Supplementary information:

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1 Supplementary information: Distinct amino acids in the C-linker domain of the plant K + subcellular localization and activity at the plasma membrane channel KAT2 determine its Nieves-Cordones et al. Part I: Supplementary Figures Part II: Supplementary Tables Part III: Supplementary Methods

2 A! KAT1! B! KAT1(aIaAa)! C! AtKC1! D! AtKC1(DIDAE)! Supplementary Information, Figure S1. Disruption of the triacidic motif DIDAE in KAT1 prevents channel targeting to the plasma membrane while introduction of this motif in AtKC1 is not sufficient to allow the resulting AtKC1(DIDAE) mutant to be expressed at the cell membrane. In KAT1, the triacidic motif DIDAE was mutated into aiaaa (replacement of each acidic aa of the motif by A). The resulting mutant channel subunit was named KAT1(aIaAa). In AtKC1, the DIDAE motif was introduced in place of the QIQAE stretch, which is the counterpart of KAT1 DIDAE motif in channel sequence alignments (Figure 5A). GFP constructs (encoding wild type or mutant channels fused to GFP) were expressed in protoplasts for confocal microscopy analysis of their ability to be localized to the plasma membrane. (A) Wild type KAT1 channel. (B) KAT1(aIaAa) mutant. (C) Wild type AtKC1. (D) AtKC1(DIDAE) mutant. A representative image of GFP and FM4-64 fluorescence is provided together with the bright field image of the corresponding protoplast (scale bar = 10 µm) for each construct. Right panel graphs: GFP (green) and FM4-64 (red) fluorescence intensity profiles across the protoplast membrane. Arrows mark the positions of the analyzed sections. Ext. and Cyt.: external and cytosolic sides, respectively.

3 A! KAT2*AtKC1-C-linker(TVRAASEFA) chimera! KAT2! KC1! N-ter! C-linker!! CNBD! TVRAASEFA! 309! M R S T V R A A S E F A S K N! 323! K HA! B! C! D! 4 µa! 0.3 s Supplementary information, Figure S2. Introduction of the KAT2 C linker TVRAASEFA motif in KAT2*AtKC1 C linker chimera does not restore channel surface expression. The KAT2*AtKC1-C-linker chimera displayed intracellular retention when expressed in tobacco protoplasts as shown in Figure 1D. The KAT2 TVRAASEFA motif was substituted for the corresponding AtKC1 AINDILRYT motif in this chimera. The resulting construct, named KAT2*AtKC1 C linker(tvraasefa) chimera, was either fused to GFP and expressed in protoplasts for subcellular localization by confocal microscopy or directly expressed in Xenopus oocytes for electrophysiological recordings. Both kinds of experiments indicate that the presence of the TVRAASEFA in the KAT2*AtKC1 C linker(tvraasefa) chimera does not result in localization of this chimera to plasma membrane. (A) Pictogram of the KAT2*AtKC1-C-linker(TVRAASEFA) chimera displaying the sequences from KAT2 and AtKC1 in blue and black, respectively. The position of the TVRAASEFA motif in the C linker sequence of the resulting KAT2*AtKC1 C linker(tvraasefa) chimera is indicated at the top right of the panel (aa from KAT2 and AtKC1 in blue and black, respectively). (B) Representative confocal microscopy image of GFP and FM4-64 fluorescence in a protoplast expressing the GFP construct together with the corresponding bright field image (scale bar = 10 µm). (C) GFP (green) and FM4-64 (red) fluorescence intensity profiles across the membrane of the protoplast shown in (B). The position of the analyzed section is indicated by the white arrow in (B). (D) Representative current traces recorded in oocytes expressing the KAT2*AtKC1 C-linker(TVRAASEFA) chimera (not fused to GFP). The external solution bathing the oocyte contained 100 mm K +.

4 EAG(Dm) ERG(H) ZELK(Dr) CNGB1(H) CNGB3(H) CNGA4(H) CNGA1(H) CNGA3(H) CNGA2(H) SpIH HCN3(R) HCN4(R) HCN1(H) HCN2(M) HCN2(H) KAT2 KAT1 SPIK AKT5 AKT1 AKT2 AtKC1 0.5 Supplementary information, Figure S3. Phylogenetic relationships between sequences encompassing the C linker and CNBD domains in plant Shaker channels and in animal channels characterized by the presence of a CNBD in their cytosolic C-terminal part. Unrooted tree constructed using the complete set of Arabidopsis inward Shaker channel subunits and some animal HCN (hyperpolarization-activated cyclic nucleotide modulated), KCNH [EAG, EAG-related gene (ERG) and EAG-like (ELK)] and CNG (cyclic nucleotide gated) channel subunits. (1) Arabidopsis inward Shaker channels [AKT1 : AEC07870, AtKC1 : AEE86098, KAT2 : AEE84021, SPIK : AEC07722, AKT5 : AEE86069, KAT1 : AED95356], (2) HCN channels [HCN1(H) (Homo sapiens AAO49469), HCN2(M) (Mus musculus EDL31671), HCN2(H) (Homo sapiens AAC28444), HCN3(R) (Rattus novegicus EDM00667), HCN4(R) (Rattus novegicus EDL95687), SpIH (SpIH NP_999729)], (3) KCNH channels [ERG(H) (Homo sapiens BAA37096), EAG(Dm) (Drosophila melanogaster NP_511158), zelk(dr) (Danio rerio 3UKV_A)] and (4) CNG channels [CNGA1(H) (Homo sapiens NP_ ), CNGA2(H) (Homo sapiens AAI26305), CNGA3(H) (Homo sapiens NP_001289), CNGA4(H) (Homo sapiens AAH40277), CNGB1(H) (Homo sapiens NP_001288), CNGB3(H) (Homo sapiens NP_061971)].

5 !" KAT2! AtKC1! 2PTM! 3BPZ! 1BL8! #"!"#$%& %'()*$+,--./$01$1+213$-1##042#01+5, *,0.78,"2"$49,"554#,+9,6"#71007#+:, :, ,25,-0373#4: ,5+4#6$3,:7,-09#-32$-: "#--93#29479-,8:$6562$-1$$4,057,170""0,*0#*1# *++,2*",19);''& &!"#$%&<=%>#7+37,1:$><%?& %'()*$+,--./$01$1+213#7+37,1:$01+5, *,0.78,"2"$49,"554#,+9,6"#71007#+:, :, ,25,-0373#4: ,5+4#6$3,:7,-09#-32$-: "#--93#29479-,8:$6562$-1$$4,057,170""0, *0#*1# *++,2*",19);''& %'()*$+,--./$01$1+213$-1##042#01+5, *,0.78,"2"$49,"554#,+9,6"#71007#+:, :, ,25,-0373#4: ,5+4#6$3,:7,-09#-32$-: "#--93#29479-,8:$6562$-1$$4,057,170""0,*0#*1# *++,2*",19);''& Supplementary information, Figure S4. Protein sequences used to create homology models of plant channels C-linker and CNBD regions. (A) Multiple-sequence alignment of the cytoplasmic regions of KAT2 (between M295 and G499), AtKC1 (M324 to L530) and the corresponding sequences of two templates (PDB code 2PTM and 3BPZ). An additional sequence of 10 residues corresponding to the end of the transmembrane segment S6 of a cristalized potassium channel from Streptomyces lividans (PDB code 1BL8) was added to guide the last transmembrane segment of KAT2. Identical amino acids are given in dark blue. Conserved residues with identical hydrophobicity properties are in blue, and conserved residues without identical hydrophobicity are in light blue. Dots depict gaps introduced for maximal sequence alignments. (B) Protein sequence of KAT2 and the chimeric proteins [KAT2( 312 AINDILRYT 320 ) and KAT2(V381F/S382P)] that were modelled for the close-ups shown in Figures 8D and 8F.

6 Supplementary Tables Primer name Sequence KAT2-F1 CAAAAAAGCAGGCTTCATGTCAATCTCTTGTACCAGAAACTTC 2. KAT2-F2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCAATCTCTTGTACCAG 3. KAT2ΔTAA-R1 CAAGAAAGCTGGGTCAGAGTTTTCATTGATGAGAATATAC 4. KAT2ΔTAA-R2 GGGGACCACTTTGTACAAGAAAGCTGGGTCAGAGTTTTCATTGATGAG 5. S1-S6KAT2 C-LINKER-KC1-F GAAATATGACCAACCTTGTGGTTCACGGCGCTCTTCGTACATTCGCCATG 6. S1-S6KAT2 C-LINKER-KC1-R CATGGCGAATGTACGAAGAGCGCCGTGAACCACAAGGTTGGTCATATTTC 7. C-LINKER-KC1 CNBDKAT2-F GGATTCCCCGAAGGCCTCCTCGTCCAGCTGGTTTCAGATATAGACGCCG 8. C-LINKER-KC1 CNBDKAT2-R CGGCGTCTATATCTGAAACCAGCTGGACGAGGAGGCCTTCGGGGAATCC 9. KAT KC1-F GAAGAGGTTCTTCAAGACTTGCCGAAAGCAATCCGGTCAAGC 10. KAT KC1-R CGGATTGCTTTCGGCAAGTCTTGAAGAACCTCTTCTTGCC 11. KAT KC1-F CAACAAGAAGCTTTAAATGGTTTACCTAAGGCCATAAGATCAAGCATTAACC 12. KAT KC1-R GATCTTATGGCCTTAGGTAAACCATTTAAAGCTTCTTGTTGTTTCAGACC 13. KAT KC1-F CCATCATCGAAGAAGCTTACCTATTTCACGGAGTTTCTCGC 14. KAT KC1-R GAAACTCCGTGAAATAGGTAAGCTTCTTCGATGATGGAGCGGAATAG 15. KAT KC1-F CCAATCGTTCAGAACGTCTATCTTTTTAAAGGATTCCCCGAAGG 16. KAT KC1-R GGAATCCTTTAAAAAGATAGACGTTCTGAACGATTGGGAAGAAGAG 17. C-LINKER-KAT2 CNBDKC1-F GGTTTCAGATATAGACGCCGAATATTTTCCGCCGAAAATGGAGATAATC 18. C-LINKER-KAT2 CNBDKC1-R GATTATCTCCATTTTCGGCGGAAAATATTCGGCGTCTATATCTGAAACC 19. CNBD-KC1 C-TERKAT2-F CATAAGTTCAAAGAAATGGTGCAGTCTCATGTCGAAGATGGACGAGTC 20. CNBD-KC1 C-TERKAT2-R GACTCGTCCATCTTCGACATGAGACTGCACCATTTCTTTGAACTTATG 21. C-LINK-KC KAT2-F GTAAGAGCTGCTTCAGAGTTTGCAAGCAAGAACAGGTTACCGGATACAATGA 22. C-LINK-KC KAT2-R AAACTCTGAAGCAGCTCTTACAGTACTCCTCATGGCGAATGTACGAAGAGCG 23. KAT KC1-F CGATCAATGATATATTGCGATACACATCAAGAAACCAACTCCCACCAAACATAC 24. KAT KC1-R GTGTATCGCAATATATCATTGATCGCATCTCTAAAGTTTCTGGTGCGGCTAGTC 25. KAT1-F1 CAAAAAAGCAGGCTTCATGTCGATCTCTTGGACTCGAAATTTC 26. KAT1-F2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCGATCTCTTGGACTCG 27. KAT1ΔTAA-R1 CAAGAAAGCTGGGTCATTTGATGAAAAATACAAATGATCACCATCC 28. KAT1ΔTAA-R2 GGGGACCACTTTGTACAAGAAAGCTGGGTCATTTGATGAAAAATACAAATG 29. AtKC1-F1 CAAAAAAGCAGGCTTCATGTCTACGACGACTACTGAGGCG 30. AtKC1-F2 GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCTACGACGACTACTG 31. AtKC1ΔTAA-R1 CAAGAAAGCTGGGTCGAAAATATATAAATGATCGTTTTCTCG 32. AtKC1ΔTAA-R2 GGGGACCACTTTGTACAAGAAAGCTGGGTCGAAAATATATAAATGATCG 33. KAT2-T7-F GCGAAATTAATACGACTCACTATAGGGAGAATGTCAATCTCTTGTACCAGAAACTTC 34. KAT2-BP-R CTTGATCTTGTTAAGAGTTTTCATTGATGAGAATATAC 35. GFP-BP-R CTTGATCTTGTTACTTGTACAGCTCGTCCATGC Supplementary information, Table S1: Bridge PCR primers used for chimera cloning and for subdomain exchange between AtKC1 and KAT2 cdnas

7 Primer name Sequence KAT2 R383E-F CACGGAGTTTCTGAAAACTTCCTATTTCAGTTGG 2. KAT2 R383E-R CCAACTGAAATAGGAAGTTTTCAGAAACTCCGTG 3. KAT2 N384G-F GGAGTTTCTCGCGGCTTCCTATTTCAG 4. KAT2 N384G-R CTGAAATAGGAAGCCGCGAGAAACTCC 5. KAT2 F385L/F387V-F GGAGTTTCTCGCGGCCTCCTAGTTCAGTTGGTTTCAG 6. KAT2 F385L/F387V-R CTGAAACCAACTGAACTAGGAGGCCGCGAGAAACTCC 7. KAT2 H379K-F GAACGTCTACCTATTTAAAGGAGTTTCTCGCAAC 8. KAT2 H379K-R GTTGCGAGAAACTCCTTTAAATAGGTAGACGTTC 9. KAT2 V381F/S382P-F CTACCTATTTAAAGGATTTCCTCGCAACTTCC 10. KAT2 V381F/S382P-R GGAAGTTGCGAGGAAATCCTTTAAATAGGTAG 11. KAT2 V381F-F CCTATTTCACGGATTTTCTCGCAACTTCC 12. KAT2 V381F-R GGAAGTTGCGAGAAAATCCGTGAAATAGG 13. KAT2 S382P-F CTATTTCACGGAGTTCCTCGCAACTTCC 14. KAT2 S382P-R GGAAGTTGCGAGGAACTCCGTGAAATAG 15. KAT2 S382A-F CCTATTTCACGGAGTTGCTCGCAACTTCC 16. KAT2 S382A-R GGAAGTTGCGAGCAACTCCGTGAAATAGG 17. KAT2 C-LINK-KC1 F381V/P382S-F CGAAGAAGCTTATCTTTTTAAAGGAGTCTCCGAAGGCCTCCTC 18. KAT2 C-LINK-KC1 F381V/P382S-R GAGGAGGCCTTCGGAGACTCCTTTAAAAAGATAAGCTTCTTCG 19. KAT2 V381A-F CCTATTTCACGGAGCTTCTCGCAACTTCC 20. KAT2 V381A-R GGAAGTTGCGAGAAGCTCCGTGAAATAGG 21. KAT2 V381M-F CGTCTACCTATTTCACGGAATGTCTCGCAACTTCCT 22. KAT2 V381M-R AGGAAGTTGCGAGACATTCCGTGAAATAGGTAGACG 23. KAT2 V381Y-F CTACCTATTTCACGGATATTCTCGCAACTTCC 24. KAT2 V381Y-R GGAAGTTGCGAGAATATCCGTGAAATAGGTAG 25. KAT1 V381F-F CATTTACCTCTTTCAAGGATTTTCTCGTAACTTCCTC 26. KAT1 V381F-R GAGGAAGTTACGAGAAAATCCTTGAAAGAGGTAAATG 27. KAT2 AIAAA-F GTTGGTTTCAGCTATAGCCGCCGCATATTTCCCACC 28. KAT2 AIAAA-R GGTGGGAAATATGCGGCGGCTATAGCTGAAACCAAC 29. KAT1 AIAAA-F1 GGTTTCAGATATAGCCGCTGCGTATTTCCCACC 30. KAT1 AIAAA-R1 GGTGGGAAATACGCAGCGGCTATATCTGAAACC 31. KAT1 AIAAA-F2 CAATTGGTTTCAGCTATAGCCGCTGCG 32. KAT1 AIAAA-R2 CGCAGCGGCTATAGCTGAAACCAATTG 33. KC1 DIDAE-F1 CAGCTGGTTTCGGATATACAAGCAGAA 34. KC1 DIDAE-R1 TTCTGCTTGTATATCCGAAACCAGCTG 35. KC1 DIDAE-F2 CCTCGTCCAGCTGGTTTCGGATATAGACGCAGAATATTTTCC 36. KC1 DIDAE-R2 GGAAAATATTCTGCGTCTATATCCGAAACCAGCTGGACGAGG Supplementary information, Table S2: Primers used for site-directed mutagenesis

8 SUPPLEMENTARY METHODS 3D reconstruction of tobacco protoplasts expressing Shaker-GFP fusions Tobacco protoplasts transformation with Shaker-GFP fusions and GFP fluorescence recording was performed as described in the Materials and Methods section. To obtain z-stacks, 40 to 60 2D confocal images covering 8 to 12 µm along the z axis were captured. Then, 3D reconstructions of such z-stacks were generated with IMARIS software (Bitplane AG). Five GFP-positive protoplasts were analyzed for each construct and representative animations are showed (Supplemental Videos S1- S5). Phylogenic Analysis Sequence alignments and phylogenic tree construction were performed using online softwares available at Multiple protein sequence alignments were performed using the MUSCLE algorithm (Edgar, 2004). Maximum likelihood phylogeny was calculated from the alignment of all protein sequences using the PHYML3.0 algorithm (Guindon and Gascuel, 2003) based on the neighbour-joining method (Saitou and Nei, 1987), with a bootstrap analysis of 1000 replicates. The graphical output of the unrooted phylogram was generated using the TreeDyn software ( REFERENCES Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucl Acids Res 32: Guindon S, Gascuel O (2003) A simple, fast, and accurate algorithm to estimate large phylogenies by maximum likelihood. Syst Biol 52: Saitou N, Nei M (1987) The neighbour-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4: