Wang H , Zhang L , Tao Y , Wang Z , Shen D , Dong H .

	Figure 1 Localization analysis of AtPIP1;1 and AtPIP2;4. (A) Subcellular localization of fluorescent proteins fused AtPIP1;1 or AtPIP2;4 expressed in Saccharomyces cerevisiae cells (left), Xenopus laevis oocytes (middle), and protoplasts of Arabidopsis thaliana (right). White scale lines represent the length of 5 μm (upper), 100 μm (middle), and 5 μm (nether). (B) Immunoblot of plasma membrane proteins (PM fraction) and intracytoplasmic proteins (including cytosolic proteins and intracellular membrane proteins, CP fraction), extracted from AtPIP1;1-6xHis– or AtPIP2;4-6xHis–transformed yeast cells, with the antibody of anti-6xHis (HT501). (C) Interaction between AtPIP1;1 and other AtPIPs using split ubiquitin yeast two hybrid (SUB Y2H) system showing AtPIP1;1 interacted with all the AtPIPs. The transformed yeast cells were series-diluted, and 5 μl of the yeast suspension was spotted on the SD-WLAH plates.
	Figure 2 Failed PM targeting of 2;4ΔTM2/1;1TM2 and 2;4ΔTM3/1;1TM3. (A) Succinct structure drawing of AtPIP1;1 and AtPIP2;4, which were divided into 11 fragments respectively (upper). Western blot of plasma membrane proteins of the mutants of AtPIP2;4 with different fragments replaced by those of AtPIP1;1 (nether). Membrane proteins were extracted from transformed yeast, and 6xHis antibody was applied. (B) Hand-drawn structure of 2;4ΔTM2/1;1TM2 and 2;4ΔTM3/1;1TM3. (C) Confocal images of AtPIP2;4-eGFP, 2;4ΔTM2/1;1TM2-eGFP, and 2;4ΔTM3/1;1TM3-eGFP expressing yeast cells (left) and Xenopus oocytes (right). White scale lines represent the length of 5 μm (left) and 100 μm (right).
	Figure 3 Successful localization of 1;1Δ(TM2+TM3)/2;4(TM2+TM3) in the PM. (A) Hand-painted structure of 1;1TM2/2;4TM2, 1;1TM3/2;4TM3, and 1;1Δ(TM2+TM3)/2;4(TM2+TM3). (B) Western blot of membrane proteins extracted from AtPIP2;4-, AtPIP1;1-, 1;1ΔTM2/2;4TM2-, 1;1ΔTM3/2;4TM3-, or 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-transformed yeast cells using 6xHis antibody. (C) Confocal images of 1;1ΔTM2/2;4TM2-eGFP, 1;1ΔTM3/2;4TM3-eGFP, and 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-eGFP expressing yeast cells (left) and Xenopus oocytes (right). White scale lines represent the length of 5 μm (left) and 100 μm (right). (D) Alignment of transmembrane helix 2 (TM2) and TM3 from AtPIP1;1 and AtPIP2;4. Red letters are the varying residues. Numbers are the sites of the residues. (E and F) Interaction of mutants of AtPIP1;1 and AtPIP2;4 using SUB Y2H system. The transformed yeast cells were series-diluted, and 5 μl of the yeast suspension was spotted on the SD-WLAH [synthetic dextrose minimal media without W (tryptophan), L (leucine), H (histidine), and A (alanine)] plates.
	Figure 4 Function analysis of AtPIP1;1, AtPIP2;4, and their mutants. (A) Bar graph of Pf values of Xenopus oocytes injected with 2 ng cRNA coding eGFP, AtPIP1;1-eGFP, AtPIP2;4-eGFP, 1;1ΔTM2/2;4TM2-eGFP, 1;1ΔTM3/2;4TM3-eGFP, or 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-eGFP. Data shown are the means ± SDs. Different letters indicate significant differences in multiple comparisons of each Pf value using an one-way ANOVA analysis, P < 0.01; for each sample, 10 technical replicates were measured, and the experiments were independently repeated three times. (B) Growth status of yeast cells transformed with AtPIP2;4, AtPIP1;1, or their variants spotted on YPDA plates plus 0 or 3 mM H2O2. (C) DCF curves of yeast cells transformed with AtPIP2;4, AtPIP1;1, or their variants with DCF stored and 3 mM H2O2 added, showing fluorescent signal detected by microplate reader. Data are the mean ± SDs. For each sample, triplicates (technical replicates) were measured, and the experiments were independently repeated three times.
	Figure 5 Subcellular localization and function analysis of AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, and their mutants. (A) Confocal images of yeast cells (left) and Xenopus oocytes (right), which were expressing AtPIP1;2-eGFP, AtPIP1;3-eGFP, AtPIP1;4-eGFP, AtPIP1;5-eGFP, and their eGFP-fused mutants. White scale lines represent the length of 5 μm (left) and 100 μm (right). (B) Bar graph of Pf values of Xenopus oocytes injected with 2 ng cRNA coding eGFP, AtPIP1;2-eGFP, AtPIP1;3-eGFP, AtPIP1;4-eGFP, AtPIP1;5-eGFP, and their eGFP-fused mutants. On bar graphs, different letters indicate significant differences in multiple comparisons of each Pf values using an one-way ANOVA analysis, P < 0.01; for each sample, 10 technical replicates were measured, and the experiments were independently repeated three times. (C) Growth status of yeast cells transformed with AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, or their mutants spotted on YPDA plates plus 0 or 3 mM H2O2. (D) DCF curves of AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, or their mutant-transformed yeast cells with DCF stored and 3 mM H2O2 added, showing fluorescent signal detected by microplate reader. Data are the mean ± SDs. For each sample, triplicates (technical replicates) were measured, and the experiments were independently repeated three times.
	Figure 6 Structure analysis and localization working model of AtPIP1;1 and AtPIP2;4. (A) Tetrameric structure of AtPIP2;4 with AtPIP1;1 aligned in. TM2s and TM3s are highlighted with cyan (AtPIP1;1) and green (AtPIP2;4). (B) Structure of four TM2s from AtPIP2;4 tetramer (green) with those from AtPIP1;1 (cyan) aligned in. The Q residue and the corresponding L residue are in the form of sticks. Red in the sticks represents oxygen atom, while blue represents nitrogen atom. (C) Sequences alignment of TM3 region of AtPIP1;1 with cyan background and AtPIP2;4 with green background (upper) and structure of AtPIP1;1 and AtPIP2;4 with TM3 highlighted in cyan (AtPIP1;1) or green (AtPIP2;4) (nether). Red letters are the varying residues, and LxxxA region is in the square frame (upper). All outfacing residues are in the form of sticks (nether). (D) Working model of the subcellular localization of plasma membrane intrinsic proteins. Other relative residues are some of the different residues except the L81 and LxxxA motif in TM2 and TM3 between PIP1s and PIP2s that might also take part in localization of PIPs.
	Supplementary figure 1: Relative expression level (amount of mRNA) of AtPIP1s, AtPIP2;4 and their mutants in transformed S. cerevisiae NMY51 estimated by a RT-qPCR.
	Supplementary figure 2: Immunoblot of proteins extracted from Xenopus oocytes injected with cRNA coding eGFP, AtPIP1; 1-eGFP, AtPIP2;4-eGFP and their mutants using anti-GFP antibody.
	Supplementary figure 3: Alignment of both TM2 and TM3 among all the AtPIPs. Quincunx points the Q88 and L81 residues; Residues with blue background are the residues extending outward in monomer; It is the LxxxA motif in the square frame.
	Supplementary figure 4: The yeast concentrations of SUB Y2H positive transformants of AtPIP1; 1-CubPLV and NubG-AtPIPs after a 48 hours culture in liquid SD-WLAH medium. Data shown are the means +- SDs. Different letters indicate significant differences in multiple comparisons of each pf values using an one-way ANOVA analysis, P< 0.01. For each sample, triplicates (technical replicates) were measured and the experiments were repeated three times.
	Supplementary figure 5: The yeast concentrations of SUB Y2H positive transformants of AtPIP1;1 mutants or AtPIP2;4 mutants after a 48 hours culture in liquid SD-WLAH medium. Data shown are the means +- SDSs. Different letters indicate significant differences in multiple comparisons of each pf values using an one-way ANOVA analysis, P< 0.05. For each sample, triplicates (technical replicates) were measured and the experiments were repeated three times.
	Supplementary figure 6: The yeast concentrations of SUB Y2H positive transformants of AtPIP1; 1-CubPLV/1;1Q88L-CubPLV and NubG-AtPIP2;4 or AtPIP2;4 mutants after a 48hours culture in liquid SD-WLAH medium. Data shown are the means +- SDs. Different letters indicate significant differences in multiple comparisons of each pf values using an one-way ANOVA analysis, P< 0.05. For each sample, triplicates (technical replicates) were measures and the experiments were repeated three times.
	Supplementary figure 7: Localization of 1;1delta(Q88L+LxxxA) in S. cereviciae (left) and X. laevis oocytes (right). White scale bars represent the length of 5 micrometers (left) and 100 micrometers (right).
	Figure 1. Localization analysis of AtPIP1;1 and AtPIP2;4. (A) Subcellular localization of fluorescent proteins fused AtPIP1;1 or AtPIP2;4 expressed in Saccharomyces cerevisiae cells (left), Xenopus laevis oocytes (middle), and protoplasts of Arabidopsis thaliana (right). White scale lines represent the length of 5 μm (upper), 100 μm (middle), and 5 μm (nether). (B) Immunoblot of plasma membrane proteins (PM fraction) and intracytoplasmic proteins (including cytosolic proteins and intracellular membrane proteins, CP fraction), extracted from AtPIP1;1-6xHis– or AtPIP2;4-6xHis–transformed yeast cells, with the antibody of anti-6xHis (HT501). (C) Interaction between AtPIP1;1 and other AtPIPs using split ubiquitin yeast two hybrid (SUB Y2H) system showing AtPIP1;1 interacted with all the AtPIPs. The transformed yeast cells were series-diluted, and 5 μl of the yeast suspension was spotted on the SD-WLAH plates.
	Figure 2. Failed PM targeting of 2;4ΔTM2/1;1TM2 and 2;4ΔTM3/1;1TM3. (A) Succinct structure drawing of AtPIP1;1 and AtPIP2;4, which were divided into 11 fragments respectively (upper). Western blot of plasma membrane proteins of the mutants of AtPIP2;4 with different fragments replaced by those of AtPIP1;1 (nether). Membrane proteins were extracted from transformed yeast, and 6xHis antibody was applied. (B) Hand-drawn structure of 2;4ΔTM2/1;1TM2 and 2;4ΔTM3/1;1TM3. (C) Confocal images of AtPIP2;4-eGFP, 2;4ΔTM2/1;1TM2-eGFP, and 2;4ΔTM3/1;1TM3-eGFP expressing yeast cells (left) and Xenopus oocytes (right). White scale lines represent the length of 5 μm (left) and 100 μm (right).
	Figure 3. Successful localization of 1;1Δ(TM2+TM3)/2;4(TM2+TM3) in the PM. (A) Hand-painted structure of 1;1TM2/2;4TM2, 1;1TM3/2;4TM3, and 1;1Δ(TM2+TM3)/2;4(TM2+TM3). (B) Western blot of membrane proteins extracted from AtPIP2;4-, AtPIP1;1-, 1;1ΔTM2/2;4TM2-, 1;1ΔTM3/2;4TM3-, or 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-transformed yeast cells using 6xHis antibody. (C) Confocal images of 1;1ΔTM2/2;4TM2-eGFP, 1;1ΔTM3/2;4TM3-eGFP, and 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-eGFP expressing yeast cells (left) and Xenopus oocytes (right). White scale lines represent the length of 5 μm (left) and 100 μm (right). (D) Alignment of transmembrane helix 2 (TM2) and TM3 from AtPIP1;1 and AtPIP2;4. Red letters are the varying residues. Numbers are the sites of the residues. (E and F) Interaction of mutants of AtPIP1;1 and AtPIP2;4 using SUB Y2H system. The transformed yeast cells were series-diluted, and 5 μl of the yeast suspension was spotted on the SD-WLAH [synthetic dextrose minimal media without W (tryptophan), L (leucine), H (histidine), and A (alanine)] plates.
	Figure 4. Function analysis of AtPIP1;1, AtPIP2;4, and their mutants. (A) Bar graph of Pf values of Xenopus oocytes injected with 2 ng cRNA coding eGFP, AtPIP1;1-eGFP, AtPIP2;4-eGFP, 1;1ΔTM2/2;4TM2-eGFP, 1;1ΔTM3/2;4TM3-eGFP, or 1;1Δ(TM2+TM3)/2;4(TM2+TM3)-eGFP. Data shown are the means ± SDs. Different letters indicate significant differences in multiple comparisons of each Pf value using an one-way ANOVA analysis, P < 0.01; for each sample, 10 technical replicates were measured, and the experiments were independently repeated three times. (B) Growth status of yeast cells transformed with AtPIP2;4, AtPIP1;1, or their variants spotted on YPDA plates plus 0 or 3 mM H2O2. (C) DCF curves of yeast cells transformed with AtPIP2;4, AtPIP1;1, or their variants with DCF stored and 3 mM H2O2 added, showing fluorescent signal detected by microplate reader. Data are the mean ± SDs. For each sample, triplicates (technical replicates) were measured, and the experiments were independently repeated three times.
	Figure 5. Subcellular localization and function analysis of AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, and their mutants. (A) Confocal images of yeast cells (left) and Xenopus oocytes (right), which were expressing AtPIP1;2-eGFP, AtPIP1;3-eGFP, AtPIP1;4-eGFP, AtPIP1;5-eGFP, and their eGFP-fused mutants. White scale lines represent the length of 5 μm (left) and 100 μm (right). (B) Bar graph of Pf values of Xenopus oocytes injected with 2 ng cRNA coding eGFP, AtPIP1;2-eGFP, AtPIP1;3-eGFP, AtPIP1;4-eGFP, AtPIP1;5-eGFP, and their eGFP-fused mutants. On bar graphs, different letters indicate significant differences in multiple comparisons of each Pf values using an one-way ANOVA analysis, P < 0.01; for each sample, 10 technical replicates were measured, and the experiments were independently repeated three times. (C) Growth status of yeast cells transformed with AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, or their mutants spotted on YPDA plates plus 0 or 3 mM H2O2. (D) DCF curves of AtPIP1;2, AtPIP1;3, AtPIP1;4, AtPIP1;5, or their mutant-transformed yeast cells with DCF stored and 3 mM H2O2 added, showing fluorescent signal detected by microplate reader. Data are the mean ± SDs. For each sample, triplicates (technical replicates) were measured, and the experiments were independently repeated three times.
	Figure 6. Structure analysis and localization working model of AtPIP1;1 and AtPIP2;4. (A) Tetrameric structure of AtPIP2;4 with AtPIP1;1 aligned in. TM2s and TM3s are highlighted with cyan (AtPIP1;1) and green (AtPIP2;4). (B) Structure of four TM2s from AtPIP2;4 tetramer (green) with those from AtPIP1;1 (cyan) aligned in. The Q residue and the corresponding L residue are in the form of sticks. Red in the sticks represents oxygen atom, while blue represents nitrogen atom. (C) Sequences alignment of TM3 region of AtPIP1;1 with cyan background and AtPIP2;4 with green background (upper) and structure of AtPIP1;1 and AtPIP2;4 with TM3 highlighted in cyan (AtPIP1;1) or green (AtPIP2;4) (nether). Red letters are the varying residues, and LxxxA region is in the square frame (upper). All outfacing residues are in the form of sticks (nether). (D) Working model of the subcellular localization of plasma membrane intrinsic proteins. Other relative residues are some of the different residues except the L81 and LxxxA motif in TM2 and TM3 between PIP1s and PIP2s that might also take part in localization of PIPs.

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