A novel fluorescent assay for sucrose transporters

When type I sucrose transporters StSUT1 from potato or AtSUC2 from Arabidopsis were expressed in yeast, the cells were able to take up esculin and became brightly fluorescent. We tested a variety of incubation times, esculin concentrations, and buffer pH values and found that for these transporters, a 1 hr incubation at 0.1 to 1 mM esculin at pH 4.0 produced fluorescent cells that were easily distinguished from vector controls. Esculin uptake was assayed by several methods including fluorescence microscopy, spectrofluorometry and fluorescence-activiated cell sorting (FACS). Expression of the type II sucrose transporter OsSUT1 from rice did not result in increased esculin uptake under any conditions tested. Results were reproduced successfully in two distinct yeast strains, SEY6210 (an invertase mutant) and BY4742.The esculin uptake assay is rapid and sensitive and should be generally useful for preliminary tests of sucrose transporter function by heterologous expression in yeast. This assay is also suitable for selection of yeast showing esculin uptake activity using FACS.Sucrose transporters (SUTs or SUCs) play a critical role in long distance transport of carbohydrates in plants. Products of photosynthesis must have an efficient means of being distributed to cells in the plant that depend on the net import of fixed carbon such as in roots, flowers, and seeds. In many plants, this is achieved by active loading of the phloem using H+-coupled sucrose transporters [1]. The first sucrose uptake transporter (SUT) was cloned from spinach by expression in the yeast strain SuSy7 [2]. SuSy7 is an invertase mutant that expresses plant sucrose synthase in the cytoplasm. Growth of SuSy7 on sucrose depends on expression of a sucrose uptake transporter. Growth assays using SuSy7 have been subsequently used to demonstrate sucrose transport activity of many cloned SUT homologs such as AtSUT4 [3], OsSUT1 and OsSUT3 [4], TaSUT1 [5], and VvSUC27 [6]. The SuSy7 growth assay is rapid a