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Structure of a eukaryotic SWEET transporter in a homotrimeric complex

Nature volume 527, pages 259–263 (2015)Cite this article

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Abstract

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.

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Figure 1: Localization and structure of OsSWEET2b.
Figure 2: Key elements of the transport pathway.
Figure 3: Structure of the OsSWEET2b trimer.
Figure 4: Trimer formation by OsSWEET2b.

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Primary accessions

Protein Data Bank

Data deposits

The crystallographic coordinates and structural factors are deposited into Protein Data Bank with accession numbers 5CTG (3.1 Å) and 5CTH (3.7 Å).

References

  1. Wright, E. M. Glucose transport families SLC5 and SLC50. Mol. Aspects Med. 34, 183–196 (2013)

    Article  CAS  Google Scholar 

  2. Cura, A. J. & Carruthers, A. Role of monosaccharide transport proteins in carbohydrate assimilation, distribution, metabolism, and homeostasis. Compr. Physiol. 2, 863–914 (2012)

    PubMed  PubMed Central  Google Scholar 

  3. Chen, L. Q. et al. Sugar transporters for intercellular exchange and nutrition of pathogens. Nature 468, 527–532 (2010)

    Article  ADS  CAS  Google Scholar 

  4. 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)

    Article  CAS  Google Scholar 

  5. Chen, L. Q., Cheung, L. S., Feng, L., Tanner, W. & Frommer, W. B. Transport of sugars. Annu. Rev. Biochem. 84, 865–894 (2015)

    Article  CAS  Google Scholar 

  6. Eom, J. S. et al. SWEETs, transporters for intracellular and intercellular sugar translocation. Curr. Opin. Plant Biol. 25, 53–62 (2015)

    Article  CAS  Google Scholar 

  7. Lin, I. W. et al. Nectar secretion requires sucrose phosphate synthases and the sugar transporter SWEET9. Nature 508, 546–549 (2014)

    Article  ADS  CAS  Google Scholar 

  8. Chen, L. Q. et al. Sucrose efflux mediated by SWEET proteins as a key step for phloem transport. Science 335, 207–211 (2012)

    Article  ADS  CAS  Google Scholar 

  9. 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)

    Article  CAS  Google Scholar 

  10. 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)

    Article  CAS  Google Scholar 

  11. 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)

    Article  CAS  Google Scholar 

  12. 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)

    Article  Google Scholar 

  13. Chen, H.-Y. et al. Vacuolar SWEET2 transporter reduces glucose efflux to limit pathogen propagation in roots. Plant J. 83, 1046–1058 (2015)

    Article  CAS  Google Scholar 

  14. Xu, Y. et al. Structures of bacterial homologues of SWEET transporters in two distinct conformations. Nature 515, 448–452 (2014)

    Article  ADS  CAS  Google Scholar 

  15. Wang, J. et al. Crystal structure of a bacterial homologue of SWEET transporters. Cell Res. 24, 1486–1489 (2014)

    Article  CAS  Google Scholar 

  16. 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)

    Article  ADS  Google Scholar 

  17. Yee, D. C. et al. The transporter-opsin-G protein-coupled receptor (TOG) superfamily. FEBS J. 280, 5780–5800 (2013)

    Article  CAS  Google Scholar 

  18. 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)

    Article  CAS  Google Scholar 

  19. Jaehme, M., Guskov, A. & Slotboom, D. J. The twisted relation between Pnu and SWEET transporters. Trends Biochem. Sci. 40, 183–188 (2015)

    Article  CAS  Google Scholar 

  20. 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)

    Article  Google Scholar 

  21. Lawrence, M. C. & Colman, P. M. Shape complementarity at protein/protein interfaces. J. Mol. Biol. 234, 946–950 (1993)

    Article  CAS  Google Scholar 

  22. Kazmier, K. et al. Conformational dynamics of ligand-dependent alternating access in LeuT. Nature Struct. Mol. Biol. 21, 472–479 (2014)

    Article  CAS  Google Scholar 

  23. Krishnamurthy, H. & Gouaux, E. X-ray structures of LeuT in substrate-free outward-open and apo inward-open states. Nature 481, 469–474 (2012)

    Article  ADS  CAS  Google Scholar 

  24. 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)

    Article  CAS  Google Scholar 

  25. Yuan, L. et al. Allosteric regulation of transport activity by heterotrimerization of Arabidopsis ammonium transporter complexes in vivo. Plant Cell 25, 974–984 (2013)

    Article  CAS  Google Scholar 

  26. Lanquar, V. et al. Feedback inhibition of ammonium uptake by a phospho-dependent allosteric mechanism in Arabidopsis. Plant Cell 21, 3610–3622 (2009)

    Article  CAS  Google Scholar 

  27. Forrest, L. R., Kramer, R. & Ziegler, C. The structural basis of secondary active transport mechanisms. Biochim. Biophys. Acta 1807, 167–188 (2011)

    Article  CAS  Google Scholar 

  28. Keller, R., Ziegler, C. & Schneider, D. When two turn into one: evolution of membrane transporters from half modules. Biol. Chem. 395, 1379–1388 (2014)

    Article  CAS  Google Scholar 

  29. 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)

    Article  CAS  Google Scholar 

  30. Otwinowski, Z. & Minor, W. in Methods Enzymol. Vol. 276 (ed. Carter, C. W. Jr) 307–326 (Academic Press, 1997)

    Google Scholar 

  31. Kabsch, W. Integration, scaling, space-group assignment and post-refinement. Acta Crystallogr. D 66, 133–144 (2010)

    Article  CAS  Google Scholar 

  32. Evans, P. R. An introduction to data reduction: space-group determination, scaling and intensity statistics. Acta Crystallogr. D 67, 282–292 (2011)

    Article  CAS  Google Scholar 

  33. Schneider, T. R. & Sheldrick, G. M. Substructure solution with SHELXD. Acta Crystallogr. D 58, 1772–1779 (2002)

    Article  Google Scholar 

  34. McCoy, A. J. et al. Phaser crystallographic software. J. Appl. Crystallogr. 40, 658–674 (2007)

    Article  CAS  Google Scholar 

  35. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    Article  CAS  Google Scholar 

  36. Emsley, P. & Cowtan, K. Coot: model-building tools for molecular graphics. Acta Crystallogr. D 60, 2126–2132 (2004)

    Article  Google Scholar 

  37. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, 213–221 (2010)

    Article  CAS  Google Scholar 

  38. McCoy, A. J. Solving structures of protein complexes by molecular replacement with Phaser. Acta Crystallogr. D 63, 32–41 (2007)

    Article  CAS  Google Scholar 

  39. Schröder, G. F., Levitt, M. & Brunger, A. T. Super-resolution biomolecular crystallography with low-resolution data. Nature 464, 1218–1222 (2010)

    Article  ADS  Google Scholar 

  40. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

    Article  CAS  Google Scholar 

  41. DeLano, W. L. The PyMOL Molecular Graphics System. DeLano Scientific LLC, Palo Alto, California, USA. http://www.pymol.org (2008)

    Google Scholar 

  42. 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)

    Article  CAS  Google Scholar 

  43. 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)

    Article  CAS  Google Scholar 

  44. 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)

    Article  CAS  Google Scholar 

  45. 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)

    Article  ADS  Google Scholar 

  46. Hou, B. H. et al. Optical sensors for monitoring dynamic changes of intracellular metabolite levels in mammalian cells. Nature Protocols 6, 1818–1833 (2011)

    Article  CAS  Google Scholar 

  47. 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)

    CAS  PubMed  Google Scholar 

  48. 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)

    Article  CAS  Google Scholar 

Download references

Acknowledgements

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).

Author information

Author notes

  1. Yuyong Tao and Lily S. Cheung: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Molecular and Cellular Physiology, 279 Campus Drive, Stanford University School of Medicine, Stanford, 94305, California, USA

    Yuyong Tao, Shuo Li, Yan Xu & Liang Feng

  2. Department of Plant Biology, Carnegie Institution for Science, 260 Panama Street, Stanford, 94305, California, USA

    Lily S. Cheung, Joon-Seob Eom, Li-Qing Chen & Wolf B. Frommer

  3. Center of Growth, Metabolism and Aging, Key Laboratory of Bio-Resource and Eco-Environment of Ministry of Education, College of Life Sciences, Sichuan University, Chengdu, 610014, China

    Shuo Li

  4. NE-CAT and Dep. of Chemistry and Chemical Biology, Cornell University, Building 436E, Argonne National Laboratory, 9700 S. Cass Avenue, Argonne, 60439, Illinois, USA

    Kay Perry

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Contributions

W.B.F. and L.F. conceived and designed experiments. Y.T. performed expression, purification, crystallization, data collection, crystallography, and biochemical experiments, S.L. performed liposome uptake experiments, Y.X. contributed to initial characterization and biochemical characterizations, L.S.C., L.-Q.C., and J.-S.E. performed functional experiments, K.P. assisted crystallography, L.F. contributed to initial characterization, data collection and crystallography. Y.T., L.C., L.-Q.C., J.-S.E., S.L., W.B.F., and L.F. analysed the data. L.F. and W.B.F. wrote the manuscript.

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Correspondence to Liang Feng.

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Extended data figures and tables

Extended Data Figure 1 Phylogenetic tree for clade I and II SWEETs from Arabidopsis and rice.

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.

Extended Data Figure 2 Functional analysis of SWEET activities by yeast growth assay.

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.

Extended Data Figure 3 Functional characterization of OsSWEET2b.

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.

Extended Data Figure 4 Sequence alignment of AtSWEETs and OsSWEET2b.

Sequences of SWEETs were aligned using Clustal Omega. Secondary structure assignment based on OsSWEET2b structure is indicated above the alignment.

Extended Data Figure 5 Experimental electron density map and crystal packing of two crystal forms.

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.

Extended Data Figure 6 Comparison of THBs of OsSWEET2b and EcSemiSWEET.

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).

Extended Data Figure 7 Comparison of SWEET, PnuC, and GPCR.

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.

Extended Data Figure 9 Membrane localization of mutants that abolish glucose transport in AtSWEET1.

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.

Extended Data Table 1 Data collection and refinement statistics
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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

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