Search for: All records

Not AvailableTransporters play key roles in regulating the movement of molecules into and out of cells. Uniporters, the simplest class of transporters, use facilitated diffusion to translocate molecules across membranes down their concentration gradient. This process can be affected by the presence of additional substrates in the intra- and extracellular environment, which can either increase the net transport rate of a molecule via trans acceleration or decrease it via competitive inhibition. In this study, we derived mathematical models to describe the net transport rate of uniporters in the presence of multiple extracellular substrates or inhibitors. Analyses of these models identified four possible states for the system when two substrates are present, with two states leading to trans acceleration and the other two states resulting in inhibition. Finally, we found that the relation between kinetic constants that controls the fraction of transporters in the inward-facing open state is responsible for these behaviors. Our theoretical results provide a mathematical framework for understanding the dynamic response of uniporters in the presence of multiple substrates and inhibitors, which could have implications for various processes, from nutrient utilization to metabolic engineering. 

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. 

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.