Structure of a eukaryotic SWEET transporter in a homotrimeric complex

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 SWEETs. SWEETs carry mono- and disaccharides across vacuolar or plasma membranes. Plant SWEETs play key roles in sugar translocation between compartments, cells, and organs, notably in nectar secretion, phloem loading for long distance translocation, pollen nutrition, and seed filling. Plant SWEETs cause pathogen susceptibility possibly by sugar leakage from infected cells. The vacuolar Arabidopsis thaliana AtSWEET2 sequesters sugars in root vacuoles; loss-of-function mutants show increased susceptibility to Pythium infection. 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.

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 model. 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 dataset. Evolutionary analyses were conducted in MEGA6. ‘n/t’ represents not-tested. b, Percentage identity and similarity between Arabidopsis and rice SWEETs in Clade I were performed using NCBI BLASTP.

Extended Data Figure 2. Functional analysis of SWEET activities by a 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. Glucose uptake activity of the OsSWEET2b/OsSWEET1a chimera (b) and OsSWEET1a (c) were reported by the co-expressed sensor FLII¹²Pglu700μδ6 (±SEM, 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 color scheme is used for TMs. b, Structural comparison of OsSWEET2b and PnuC. OsSWEET2b is shown in green, and PnuC is in purple. c, Structural comparison of OsSWEET2b and a GPCR (metabotropic glutamate receptor). 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 residues 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.

Figure 1. Localization and structure of OsSWEET2b

a, OsSWEET2b-EGFP localizes to the vacuolar membrane in yeast. b, Transport activity of OsSWEET2b was examined by a liposome uptake assay. Glucose uptake is shown as bars in blue. Liposomes with OsSWEET2b showed elevated glucose uptake compared with control liposomes without protein. Mutation of two putative pore-lining asparagine residues abolished transport activities (±SEM, n=4). Fructose uptake is shown as bars in grey (±SEM, n=3). c, Membrane topology diagram of OsSWEET2b. TM4 and THB1 constitute the N-terminal domain (NTD) while THB2 forms the C-terminal domain (CTD). d, Ribbon representation of the OsSWEET2b protomer. THB1 is coloured in blue, THB2 in green, and TM4 in yellow. e, Slab view of OsSWEET2b in an inward (cytoplasmic) open conformation. Surface is coloured according to electrostatic potential.

Figure 2. Key elements of the transport pathway

a, Comparison of the substrate binding pocket of OsSWEET2b (green) to that of EcSemiSWEET (gray). Key residues in the binding pocket are shown as sticks. b, AtSWEET1 complements the defective growth in glucose of EBY4000, a hexose transport-deficient yeast strain. W176A, N73A, and N192A mutations in AtSWEET1 abolished glucose-dependent growth. Growth on maltose was not affected by the different mutants. c, Extrafacial (extracellular) gate. Residues that cap the aqueous cavity from the extrafacial side form the extrafacial gate with side chains shown as sticks in yellow. Residues in the substrate-binding pocket are coloured in grey. The surface of the protein is shown as pale blue mesh. d, Effect of mutations in the extrafacial gate of AtSWEET1. Alanine substitution of V188, but not the conserved D185, in the extrafacial gate of AtSWEET1 abolished growth in glucose. Alanine substitution of Y57 leads to a growth defect on glucose, possibly due to mistargeting. e, Intrafacial hinge points. The four proline residues (shown as sticks) lining the transport pathway are near the same level in the membrane. f, The intrafacial (cytosolic) gate is required for sugar transport. Mutations in the conserved prolines that form the intrafacial gate of AtSWEET1 (P23A, P43A, P145A and P162A) eliminate the growth of the EBY4000 yeast strain in synthetic medium supplemented with glucose.

Figure 3. Structure of the OsSWEET2b trimer

a, Two orthogonal views of the OsSWEET2b trimer in ribbon representation. b, Surface representations of OsSWEET2b viewed from the intrafacial (cytosolic; left) and extrafacial (luminal; right) side. c, Close-up view of the trimer interface. TM4 from one protomer and TM5 and TM7 from the neighbouring protomer are shown as ribbon representation. Side chains of residues participating in the interaction are shown as sticks. d, Cross-section view of the trimer interface. The two interacting protomers are presented as blue and green surface meshes.

Figure 4. Trimer formation by OsSWEET2b

a, Co-immunoprecipitation (Co-IP) of OsSWEET2b and AtSWEET1. Left, c-Myc-tagged OsSWEET2b pulls down HA-tagged OsSWEET2b; right, c-Myc-tagged AtSWEET1 pulls down HA-tagged AtSWEET1. b, Cross-link of purified OsSWEET2b in detergent micelle. Increasing amounts of glutaraldehyde (0, 0.5, 1, 5, 10 ppm) were incubated with the purified OsSWEET2b. The samples were analysed by SDS-PAGE. c, The design of cysteine pair mutations at the interface of the trimer. M102 and I158 were mutated to cysteines (side chains shown as sticks). d, Cysteine-directed cross-linking of OsSWEET2b in detergent solution. GFP-tagged OsSWEET2b with M102C/I158C mutations was purified and oxidized in air or with copper phenanthroline (CuP). Proteins separated on SDS-PAGE were imaged by in-gel fluorescence of GFP. e, Cysteine-directed cross-linking of OsSWEET2b in membrane. Cell membranes containing M102C/I158C mutant were untreated or treated with CuP. OsSWEET2b-GFP was visualized by in-gel fluorescence. f, Mutations in the residues at the gates of AtSWEET1 have a dominant negative effect on transport. EBY4000 yeast co-expressing wild-type AtSWEET1 and either an empty vector control or wild-type AtSWEET1 were able to grow on medium supplemented with glucose as the sole carbon source. Co-expression with an AtSWEET1-Y57A or G58D mutant prevents growth on glucose. Similarly, co-expression with mutants that abolish transport and that are localized in the intrafacial (P23T) or extrafacial (V188A) gates prevent growth on glucose. As a control, growth on maltose was not affected.