Eukaryotes rely on efficient distribution of energy and carbon skeletons between organs in the form of sugars. Glucose in animals and sucrose in plants serve as the dominant distribution forms. Cellular sugar uptake and release require vesicular and/or plasma membrane transport proteins. Humans and plants use proteins from three superfamilies for sugar translocation: the major facilitator superfamily (MFS), the sodium solute symporter family (SSF; only in the animal kingdom), and SWEETs1,2,3,4,5. SWEETs carry mono- and disaccharides6 across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion7, phloem loading for long distance translocation8, pollen nutrition9, and seed filling10. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells3,11,12. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection13. Here we show that its orthologue, the vacuolar glucose transporter OsSWEET2b from rice (Oryza sativa), consists of an asymmetrical pair of triple-helix bundles, connected by an inversion linker transmembrane helix (TM4) to create the translocation pathway. Structural and biochemical analyses show OsSWEET2b in an apparent inward (cytosolic) open state forming homomeric trimers. TM4 tightly interacts with the first triple-helix bundle within a protomer and mediates key contacts among protomers. Structure-guided mutagenesis of the close paralogue SWEET1 from Arabidopsis identified key residues in substrate translocation and protomer crosstalk. Insights into the structure–function relationship of SWEETs are valuable for understanding the transport mechanism of eukaryotic SWEETs and may be useful for engineering sugar flux.
Subscribe to Journal
Get full journal access for 1 year
only $3.90 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
Wright, E. M. Glucose transport families SLC5 and SLC50. Mol. Aspects Med. 34, 183–196 (2013)
Cura, A. J. & Carruthers, A. Role of monosaccharide transport proteins in carbohydrate assimilation, distribution, metabolism, and homeostasis. Compr. Physiol. 2, 863–914 (2012)
Chen, L. Q. et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527–532 (2010)
Xuan, Y. H. et al. Functional role of oligomerization for bacterial and plant SWEET sugar transporter family. Proc. Natl Acad. Sci. USA 110, E3685–E3694 (2013)
Chen, L. Q., Cheung, L. S., Feng, L., Tanner, W. & Frommer, W. B. Transport of sugars. Annu. Rev. Biochem. 84, 865–894 (2015)
Eom, J. S. et al. SWEETs, transporters for intracellular and intercellular sugar translocation. Curr. Opin. Plant Biol. 25, 53–62 (2015)
Lin, I. W. et al. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 508, 546–549 (2014)
Chen, L. Q. et al. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335, 207–211 (2012)
Sun, M. X., Huang, X. Y., Yang, J., Guan, Y. F. & Yang, Z. N. Arabidopsis RPG1 is important for primexine deposition and functions redundantly with RPG2 for plant fertility at the late reproductive stage. Plant Reprod. 26, 83–91 (2013)
Chen, L. Q. et al. A cascade of sequentially expressed sucrose transporters in the seed coat and endosperm provides nutrition for the Arabidopsis embryo. Plant Cell 27, 607–619 (2015)
Zhou, J. et al. Gene targeting by the TAL effector PthXo2 reveals cryptic resistance gene for bacterial blight of rice. Plant J. 82, 632–643 (2015)
Cohn, M. et al. Xanthomonas axonopodis virulence is promoted by a transcription activator-like effector-mediated induction of a SWEET sugar transporter in cassava. Mol. Plant Microbe Interact. 27, 1186–1198 (2014)
Chen, H.-Y. et al. Vacuolar SWEET2 transporter reduces glucose efflux to limit pathogen propagation in roots. Plant J. 83, 1046–1058 (2015)
Xu, Y. et al. Structures of bacterial homologues of SWEET transporters in two distinct conformations. Nature 515, 448–452 (2014)
Wang, J. et al. Crystal structure of a bacterial homologue of SWEET transporters. Cell Res. 24, 1486–1489 (2014)
Lee, Y., Nishizawa, T., Yamashita, K., Ishitani, R. & Nureki, O. Structural basis for the facilitative diffusion mechanism by SemiSWEET transporter. Nat. Commun. 6, 6112 (2015)
Yee, D. C. et al. The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 280, 5780–5800 (2013)
Jaehme, M., Guskov, A. & Slotboom, D. J. Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homolog. Nature Struct. Mol. Biol. 21, 1013–1015 (2014)
Jaehme, M., Guskov, A. & Slotboom, D. J. The twisted relation between Pnu and SWEET transporters. Trends Biochem. Sci. 40, 183–188 (2015)
Niittylä, T., Fuglsang, A. T., Palmgren, M. G., Frommer, W. B. & Schulze, W. X. Temporal analysis of sucrose-induced phosphorylation changes in plasma membrane proteins of Arabidopsis. Mol. Cell. Proteomics 6, 1711–1726 (2007)
Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)
Kazmier, K. et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nature Struct. Mol. Biol. 21, 472–479 (2014)
Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012)
De Zutter, J. K., Levine, K. B., Deng, D. & Carruthers, A. Sequence determinants of GLUT1 oligomerization: analysis by homology-scanning mutagenesis. J. Biol. Chem. 288, 20734–20744 (2013)
Yuan, L. et al. Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo. Plant Cell 25, 974–984 (2013)
Lanquar, V. et al. Feedback inhibition of ammonium uptake by a phospho-dependent allosteric mechanism in Arabidopsis. Plant Cell 21, 3610–3622 (2009)
Forrest, L. R., Kramer, R. & Ziegler, C. The structural basis of secondary active transport mechanisms. Biochim. Biophys. Acta 1807, 167–188 (2011)
Keller, R., Ziegler, C. & Schneider, D. When two turn into one: evolution of membrane transporters from half modules. Biol. Chem. 395, 1379–1388 (2014)
Rypniewski, W. R., Holden, H. M. & Rayment, I. Structural consequences of reductive methylation of lysine residues in hen egg white lysozyme: an X-ray analysis at 1.8-A resolution. Biochemistry 32, 9851–9858 (1993)
Otwinowski, Z. & Minor, W. in Methods Enzymol. Vol. 276 (ed. Carter, C. W. Jr) 307–326 (Academic Press, 1997)
Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)
Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011)
Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)
McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)
Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)
Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)
Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)
McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)
Schröder, G. F., Levitt, M. & Brunger, A. T. Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)
Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)
DeLano, W. L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, California, USA. http://www.pymol.org (2008)
Veenhoff, L. M. & Poolman, B. Substrate recognition at the cytoplasmic and extracellular binding site of the lactose transport protein of Streptococcus thermophilus. J. Biol. Chem. 274, 33244–33250 (1999)
Wieczorke, R. et al. Concurrent knock-out of at least 20 transporter genes is required to block uptake of hexoses in Saccharomyces cerevisiae. FEBS Lett. 464, 123–128 (1999)
Gietz, R. D. & Schiestl, R. H. Large-scale high-efficiency yeast transformation using the LiAc/SS carrier DNA/PEG method. Nature Protocols 2, 38–41 (2007)
Loqué, D., Lalonde, S., Looger, L. L., von Wiren, N. & Frommer, W. B. A cytosolic trans-activation domain essential for ammonium uptake. Nature 446, 195–198 (2007)
Hou, B. H. et al. Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells. Nature Protocols 6, 1818–1833 (2011)
Jones, D. T., Taylor, W. R. & Thornton, J. M. The rapid generation of mutation data matrices from protein sequences. Comput. Appl. Biosci. 8, 275–282 (1992)
Tamura, K., Stecher, G., Peterson, D., Filipski, A. & Kumar, S. MEGA6: Molecular Evolutionary Genetics Analysis version 6.0. Mol. Biol. Evol. 30, 2725–2729 (2013)
We thank the staff at Beamline 23ID-B and 23ID-D (APS, Argonne National Laboratory) and at Beamline 5.0.2 (Advanced Light Source) for assistance at the synchrotron. This work was made possible by support from Stanford University, the Harold and Leila Y. Mathers Charitable Foundation and the Alfred P. Sloan Foundation to L.F. and the Division of Chemical Sciences, Geosciences and Biosciences, Office of Basic Energy Sciences at the US Department of Energy (DOE DE-FG02-04ER15542) to W.B.F. The functional characterization of OsSWEET2b was supported by grants from the National Science Foundation (IOS-1258018) to W.B.F. L.F. is a Klingenstein-Simons Fellow. L.S.C. was supported by the National Science Foundation Postdoctoral Research Fellowship in Biology (1401855). S.L. was supported by the National Natural Science Foundation of China (31300618). Part of this work was conducted at the Advanced Photon Source on the Northeastern Collaborative Access Team, supported by a grant from the National Institute of General Medical Sciences (NIH, P41 GM103403). Use of the Advanced Photon Source, an Office of Science User Facility operated for the US Department of Energy (DOE) and the Office of Science by Argonne National Laboratory, was supported by US DOE (DE-AC02-06CH11357).
The authors declare no competing financial interests.
Extended data figures and tables
a, Molecular phylogenetic analysis was performed by the maximum likelihood method. The evolutionary history was inferred using the maximum likelihood method based on the JTT matrix-based model47. The tree with the highest log likelihood (−6353.2408) is shown. The percentage of trees in which the associated taxa clustered together is shown next to the branches. Initial tree(s) for the heuristic search were obtained automatically by applying neighbour-joining and BioNJ algorithms to a matrix of pairwise distances estimated using a JTT model, and then selecting the topology with the superior log likelihood value. The analysis involved 24 amino acid sequences. All positions with less than 95% site coverage were eliminated. That is, fewer than 5% alignment gaps, missing data, and ambiguous bases were allowed at any position. There were a total of 208 positions in the final data set. Evolutionary analyses were conducted in MEGA6 (ref. 48). ‘n/t’ represents not-tested; PM, plasma membrane; VM, vacuole membrane. b, Percentage identity and similarity between Arabidopsis and rice SWEETs in clade I were calculated using NCBI BLASTP.
When tested for complementation of the growth defect of the EBY4000 mutant strain, OsSWEET1a and OsSWEET1b showed limited growth on glucose, while OsSWEET2a and OsSWEET2b did not show growth. When tested on the toxic glucose analogue 2-deoxyglucose, only OsSWEET1a and OsSWEET1b failed to grow, suggesting that they may be glucose transporters. In contrast, OsSWEET2a and OsSWEET2b were able to grow in the presence of the 2-deoxyglucose, possibly because they are localized to the vacuole membrane and are not able to mediate uptake of the sugar analogue.
a, HEK293T cells expressing the FRET glucose sensor FLII12Pglu700μδ6 by itself served as negative controls. b, c, Glucose uptake activity of the OsSWEET2b/OsSWEET1a chimaera (b) and OsSWEET1a (c) were reported by the co-expressed sensor FLII12Pglu700μδ6 (± s.e.m., n = 12). The experiments were repeated four times. Representative results from one experiment are shown.
Sequences of SWEETs were aligned using Clustal Omega. Secondary structure assignment based on OsSWEET2b structure is indicated above the alignment.
a, The electron density map is contoured at σ1.5 and coloured in blue. b, Crystal lattice structure of OsSWEET2b in the P21 space group (form I). c, Crystal lattice structure of OsSWEET2b in the P212121 space group (form II). Each protomer within a trimer is shown in blue, purple or cyan.
a, Comparison of THB1 and THB2 of OsSWEET2b. THB1 of OsSWEET2b (yellow) was superimposed onto THB2 of OsSWEET2b (blue). The inversion linker TM4 is coloured in grey. b, Superposition of OsSWEET2b (THB1 in yellow, THB2 in blue, and TM4 in grey) to EcSemiSWEET (green).
a, Membrane topology diagram of SWEET (left), GPCR (middle), and PnuC (right) is shown with ribbon representations of their respective three TM unit (bottom). The same colour scheme is used for TMs. b, Structural comparison of OsSWEET2b and PnuC. OsSWEET2b is shown in green, and PnuC (4QTN) is in purple. c, Structural comparison of OsSWEET2b and a GPCR (4OR2). OsSWEET2b is shown in green, and GPCR in orange.
Extended Data Figure 8 Conservation of SWEET and key residues in the transport pathway of OsSWEET2b.
a, Conservation surface mapping of OsSWEET2b, which is coloured according to the degree of conservation of the surface residues of 527 analysed SWEET sequences. b, The cut-away view of OsSWEET2b shows the degree of the conservation of residues lining the transport route. Two clusters with higher conservation are labelled and correspond with the presumptive sugar binding site (I) and the intrafacial gate (II). c, Ribbon representation of OsSWEET2b with selected residues in the transport pathway are shown as sticks. Extrafacial gate residues are coloured in yellow, substrate binding pocket residues in green, and intrafacial hinge residues in cyan. d, Amino acids flanking the critical prolines in the intrafacial gate are also essential for AtSWEET1 activity. Alanine substitution of residues immediately above and below the conserved prolines that form the intrafacial gate in AtSWEET1 reduce the transport of glucose. Growth of the EBY4000 strain is unaffected in maltose. These results suggest that mutations in residues flanking the intrafacial gate have a similar effect as mutations of the conserved prolines.
a–o, Fluorescence and overlaid transmitted light images of yeast expressing an AtSWEET1-EGFP fusion (b) and its mutants that did not grow on glucose (c–o). The mutants failed to complement the growth defect of the EBY4000 strain despite proper targeting to the plasma membrane.
About this article
Cite this article
Tao, Y., Cheung, L., Li, S. et al. Structure of a eukaryotic SWEET transporter in a homotrimeric complex. Nature 527, 259–263 (2015). https://doi.org/10.1038/nature15391
Plant Communications (2020)
Trends in Plant Science (2020)
Biochimica et Biophysica Acta (BBA) - Biomembranes (2020)
Molecular basis for KDEL-mediated retrieval of escaped ER-resident proteins – SWEET talking the COPs
Journal of Cell Science (2020)
Plant Physiology and Biochemistry (2020)