10.1105/tpc.113.113621)
