The Arabidopsis SWEET1 and SWEET2 uniporters recognize similar substrates despite differences in subcellular localization

Sugars Will Eventually be Exported Transporters (SWEETs) are central for sugar allocation in plants. The SWEET family is vast, with approximately 20 homologs in most plant genomes. Despite extensive research on their structures and molecular functions, it is still unclear how diverse SWEETs recognize their 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 transporters 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 non-specific interactions involving key residues in the binding pocket. Furthermore, SweetTrac2 can be an effective tool for monitoring sugar transport at vacuolar membranes that would be otherwise challenging to study.