The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates while differing in subcellular localization.

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family has approximately 20 homologs in most plant genomes, and despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize different substrates. Previous work using SweetTrac1, a biosensor constructed by the intramolecular fusion of a conformation-sensitive fluorescent protein in the plasma membrane transporter SWEET1 from Arabidopsis thaliana, identified common features in the transporter's substrates. Here, we report SweetTrac2, a new biosensor based on the Arabidopsis vacuole membrane transporter SWEET2, and use it to explore the substrate specificity of this second protein. Our results show that SWEET1 and SWEET2 recognize similar substrates but some with different affinities. Sequence comparison and mutagenesis analysis support the conclusion that the differences in affinity depend on nonspecific interactions involving previously uncharacterized residues in the substrate-binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.

Exploring the Substrate Specificity of a Sugar Transporter with Biosensors and Cheminformatics.

Sugars will eventually be exported transporters (SWEETs) are conserved sugar transporters that play crucial roles in plant physiology and biotechnology. The genomes of flowering plants typically encode about 20 SWEET paralogs that can be classified into four clades. Clades I, II, and IV have been reported to favor hexoses, while clade III SWEETs prefer sucrose. However, the molecular features of substrates required for recognition by members of this family have not been investigated in detail. Here, we show that SweetTrac1, a previously reported biosensor constructed from the Clade I Arabidopsis thaliana SWEET1, can provide insight into the structural requirements for substrate recognition. The biosensor translates substrate binding to the transporter into a change in fluorescence, and its application in a small-molecule screen combined with cheminformatics uncovered 12 new sugars and their derivatives capable of eliciting a response. Furthermore, we confirmed that the wild-type transporter mediates cellular uptake of three of these species, including the diabetes drugs 1-deoxynojirimycin and voglibose. Our results show that SWEETs can recognize different furanoses, pyranoses, and acyclic sugars, illustrating the potential of combining biosensors and computational techniques to uncover the basis of substrate specificity.

Development and quantitative analysis of a biosensor based on the Arabidopsis SWEET1 sugar transporter.

SWEETs are transporters with homologs in Archeae, plants, some fungi, and animals. As the only transporters known to facilitate the cellular release of sugars in plants, SWEETs play critical roles in the allocation of sugars from photosynthetic leaves to storage tissues in seeds, fruits, and tubers. Here, we report the design and use of genetically encoded biosensors to measure the activity of SWEETs. We created a SweetTrac1 sensor by inserting a circularly permutated green fluorescent protein into the Arabidopsis SWEET1, resulting in a chimera that translates substrate binding during the transport cycle into detectable changes in fluorescence intensity. We demonstrate that a combination of cell sorting and bioinformatics can accelerate the design of biosensors and formulate a mass action kinetics model to correlate the fluorescence response of SweetTrac1 with the transport of glucose. Our analysis suggests that SWEETs are low-affinity, symmetric transporters that can rapidly equilibrate intra- and extracellular concentrations of sugars. This approach can be extended to SWEET homologs and other transporters.