Measuring Sucrose Transporter Activities Using a Protoplast-Esculin Assay
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Sucrose transport across membranes requires the activity of transport proteins. Sucrose-specific SWEET proteins mediate sugar efflux out of the cytosol and SUC proteins catalyze the uptake of sucrose from the apoplast. Both transport processes are involved in phloem loading in source leaves as well as in the post-phloem pathway in sink tissues. An important step during the characterization of new sucrose transporters is to analyze their transport activity. This is usually achieved by heterologous expression of the respective gene in yeast cells or Xenopus oocytes and subsequent uptake measurements. However, in some cases, mistargeting to internal membranes or the lack of protein modifications and/or interaction partners in the heterologous system can interfere with uptake analyses. Therefore, a new in planta method was developed that is based on mesophyll protoplasts as expression system and the fluorescent sucrose analog esculin to monitor uptake activities by confocal microscopy. In this chapter we describe the design of constructs required to analyze sucrose transporters in protoplasts, the experimental setup of the protoplast-esculin assay, and the quantitative evaluation of the obtained data. The quantification of esculin uptake allows the application of the new assay to a variety of questions, e.g., by comparison of point mutants, splice variants, or transporters with and without interaction partners.
Key wordsSucrose Sugar transport proteins Uptake measurement Esculin Protoplasts Confocal microscopy Phloem
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