MCB-9420599 Boorer The cloned H+/hexose transporters from Arabidopsis (STP), H+/sucrose transporters from spinach (SoSUC1) and potato (StSUC1) and a H+/amino acid transporter from Arabidopsis (NAT2) will be expressed in Xenopus oocytes. Their transport kinetics will be investigated using the 2- electrode voltage-clamp and the cut-open oocyte vaseline gap methods to determine the external and internal transport reaction steps of the transporters. The dependence of maximal rate of transport, imax, and the apparent affinity K0.5 for ligands on voltage and the concentration of ligands will be determined. Measurement of reversal potentials and the simultaneous measurement of the flux of labelled substrates and H+ currents will be used to determine the stoichiometry of transport. Analysis of the presteady- state kinetics will provide unique insights in both partial reaction steps of the transport reaction cycle and the number of transporters in the membrane. This kinetic information will be used to formulate and examine transport models to describe the kinetic properties of the sugar and amino acid transporters. Mutant transporters which have been tested for function by complementing yeast amino acid transport mutants will be expressed in oocytes and a detailed kinetic characterization of the transporters will be undertaken. This will allow transport models to be tested. For SoSUC1 and STSUC1 specific interactions between transported substrates and inhibitors of transport will be investigated and the optimal 3D structure for recognition by the transporter determined by energy minimization and molecular model building. In the long term, a concensus structure for transported analogues may be used to target the sucrose transporter in the design of novel herbicides and pesticides. Also, the mutants transtporters will be used to alter the partitioning of carbon and nitrogen in transgenic plants which will, in turn, lead to the introduction of favorable agron omic traits into crop plants. %%% The goal of this study is to understand the molecular mechanisms underlying the transport of amino acids and sugars into and out of plant cells. Amino acids and sugars are essential nutrients and are transported into plant cells via H+-coupled cotransporters which are integral membrane protiens that couple the uptake of substrates to the electrochemical gradient for H+ generated by the H+- pumping ATPase. These transporters play a vital role in the partitioning of assimilates between source and sink tissues which is a major determinant of crop yield. The approach will be to express the cloned H+-dependent hexose, sucrose and amino acid transporters in Xenopus oocytes and to use electrophysiological and radiotracer flux techniques to characterize the biophysical properties and kinetics (ligand specificity, ligand stoichiometry, voltage-dependence, reversiblity) of these cotransporters. These studies will be combined with molecular biological techniques to characterize mutant transporters which will allow the identification of specific amino acid residues involved in H+ and substrate-binding and transport mechanisms. ***
