Single Mutations in the Transmembrane Domains of Maize Plasma Membrane Aquaporins Affect the Activity of Monomers within a Heterotetramer  Marie C. Berny, Dimitri Gilis, Marianne Rooman, François Chaumont  Molecular Plant  Volume 9, Issue 7, Pages 986-1003 (July 2016) DOI: 10.1016/j.molp.2016.04.006 Copyright © 2016 The Author Terms and Conditions

Figure 1 PIP1;2-PIP2;5 Hetero-Oligomers Are Formed by Homo- and Heterodimers. (A and B) Principle of the experiment. PIP2;5 (in blue) and PIP1;2 (in red) monomers have a molecular mass of about 30 kDa while the mCFP or mYFP (in green) has a mass of about 25 kDa. The presence of the mYFP or mCFP increases the molecular mass of the dimer and allows the homodimers (bands at ∼55 kDa) and heterodimers (bands at ∼70 kDa) to be distinguished (A). The black curve represents a disulfide bridge. After affinity chromatography purification, migration on a polyacrylamide gel in the absence of DTT to preserve the disulfide bridge, and immunodetection with antibodies (αPIP), the position of the homo- and heterotetramers are shown (B). The different fractions are indicated. (C–F) Microsomes from Xenopus oocytes co-expressing the indicated tagged PIP isoforms were used to solubilize and purify 6His-PIP1;2 or 6His-PIP2;5 on the Ni2+-NTA column. The panels show the tagged PIP1;2 and PIP2;5 in oocyte microsomes (M), flow-through (Ft), last wash (Lw), and eluted fraction (E), after immunodetection using specific antibodies raised against Zm PIP1;2 (αPIP1;2), Zm PIP2;5 (αPIP2;5), or GFP (αGFP). The oocytes were injected with water (C), or co-injected with PIP1;2 and mCFP-PIP2;5 cRNA (negative control for the purification) (D), mYFP-PIP1;2 and 6His-PIP2;5 cRNA (E), or 6His-PIP1;2 and mCFP-PIP2;5 cRNA (F). The molecular mass markers are indicated on the left. The samples were loaded into an 8% polyacrylamide gel in the absence of DTT. Small pictograms show the PIP2;5 monomer in blue, the PIP1;2 monomer in red, and mCFP or mYFP in green; the black curve represents a disulfide bridge. (G and H) PIP association in homo- and/or heterodimers leads to the formation of heterotetramers of different compositions. A disulfide bridge between the Cys residues located in loops A (black curve) binds two adjacent monomers, forming homo- and heterodimers (G). Several combinations of their association leads to the formation of heterotetramers, which can contain from one to three units of PIP1;2 (in red) or PIP2;5 (in blue) isoforms (H). Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 2 Localization of the Putative Interacting Residues in the PIP1;2–PIP2;5 Heterotetrameric Structure. (A and B) Extracytosolic (A) and side (B) views of the a PIP1;2–PIP2;5 heterotetramer model (in light pink and blue, respectively) composed of two homodimers, showing the localization of the putative interacting residues. Leu81/Gln91, Trp85/Trp95, Phe92/Phe102, and Phe210/Phe220 in PIP2;5 and PIP1;2, respectively, are buried close to the central channel of the tetramer while Lys80 and Phe156 in PIP1;2 as well as Tyr268 in PIP2;5 are located at the extremities of the TMs near the membrane interface. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 3 Characterization of the PIP2;5W85A, PIP2;5F92A, and PIP2;5F210A Isoforms. (A) Pf measurements of Xenopus oocytes expressing PIP2;5WT, PIP2;5W85A, PIP2;5F92A, and PIP2;5F210A alone or with PIP1;2WT. Oocytes injected with water were used as negative controls. Chart bars are expressed as the means of measurements of 12 oocytes. Error bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (B) PIP protein abundance in microsomes from oocytes expressing PIP2;5WT, PIP2;5W85A, PIP2;5F92A, and PIP2;5F210A alone or with PIP1;2WT. Immunodetection was performed using specific antibodies raised against PIP1;2 and PIP2;5 after SDS-PAGE migration in reducing conditions. Colloidal blue staining was used as a loading control, and microsomes from oocytes injected with water as a negative control. Bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. A band with a molecular mass of 37 kDa was sometimes detected using anti-PIP2;5 antibodies. Its origin is unknown. (C) Confocal images of fixed oocytes expressing mCFP-PIP2;5WT, mCFP-PIP2;5W85A, mCFP-PIP2;5F92A, and mCFP-PIP2;5F210A. Similar parameters were used for all the acquisitions. Scale bars, 50 μm. (D) Relative intensity of the mCFP fluorescence in the plasma membrane and the cytosol (M/C) of oocytes expressing WT and mutated mCFP-PIP2;5. The fluorescence intensities were quantified using ImageJ software. The results are expressed as the means of eight oocytes. Bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (E) Subcellular localization of mCFP-PIP2;5W85A, mCFP-PIP2;5F92A, and mCFP-PIP2;5F210A transiently expressed in maize leaf epidermal cells. On the left, acquisitions were performed using the Z-stack mode. On the right, one optical section is shown. Scale bars, 10 μm. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 4 Expression of WT and Mutated PIP1;2 Alone or with PIP2;5W85A. (A) Pf measurements of Xenopus oocytes expressing PIP1;2WT, PIP1;2F156A, or PIP1;2F220A alone or with PIP2;5W85A. Oocytes injected with water were used as negative controls. Chart bars are expressed as the means of measurements of 12 oocytes. Error bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (B) Co-purification of WT and mutated 6His-PIP1;2 isoforms co-expressed with PIP2;5W85A. Microsomes from Xenopus oocytes co-expressing the indicated tagged PIP isoforms were used to solubilize and purify 6His-PIP1;2WT, PIP1;2F156A, or 6His-PIP1;2F220A on Ni2+-NTA columns. The panels show the PIP1;2 and PIP2;5 isoforms in oocyte microsomes (M), flow-through (FT), last wash (LW), and eluted fraction (E) after SDS-PAGE migration in reducing conditions and immunodetection using specific antibodies raised against PIP1;2 (αPIP1;2) and PIP2;5 (αPIP2;5). The molecular mass markers are indicated on the left. The bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 5 Characterization of the PIP1;2W95A, PIP1;2W102A, and PIP1;2W156A Isoforms. (A) Pf measurements of Xenopus oocytes expressing PIP1;2WT, PIP1;2W95A, PIP2;5F102A, and PIP2;5F156A alone or with PIP2;5WT. Oocytes injected with water or PIP2;5 cRNA were used as negative and positive controls, respectively. Chart bars are expressed as the means of measurements of 12 oocytes. Error bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. Signals of the PIP1;2 isoforms were quantified and normalized according to the loading control (“Quantif.,” in a.u.). The monomer over dimer ratio is indicated (M/D). (B) PIP protein abundance in microsomes from oocytes expressing PIP1;2WT, PIP1;2W95A, PIP2;5F102A, and PIP2;5F156A alone or with PIP2;5WT. Immunodetection was performed using specific antibodies raised against PIP1;2 and PIP2;5 after SDS-PAGE migration in reducing conditions. Colloidal blue staining was used as a loading control, and microsomes from oocytes injected with water as a negative control. Bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. A band with a molecular mass of 37 kDa was sometimes detected using anti-PIP2;5 antibodies. Its origin is unknown. (C) Confocal images of fixed oocytes expressing mYFP-PIP1;2WT, mYFP-PIP1;2W95A, mYFP-PIP2;5F102A, and mYFP-PIP2;5F156A alone or with mCFP-PIP2;5WT. The oocytes were previously fixed with paraformaldehyde. Similar parameters were used for all the YFP or CFP acquisitions. Scale bars, 50 μm. (D and E) Relative intensity of the fluorescence in the plasma membrane and the cytosol (M/C) of oocytes expressing WT and mutated mYFP-PIP1;2 alone or with mCFP-PIP2;5WT. The fluorescence intensities were quantified using ImageJ software. The results are expressed as the means of eight oocytes. Bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 6 Characterization of the PIP1;2Q91L and PIP1;2W220A Isoforms. (A) Pf of Xenopus oocytes expressing PIP1;2WT, PIP1;2Q91L, and PIP1;2F220A alone or with PIP2;5WT. Oocytes injected with water or PIP2;5 cRNA were used as negative and positive controls, respectively. Chart bars are expressed as the means of measurements of 12 oocytes. Error bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (B) PIP protein abundance in microsomes from oocytes expressing PIP1;2WT, PIP1;2Q91L, and PIP1;2F220A alone or with PIP2;5WT. Immunodetection was performed using specific antibodies raised against PIP1;2 and PIP2;5 after SDS-PAGE migration in reducing conditions. Colloidal blue staining was used as a loading control, and microsomes from oocytes injected with water as a negative control. Bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. (C) Confocal images of fixed oocytes expressing mYFP-PIP1;2WT, mYFP-PIP1;2Q91L, and mYFP-PIP2;5F220A alone or with mCFP-PIP2;5WT. The oocytes were previously fixed with paraformaldehyde. Similar parameters were used for all the YFP or CFP acquisitions. Scale bars, 50 μm. (D) Relative intensity of the fluorescence in the plasma membrane and the cytosol (M/C) of oocytes expressing WT and mutated mYFP-PIP1;2 alone or with mCFP-PIP2;5WT. The fluorescence intensities were quantified using ImageJ software. The results are expressed as the means of eight oocytes. Bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (E) Co-purification of 6His-PIP1;2F220A and PIP2;5WT. Microsomes from Xenopus oocytes co-expressing the indicated tagged PIP isoforms were used to solubilize and purify 6His-PIP1;2WT or 6His-PIP1;2F220A on Ni2+-NTA columns. The panels show the PIP1;2 and PIP2;5 isoforms in oocyte microsomes (M), flow-through (Ft), last wash (Lw), and eluted fraction (E) after SDS-PAGE migration in reducing conditions and immunodetection using specific antibodies raised against PIP1;2 (αPIP1;2) and PIP2;5 (αPIP2;5). The molecular mass markers are indicated on the left. The bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 7 Subcellular Localization of the WT and Mutated mYFP-PIP1;2 Isoforms Transiently Expressed Alone or with mCFP-PIP2;5WT in Maize Leaf Epidermal Cells and Mesophyll Protoplasts. (A and B) Representative confocal images of maize epidermal cells transiently expressing mYFP-PIP1;2WT, mYFP-PIP1;2K80H, mYFP-PIP1;2Q91L, mYFP-PIP1;2F102A, mYFP-PIP1;2F156A, or mYFP-PIP1;2F220A alone (A) or with mCFP-PIP2;5WT (B). One optical section and Z-stack acquisitions are shown on the top and bottom panels, respectively. Scale bars, 10 μm. (C) Representative confocal images of maize mesophyll protoplasts transiently co-expressing mYFP-PIP1;2WT, mYFP-PIP1;2Q91L, mYFP-PIP1;2F156A, or mYFP-PIP1;2F220A with the ER marker mCFP-HDEL and labeled with the FM4-64 dye used here as a plasma membrane marker. Scale bars, 5 μm. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 8 Characterization of PIP1;2F220A Co-expressed with PIP1;2WT. (A) Pf measurements of Xenopus oocytes expressing PIP1;2F220A alone or with PIP1;2WT. Oocytes injected with water, PIP1;2WT, and/or PIP2,5WT cRNA were used as controls. Chart bars are expressed as the means of measurements of 12 oocytes. Error bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. (B) PIP protein abundance in microsomes from the oocytes described in (A). Immunodetection was performed using specific antibodies raised against PIP1;2 and PIP2;5 after SDS-PAGE migration in reducing conditions. Colloidal blue staining was used as a loading control, and the bands at about 30 and 60 kDa correspond to the monomeric (M) and dimeric (D) forms of PIPs, respectively. Signals of the PIP1;2 isoforms were quantified and normalized according to the loading control (“Quantif.,” in a.u.). The monomer over dimer ratio is indicated (M/D). (C) Confocal images of fixed oocytes expressing mCFP-PIP1;2WT, mYFP-PIP1;2F220A, or both proteins. Similar parameters were used for all the acquisitions. Scale bars, 50 μm. (D) Relative intensity of the mCFP and YFP fluorescence in the plasma membrane and the cytosol (M/C) of oocytes expressing mCFP-PIP1;2 alone or mYFP-PIP1;2F220A alone or with CFP-PIP1;2WT. The fluorescence intensities were quantified using ImageJ software. The results are expressed as the means of eight oocytes. Bars represent 95% CIs. Letters represent groups of data with means significantly different (p < 0.05) according to ANOVA and Bonferroni post hoc test. Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions

Figure 9 Predicted Interactions Involving the Phe220 Residue of PIP1;2 Before and After Mutation into Ala. (A) PIP1;2F220 (red) interacts with PIP2;5W85 (blue) and PIP2;5F92 (green) within the PIP1;2-PIP2;5 heterotetramer (in pink and light blue, respectively). PIP1;2F220 is involved in aromatic–aromatic and hydrophobic interactions (purple and orange arrows, respectively). (B) After mutation of Phe220 into Ala, the residue interacts through only hydrophobic interactions with Phe92 and also Ile93 (magenta). Molecular Plant 2016 9, 986-1003DOI: (10.1016/j.molp.2016.04.006) Copyright © 2016 The Author Terms and Conditions