SUPPLEMENTARY INFORMATION

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doi:10.1038/nature09606 Chen Li-Qing et al. A novel class of sugar transporters. Supplementary Figure 1. Confocal imaging of FRET sensor localization in HEK293T cells.

1 doi: /nature09606 Chen Li-Qing et al. A novel class of sugar transporters. Supplementary Figure 1. Confocal imaging of FRET sensor localization in HEK293T cells. The subcellular localization of the FRET sensors was not affected by coexpression of SWEET1 in HEK293T cells. (a) Cytosolic FRET sensor FLIPglu600µΔ13V in cells that do not coexpress SWEET1. (b) Cytosolic FRET sensor FLIPglu600µΔ13V in cells that coexpress SWEET1. (c) ER-targeted FRET sensor FLIPglu600µΔ13V ER in cells that do not coexpress SWEET1. (d) ER-targeted FRET sensor FLIPglu600µΔ13V ER in cells that coexpress SWEET1. WWW NATURE.COM/NATURE 1

Supplementary Figure 2. Glucose uptake in HEK293T cells mediated by SWEET1- GFP fusions. (a) Plasma membrane localization of the SWEET1-GFP fusion protein in HEK293T cells by confocal microscopy.

2 Supplementary Figure 2. Glucose uptake in HEK293T cells mediated by SWEET1- GFP fusions. (a) Plasma membrane localization of the SWEET1-GFP fusion protein in HEK293T cells by confocal microscopy. (b) SWEET1-GFP mediates glucose uptake in HEK293T cells. (c) SWEET1-GFP mediates glucose efflux into the ER of HEK293T cells. (d) Intracellular localization of the GFP-SWEET1 fusion in HEK293T cells. (e) GFP-SWEET1 did not mediate measurable glucose uptake in HEK293T cells. (f) HEK293T cells expressing only the cytosolic FRET sensor did not show significant glucose uptake. (g) HEK293T cells expressing only the ER-targeted FRET sensor did not show significant glucose efflux into the ER. WWW NATURE.COM/NATURE 2

Supplementary Figure 3. Mutant forms of SWEET1 do not induce or mediate glucose-dependent changes in FRET sensor responses and do not complement the yeast glucose uptake mutant EBY4000.

3 Supplementary Figure 3. Mutant forms of SWEET1 do not induce or mediate glucose-dependent changes in FRET sensor responses and do not complement the yeast glucose uptake mutant EBY4000. (a) Negative control: HEK293T cell expressing only the FRET sensor FLIPglu600µ 13V. (b) Positive control: HEK293T cell coexpressing FLIPglu600µ 13V and SWEET1. (c) HEK293T cell coexpressing FLIPglu600µ 13V and the truncated SWEET1 with a premature stop codon at leucine 198 (L198*). (d) HEK293T cell coexpressing FLIPglu600µ 13V and the truncated SWEET1 (deleted after leucine 197) fused to GFP (SWEET1Δ198-GFP). (e) Localization of SWEET1-GFP in yeast cells. (f) Localization of the truncated SWEET1Δ198-GFP in yeast cells. (g, h) Functional complementation of the glucose uptake deficiency of EBY4000 by SWEET1 and SWEET1-GFP, but not by SWEET1Δ198-GFP. WWW NATURE.COM/NATURE 3

4 Supplementary Figure 4. SWEET1-mediated glucose transport is independent of GLUT activity in HEK293T cells. (a) Inhibition of GLUT1 activity by 20 µm cytochalasin B (Cyt) analyzed using the FLIPglu600µΔ13V sensor co-transfected with GLUT1. (b) Insensitivity of SWEET1 activity to 20 µm cytochalasin B analyzed using the FLIPglu600µΔ13V sensor co-transfected with SWEET1. (c) Expression level of SLC2 (GLUT) and SLC5 (SGLT) glucose transporter genes in HepG2 cells, HEK293T cells, and HEK293T cells coexpressing FLIPglu600µ 13V with or without SWEET1. WWW NATURE.COM/NATURE 4

Supplementary Figure 5. Biochemical properties of SWEET1. (a) Complementation of glucose uptake-deficiency of EBY4000 is at acidic and neutral ph.

5 Supplementary Figure 5. Biochemical properties of SWEET1. (a) Complementation of glucose uptake-deficiency of EBY4000 is at acidic and neutral ph. (b) ph optimum for SWEET1 glucose transport activity. Radiotracer uptake was measured at different ph. The ph optimum for uptake is about ph 8.5. Data are mean± S.D, n=3. WWW NATURE.COM/NATURE 5

6 Supplementary Figure 6. Substrate specificity of SWEET1 from Arabidopsis. SWEET1 partially complements the mannose, fructose and galactose uptake in the yeast mutant EBY4000. OsSWEET11 and HsSWEET1/RAG1AP1 do not show detectable complementation of the growth deficiency. WWW NATURE.COM/NATURE 6

7 Supplementary Figure 7. Tissue specific expression of SWEET1 and SWEET8 genes in Arabidopsis. The analysis is based on microarray studies from WWW NATURE.COM/NATURE 7

8 Supplementary Figure 8. Phylogenetic tree for SWEETs from different species. (a) Phylogenetic tree of the SWEET superfamily (PFAM PFO3083). Distances were calculated from a multiple sequence alignment (ClustalW) using the neighbor-joining method. The tree displays bootstrap values (percentage of 1000). SWEET genes fall into four clades. All sequences were obtained from NCBI ( or the Aramemnon database ( The family members were color-coded indicating the species: At, Arabidopsis thaliana (light green); Os, Oryza sativa (blue); Mt, Medicago trunculata (cyan); Chlamydomonas reinhardtii (dark green), Physcomitrella patens (orange). (b) Pairwise comparison of amino acid identity of 17 Arabidopsis SWEETs, OsSWEET11 and OsSWEET14. a WWW NATURE.COM/NATURE 8

Supplementary Figure 9. Prediction of transmembrane helices in SWEETs from different species. The TMHMM2.0 program (www.cbs.dtu.

9 Supplementary Figure 9. Prediction of transmembrane helices in SWEETs from different species. The TMHMM2.0 program ( was used to predict transmembrane helices in (a) SWEET1, (b) SWEET12, (c) SWEET13 from Arabidopsis, (d) CeSWEET1 from C. elegans, (e) HsSWEET1/ RAG1AP1 from Human, and (f) CiSWEET1/CiRGA from Ciona intestinalis. OsSWEET11 and OsSWEET14 also contain seven predicted TMHs (data not shown). WWW NATURE.COM/NATURE 9

10 Supplementary Figure 10. Functional analysis of SWEET8 in heterologous systems. (a) Cells coexpressing the cytosolic FRET glucose sensor FLIPglu600µΔ13V and SWEET8 accumulated glucose in the cytosol as evidenced by a negative cytosolic FRET ratio change in HEK293T cells 1. (b) Cells efflux glucose from the cytosol into the ER when coexpressing the ER FRET glucose sensor FLIPglu-600µ 13V ER and SWEET8 in HEK293T cells Data points are mean ± SD (n>10). (c) Relative uptake rate of SWEET1, SWEET8 and vector control in the yeast glucose transport-deficient mutant EBY4000 (2 min; 10 mm D-glucose; 0.1µCi [ 14 C]-D-glucose). Values are normalized to SWEET1 (100%). Data are mean ± S.D, n=3. (d) Confocal imaging of SWEET8 localization in leaves using stably transformed Arabidopsis plants (T1 generation). WWW NATURE.COM/NATURE 10

11 Supplementary Figure 11. Arabidopsis SWEET transporters function in glucose uptake in yeast. The five Arabidopsis SWEETs (SWEET1, 4, 5, 7, and 8) function as glucose transporters when expressed in the yeast mutant EBY4000. Note that activity of all Arabidopsis SWEETs that function in EBY4000 can be detected in HEK293T cells, except SWEET13. The other 11 Arabidopsis SWEETs did not complement the yeast mutant to a significant extent (data not shown). WWW NATURE.COM/NATURE 11

12 Supplementary Figure 12. Arabidopsis SWEET transporters function in glucose uptake in HEK293T cells. Five Arabidopsis SWEETs (SWEET1, 4, 5, 7, and 13) function as glucose transporters when expressed in HEK293T cells. Note that SWEET13 did not show significant complementation of the EBY4000 yeast sugar transport mutant. HEK293T cells coexpressed the glucose FRET sensor FLIPglu600µΔ13V with or without a respective SWEET gene. For details on method see Figure 1. In several experiments, SWEET12 showed low glucose uptake activity and the other 10 Arabidopsis SWEETs did not show significant FRET responses relative to the negative control (data not shown). WWW NATURE.COM/NATURE 12

13 Supplementary Figure 13. PthXo1 drives TAL effector-specific host gene induction. (a) Promoter fragments used for transient assays extended eighteen bases upstream of the predicted PthXo1 promoter binding site (underlined) to the start codon of OsSWEET11/Os8N3. Triangle indicates site of insertion/deletion (sequence of insertion is not shown) in the OsSWEET11/Os8N3 promoter of the IRBB13 allele of the xa13 locus. Altered bases in the mutant version of the promoter Os8N3pM1 (OsWEET11pM1) are in lower case letters. Each fragment was fused to the uida gene for β-glucuronidase (GUS) at the ATG codon. (b) Agrobacterium-mediated transient GUS assays of the promoter constructs (from Supplementary Fig. 13a) in N. benthamiana. Each construct was codelivered with 35S pthxo1 (left half of the leaf shown) or empty binary vector (right half). Sites 1 and 7 denote the sites where Os8N3pBB13 (OsSWEET11) was tested; sites 2 and 8, Os8N3pδ1; sites 3 and 9, Os8N3pδ2; sites 4 and 10, Os8N3pM1; and sites 5 and 11, Os8N3pWT. Site 6 was inoculated with 35S pthxo1 alone. (c) GUS activity in leaves co-expressing 35S pthxo1 (Supplementary Fig. 13b). Each combination represents the average of three inoculations and is expressed as µm 4-methylumbelliferyl β-dglucuronide (MUG) min -1 µg protein -1. I-bars indicate one standard deviation. The label Os8N3pWt/AvrXa7 indicates the use of a combination of the wild type OsSWEET11/Os8N3 promoter and 35S-avrXa7 in place of 35S-pthXo1. Vector indicates combination of Os8N3pWt promoter and the CaMV 35S expression. Vector indicates combination of Os8N3pWt promoter and the CaMV 35S expression vector with a TAL effector gene. Assays were performed 48 h after inoculation. Each disk was excised from independent plants and assayed as described 2. WWW NATURE.COM/NATURE 13

14 Supplementary Figure 14. OsSWEET14-mediated [ 14 C]-glucose uptake into Xenopus oocytes. Time-dependent glucose uptake mediated by OsSWEET14 in oocytes (control OsSWEET-F203* mutant; mean ± SD, n 7). WWW NATURE.COM/NATURE 14

15 Supplementary Figure 15. Functionality of GFP fusions of the C. elegans CeSWEET1 transporter. Both N- and C-terminal GFP fusions of CeSWEET1 were functional in cellular glucose uptake in HEK293T cells. WWW NATURE.COM/NATURE 15

16 Supplementary Figure 16. Control experiment testing for HsSWEET1- and CeSWEET1-induced leakiness of oocytes. Efflux measurements were performed with oocytes injected with [ 14 C]-sucrose. Sucrose efflux was unaffected by expression of HsSWEET1 or CeSWEET1. WWW NATURE.COM/NATURE 16

doi:10.1038/nature09606 Supplementary Figure 17. Immunolocalization of HsSWEET1/RAG1AP1 in the Golgi of HEK293T cells.

17 doi: /nature09606 Supplementary Figure 17. Immunolocalization of HsSWEET1/RAG1AP1 in the Golgi of HEK293T cells. Immunolocalization in fixed HEK293T cells was performed with a primary HsSWEET1 antiserum (Abcam AB5271; red channel). The Golgi was localized with an anti-human Golgin97 antiserum (Invitrogen A21270, green channel). The nuclei were stained with Hoechst (blue channel). The overlay is derived from all three channels with the transmission channel. Fluorescence imaging was performed on a Leica SP5 confocal microscope. Shown are z-sections. Secondary antiserum alone served as control. Localization was performed either on un-transfected HEK293T cells or HEK293T cells transfected with HsSWEET1 in pcdna3/v5-dest. WWW NATURE.COM/NATURE 17

doi:10.1038/nature09606 Supplementary Figure 18. Immunolocalization of HsSWEET1/RAG1AP1 variants/mutants in the Golgi of HEK293T cells.

18 doi: /nature09606 Supplementary Figure 18. Immunolocalization of HsSWEET1/RAG1AP1 variants/mutants in the Golgi of HEK293T cells. Endogenous HsSWEET1 or overexpressed HsSWEET1 were immunolocalized as in Supplementary Fig. 17. The dileucine motif mutant HsSWEET1m (Y216A, L218A, L219A) and the splice variants HsSWEET1-2 (Open Biosystems Clone Id ) were localized using the same approach. Mutation of the potential di-leucine motif at the C-terminus did not lead to increased plasma membrane localization or increased transporter activity. WWW NATURE.COM/NATURE 18

Supplementary Figure 19. Localization of HsSWEET1/RAG1AP1-GFP fusions in HEK293T cells. Top panel: GFP-HsSWEET1 fusion (green channel); bottom panel: HsSWEET-GFP fusion (green channel).

19 Supplementary Figure 19. Localization of HsSWEET1/RAG1AP1-GFP fusions in HEK293T cells. Top panel: GFP-HsSWEET1 fusion (green channel); bottom panel: HsSWEET-GFP fusion (green channel). The Golgi was localized with an anti-human Golgin97 antiserum (Invitrogen A21270, red channel). The nuclei were stained with Hoechst (blue channel). The overlay is derived from all three channels. Fluorescence imaging was performed on a Leica SP5 confocal microscope. Shown are z- sections. HsSWEET1-GFP fusions localize to the ER and Golgi in HEK293T cells. WWW NATURE.COM/NATURE 19

20 Supplementary Figure 20. Expression of HsSWEET1 in human cell lines and tissues. Microarray data suggest ubiquitous expression throughout human tissues and cell lines, with highest expression in the oviduct, the epididymis, and the intestine. Metaprofile analysis was performed using the Anatomy function in Geneinvestigator 3 WWW NATURE.COM/NATURE 20

21 Supplementary Figure 21. Expression of the mouse homolog MmSWEET1 in mammary glands. Expression was induced in the mammary gland during lactation as shown by microarray studies 4. WWW NATURE.COM/NATURE 21

22 Supplementary Figure 22. Cellular glucose efflux model. Glucose is taken up into cell either via the Na + -coupled glucose cotransporters of the SGLT family or via GLUT glucose uniporters. Cytosolic glucose is phosphorylated by hexokinase or directly exported from the cell, either via a GLUT uniporter of via a vesicular pathway as suggested by Hosokawa et al (2002) 1. Golgi-localized SWEETs may function in uptake of glucose into the Golgi as part of a vesicular efflux route. Similarly, glycogenolysisderived glucose can enter the ER, where is dephosphorylated by a membrane-bound phosphatase. Subsequently glucose is exported back into the cytosol, potentially by GLUT uniporters that transit through the ER on the way to the plasma membrane (Takanaga & Frommer 2010) 5. Subsequently, cytosolic glucose can be exported by either of the two potential efflux routes. The model may be relevant for glucose efflux from intestinal, liver or epididymis cells. WWW NATURE.COM/NATURE 22

23 Supplemental Tables Supplementary Table 1. qpcr primers used for amplification of Arabidopsis thaliana SWEET genes (SWEET names and corresponding gene ID). name gene ID primers length SWEET1 AT1G21460 SWEET1F CTTCTCCACTCTCCATCATGAGATT 232 SWEET1R CATCTGCAGATTTCTCTCCTTTGT SWEET2 AT3G14770 SWEET2F AACAGAGAGTTTAAGACAGAGAGAAG 145 SWEET2R ATCCTCCTAAACGTTGGCATTGGT SWEET3 AT5G53190 SWEET3F CCAACTTTTCCCTAATCTTTGTTCTTC 133 SWEET3R AACACCCTTGAAAATGTTACTATTGGA SWEET4 AT3G28007 SWEET4F CCATCATGAGTAAGGTGATCAAGA 128 SWEET4R CAAAATGAAAAGGTCGAACTTAATAAGT G SWEET5 AT5G62850 SWEET5F TGACCCTTATATTTTGATTCCAAATGGT 151 SWEET5R GCCAAGTTCGATTCCAGCATTC SWEET6 AT1G66770 SWEET6F GACTCGGTTACGTTGGTGAAGT 96 SWEET6R CAAACGCCGCTAACTCTTTTGTTTAA SWEET7 AT4G10850 SWEET7F GACCCATTCATGGCTATACCAAAT 240 SWEET7R ATCCCATAATCCGAAGTTTAATAACACT SWEET8 AT5G40260 SWEET8F TTGCTCTCTTCTTCATCAATCTCTCT 150 SWEET8R AGATCCTCCAGAAAGTCTTCGCT SWEET9 AT2G39060 SWEET9F GCAAGAGAAAGAGAGAAAAGTGAAGA 124 SWEET9R CCCATAAAACGTTGGCACTGGT SWEET10 AT5G50790 SWEET10F TAGAGGAAGAGAGAGGGAGAGAGT 201 SWEET10R ATATACGAACGAACGTCGGTATTG SWEET11 AT3G48740 SWEET11F TCCTTCTCCTAACAACTTATATACCATG 131 SWEET11R SWEET12 AT5G23660 SWEET12F SWEET12R TCCTATAGAACGTTGGCACAGGA AAAGCTGATATCTTTCTTACTACTTCGA A 204 CTTACAAATCCTATAGAACGTTGGCAC SWEET13 AT5G50800 SWEET13F CTTCTACGTTGCCCTTCCAAATG 309 SWEET13R CTTTGTTTCTGGACATCCTTGTTGA SWEET14 AT4G25010 SWEET14F ACTTCTACGTTGCGCTTCCAAATA 334 SWEET14R CAGTTCAACATTAAAGTCAATCACTAAT TC WWW NATURE.COM/NATURE 23

24 SWEET15 AT5G13170 SWEET15F CAATGACATATGCATAGCGATTCCAA 153 SWEET15R GGACTCATCACGACAATACTCTTAAG SWEET16 AT3G16690 SWEET16F GAGATGCAAACTCGCGTTCTAGT 114 SWEET16R GCACACTTCTCGTCGTCACA SWEET17 AT4G15920 SWEET17F AGTGACAACAAAGAGCGTGAAATAC 211 SWEET17R ACTTAAACCGTTGCTTAAACCAACC ACTIN8F CACTTTCCAGCAGATGTGGATC 58 ACTIN8R AATGCCTGGACCTGCTTCAT Supplementary Table 2. Naming of SWEET genes from Oryza sativa corresponding to gene ID OsSWEET1a OsSWEET1b OsSWEET2a OsSWEET2b OsSWEET3a OsSWEET3b OsSWEET4 OsSWEET5 OsSWEET6a OsSWEET6b OsSWEET7a OsSWEET7b OsSWEET7c OsSWEET7d OsSWEET7e OsSWEET11 / Os8N3 OsSWEET12 OsSWEET13 OsSWEET14/Os11N3 OsSWEET15 OsSWEET16 Os01g65880 Os05g35140 Os01g36070 Os01g50460 Os05g12320 Os01g12130 Os02g19820 Os05g51090 Os01g42110 Os01g42090 Os09g08030 Os09g08440 Os12g07860 Os09g08490 Os09g08270 Os08g42350 Os03g22590 Os12g29220 Os11g31190 Os02g30910 Os03g22200 WWW NATURE.COM/NATURE 24

25 Supplementary Table 3. qpcr primers used for analysis immune-precipitation of Os8N3/TAL effector complexes. Primer name Os8N3-5 -F Os8N3-5 -R Os8N3-3 -F Os8N3-3 -R Os11N3-5 -F Os11N35 -R Primer sequence CAGAGTGAAAAAGAAATATCAAGC GGTGTTAATCAGTGAGAAGG GGAGAGTGCTGTATGGCGA GTCTGCCCTGTTCAAACACA GTCAGCAGCTGGTCATGTGT GTTTGGTGGGAGGAGATCA WWW NATURE.COM/NATURE 25

26 Supplementary References References 1. Takanaga, H. & Frommer, W. B. Facilitative plasma membrane transporters function during ER transit. FASEB J 24, , (2010). 2. Gallagher, S. R. in The GUS reporter system as a tool to study plant gene expression (eds T. Martin et al.) (San Diego, 1992). 3. Hruz, T. et al. Genevestigator v3: a reference expression database for the metaanalysis of transcriptomes. Adv. Bioinformatics 2008, , (2008). 4. Anderson, S. M., Rudolph, M. C., McManaman, J. L. & Neville, M. C. Key stages in mammary gland development. Secretory activation in the mammary gland: it's not just about milk protein synthesis! Breast Cancer Res 9, 204, (2007). 5. Hosokawa, M. & Thorens, B. Glucose release from GLUT2-null hepatocytes: characterization of a major and a minor pathway. Am. J. Physiol. Enocriol. Metab., E794-E801, (2002). WWW NATURE.COM/NATURE 26

Supplemental Data. Gao et al. (2012). Plant Cell /tpc

Supplemental Data. Gao et al. (2012). Plant Cell /tpc Supplemental Figure 1. Plant EMP Proteins. (A) The Accession numbers of the 12 EMP members from Arabidopsis. (B) Phylogenetic analysis of EMP proteins from Arabidopsis, human and yeast using the Mac Vector