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Figure 1: Exogenous application of membrane permeable cAMP and cGMP analogues as kinase stimulators and the kinase inhibitor H7 influence ionic conductance of AtPIP2;1 injected oocytes.Oocytes were either untreated or were pre-treated in Low Na+ Ringers solution that contained 1 mM 8-Br-cAMP (cAMP), 1 mM 8-Br-cGMP (cGMP) or 10 µM H7 dihydrochloride (H7) or H7 followed by cAMP/cGMP. TEVC was performed in a ‘Na50’ solution. The ionic conductance of treated water injected and AtPIP2;1 cRNA injected oocytes were normalised to untreated water injected andAtPIP2;1 cRNA injected oocytes respectively. (a)Relative ionic conductance of control oocytes. (b) Relative ionic conductance of AtPIP2;1 injected oocytes. Data was compiled from at least two independent oocytes batches with the exception of the H7 + cNMP treatment where data from one batch of oocytes is represented. Data is represented as mean relative conductance ± SEM where each point represents a single oocyte. Significant differences (P < 0.001) are indicated by different letters using one-way ANOVA, Fisher’s post test, or by an * (un-paired t-test).
Figure 2: Phosphorylation mimic of AtPIP2;1 S280 and S283 residues influence AtPIP2;1 facilitated cation transport. Oocytes were injected with 46 nL water (Control) or with 46 nL water containing 23 ngAtPIP2;1 WT (WT) or S280A, S280D, S283A, S283D, A/A, D/A, A/D or D/D cRNA. Representative superimposed currents as a function of time of(a) AtPIP2;1 single phosphorylation mutants in ‘Na100’ (Na+) and ‘K100’ (K+), and(b) AtPIP2;1 double phosphorylation mutants in ‘Na100’ (Na+). Currents were recorded starting from -40 mV holding potential for 0.5 s and ranging from 40 mV to –120 mV with 20 mV decrements for 0.5 s before following a –40 mV pulse for another 0.5 s. Ionic conductance of oocytes expressing (c) AtPIP2;1 single phosphorylation mutants in ‘Na100’ (Na+) and ‘K100’ (K+), and (d) AtPIP2;1 double phosphorylation mutants in ‘Na100’ (Na+). Ionic conductance was calculated by taking the slope of a regression of the linear region across the reversal potential (–60 mV to +40 mV). (e)Na+ content of oocytes incubated in ‘Na100’ for 24 h. Data in (c-e) is compiled from three independent oocyte batches and is shown as mean ± SEM where each data point represents an individual oocyte. Significant differences (P<0.05) are indicated by different letters (one-way ANOVA, Fisher’s post-test), or by an * (un-paired t-test).
Figure 3.Phosphorylation mimics of AtPIP2;1 S280 and S283 residues influences its osmotic water permeability and the relationship between osmotic water permeability and ionic conductance. Osmotic water permeability (Pos) and ionic conductance of water injected (n= 13) and AtPIP2;1 Wild-type (n=37), S280D (n=20) , S280A (n= 13), S283D (n= 19), S283A (n= 17), A/A(n= 25), D/A (n= 16), A/D (n= 27) or AtPIP2;1 D/D (n= 30) cRNA injected oocytes was determined via the swelling assay and TEVC, respectively. (a) Ionic conductance collected from multiple batches were allocated into 10 µS bins and the mean ± SEM of each binned group and corresponding Pos is plotted. Individual conductance was plotted against the corresponding Pos for each oocyte (data shown in Figure S3). A single exponential decay best fit the combined data (P< 0.005). The red and blue dashed lines indicate the mean ionic conductance and Pos of water injected (control) oocytes. (b)Frequency histogram of Posfor each of the phospho-mimics in decreasing order of the mean Pos from left to right. The blue dashed line in each histogram indicated the mean of Pos in water injected (control) oocytes. (c) Frequency histogram of ionic conductance for each of the phospho-mimics in increasing order of the mean from left to right. The red dashed line in each histogram indicated the mean of ionic conductance in water injected (control) oocytes.(d) Comparison of the order of decreasing Posand increasing ionic conductance. Genotypes marked by shaded boxes follow the same relative order for the change in mean Pos and ionic conductance.
Figure 4: Intracellular Na+ accumulation varied in yeast expressing AtPIP2;1 CTD phosphorylation mimic mutants.Empty vector, AtPIP2;7, AtPIP2;1WT and all versions of CTD of AtPIP2;1 mutants were each expressed in the B31 yeast mutant strain. After suspension in NaCl uptake buffer (70 mM NaCl, 10 mM MES, 10 mM EGTA, pH5.6) for 40 min, intracellular Na+ contents were measured. Data are compiled from three independent experimental batches each comprised of three independent replicate cultures, and is represented as mean ± SEM. Significant differences (P<0.05) are indicated by different letters (one-way ANOVA, Fisher’s post-test). N= Empty (10), AtPIP2;7 (7), AtPIP2;1 WT (10), S280A (7), S280D (7), S283A (7), S283D (10), A/D (7), D/A (7), AtPIP2;1 A/A (7) and D/D (7).
Figure 5: Subcellular localisation of AtPIP2;1 wild-type and S280/S283 phospho-mutants in yeast. (a) A control showing that GFP alone results in a diffuse cytosolic localised signal. (b) SEC63::RFP endoplasmic reticulum marker. The yeast ER network consists of the prominent nuclear envelope ER domain (nER) and a peripheral or cortical ER domain (cER). The cER lies just beneath the plasma membrane but is not continuous around the perimeter with gaps distinguishing it from plasma membrane localisation (solid triangle). Cytoplasmic tubules link the two ER domains (*). (c) Wild-type AtPIP2;1::eGFP localises to a distinct continuous ring of expression around the cell perimeter coinciding with the plasma membrane (PM). GFP signal is also weakly present in the tonoplast of the vacuole (V). In this example, no expression is detected in the nER. (d-e) The single phospho-mimetic S280D mutant commonly shows a continuous ring of PM localisation along with a substantially stronger GFP signal co-localised with the ER marker in both the peripheral (open arrow heads) and internal ER networks (nER). (f) The single phospho-mimetic S283D mutant shows a clean sharp localisation around the PM with little to no ER co-localisation. Weak GFP signal is occasionally observed in the periphery of the vacuoles (V). (g-h) The localisation of the double phosphorylated mimetic D/D mutant occurs almost exclusively in the PM with comparably weak signal detectable in the tonoplast of the vacuole (V) and little to no signal in the ER. (i) The double A/D mutant localises to the PM. Approximately half the yeast cells examined also exhibit strong co-localisation to the ER. (j) The frequency of yeast cells with GFP signal detected in the PM only versus co-localisation in both the PM and ER. Asterisks (*) denote statistically significant difference (Fisher’s exact test p ≤ 0.05). N = WtAtPIP2;1(53), S280A(57), S283S(161), S283A(32), S283D(94), A/A(117), D/A(64), A/D(139) , D/D(83).