Amino Acid Positions Important For Substrate Specificity in Plant Sucrose Transporters

Abstract

Plant sucrose transporters (SUTs) are members of the GPH family within the Major Facilitator Superfamily (MFS). They are proton‐coupled symporters in the plasma membrane or vacuole (lysosome) membrane and function in sucrose uptake into the cytoplasm. SUTs function throughout the plant but are especially important for sucrose uptake into vascular tissue. Plants contain small gene families of SUTs that can be classified into three basic types (I, II, III) both by their amino acid sequence and by their substrate specificity. Type I SUTs are less selective and transport a range of beta glucosides, including the fluorescent coumarin glucosides esculin and fraxin, in addition to sucrose. Type II SUTs are more selective for sucrose and do not transport esculin or fraxin. To identify amino acid positions that control substrate specificity, we used gene shuffling between a Type II SUT OsSUT1 and a non‐functional version of OsSUT1 in which 64 positions had been mutated to the corresponding positions in the Type I SUT StSUT1. Gene shuffling libraries were expressed in yeast and assayed for their ability to confer uptake of esculin using fluorescence activated cell sorting (FACS). Mutation of five amino acids in OsSUT1 resulted in esculin transport. The amino acid changes clustered in two areas of the OsSUT1 protein: in the first loop and the top of TMS2 (T80L and A86K) and in TMS5 (S220A, S221A, and T224Y). The substrate specificity of this OsSUT1 variant was analyzed by expression in Xenopus oocytes and electrophysiology and was almost identical to that of type I SUTs. We are now investigating the role of these positions in more detail.