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Volume 1, Issue 6, Pages 938-949 (November 2008) Targeting of Vacuolar Membrane Localized Members of the TPK Channel Family Dunkel Marcel , Latz Andreas , Schumacher Karin , Müller Thomas , Becker Dirk , Hedrich Rainer Molecular Plant Volume 1, Issue 6, Pages 938-949 (November 2008) DOI: 10.1093/mp/ssn064 Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 1 Golgi-Dependent Trafficking of TPK1. Onion epidermal cells were co-transfected with the Golgi-marker ST-GFP (A, D) and TPK1-mRFP1 (B, E). The cells were subsequently incubated without (A–C) and with the fungal toxin Brefeldin A (D–F). (C) Overlay of the confocal images A and B. (F) Overlay of the confocal images (D) and (E). Note that in turgid plant cells, the vacuole comprises >90% of the intra-cellular volume, making it difficult to distinguish microscopically between the vacuolar membrane and the surrounding Golgi stacks. Scale bar = 20 μm. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 2 Sub-Cellular Localization of TPK1 Mutants Bearing Mutations in the 14-3-3 Binding Motif and TPK1/TPK4 Chimeras in Onion Epidermal Cells. Bright field images and confocal GFP images were merged in order to allow sub-cellular localization of the 14-3-3 binding motif mutants TPK1S42A (A) and TPK1S42E (B)the N-and C-terminal chimeras TPK1-TPK4NT (C) and TPK1-TPK4CT (D) as well as the pore chimeras TPK1-P1-TPK4-P2 (E) and TPK4-P1-TPK1-P2 (F). CT, carboxy-terminus. Scale bar = 20 μm. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 3 Alignment of TPK/KCO3 C-Termini and a Structural Model of the TPK1 CT. (A) Alignment of the C-terminal TPK1, TPK2, TPK3, TPK4, TPK5, and KCO3 amino acid sequence with the putative α-helical structures of the TPK1 EF-hands indicated (H1-4) and the diacidic ER-export motif of TPK1 in the vertical box. (B) Structural modelling of TPK1-CT showing the α-helical extension of the last transmembrane (TMD4) and the two EF-hand helix-loop-helix motifs (H1-L1-H2/H3-L3-H4), each coordinating one Ca2+-ion. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 4 Representative Confocal Images of Cells Expressing C-Terminal Truncation Mutants of TPK1 24h after Transfection. The mutant channels TPK1ΔCT (A), TPK1ΔL1 (D), TPK1ΔH2 (G), TPK1ΔL2 (J), TPK1ΔH3 (M)and TPK1ΔL3 (P) were fused to either mGFP4 (green) or mRFP1 (red) and were co-expressed with various markers of the secretory pathway. Markers used were ER localized GFP (B, E)ST-mRFP1 for Golgi-stacks (K) and TPK1-mRFP1 for the tonoplast (H, N, Q). (C, F, I, L, O, R) These images resulted from merging GFP-and RFP-channel with the respective bright field image. Scale bar = 20 μm. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 5 Sub-Cellular Localization of TPK1 Channels Mutated in C-Terminal Diacidic Motifs. (A) In order to illustrate the difference in targeting ability, the TPK1 mutant in diacidic motif one (TPK1-D296G/E298G-mGFP4) was co-expressed with the wild-type TPK1-mRFP1. (B) The localization of the TPK1 mutant in diacidic motif two and three (TPK1-D301G/D303G) was visualized by mGFP4. Scale bar = 20 μm. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

Figure 6 Sub-Cellular Localization of TPK3 without CT, TPK1, and TPK3-TMD4. (A) Heterologous expression and sub-cellular localization of TPK3ΔCT in onion epidermal cells is marked by mRFP1 fluorescence. (C) overlay of RFP-channel (A) and bright field image (B). Similarly, sub-cellular localization of TPK1-TMD4 (D)TPK1-TMD4+CT (G) TPK3-TMD4 (J) is visualized by mRFP1 fluorescence. This TMD4 constructs were co-expressed with the ER-/Golgi-marker ERD2 (EHK) to allow accurate sub-cellular localization by image overlay (F, I, L). Scale bar = 20 μm. Molecular Plant 2008 1, 938-949DOI: (10.1093/mp/ssn064) Copyright © 2008 The Authors. All rights reserved. Terms and Conditions