Yeast Na+ Content
The full-length cDNA of AtPIP2;1 and AtPIP2;1 single- and double-point C-terminal phosphorylation mutations (Table 1) generated in gateway enabled entry vectors were sub-cloned into pYES-DEST 52 (Invitrogen) yeast expression destination vector driven by GAL1 promoter through LR reaction. Empty vector and AtPIP2;7 (following Kourghiet al., 2018) were also included as negative controls. Confirmed constructs were transformed into S. cerevisiae strain B31 (Δena1::HIS3::ena4, Δnha1::LEU2 ) (Bañuelos et al., 1998) using Frozen-EZ Yeast Transformation II kit (Zymo Research). Successful transformants grown on selective media (Amino acids supplements drop-out without uracil (Sigma), 0.67 % (W/V) Na+-free Yeast nitrogen base (Formedium), 10 mM MES, 2% glucose (W/V), 2% agar (W/V) and pH 5.6) were inoculated into same liquid media for overnight incubation at 30 °C with shaking. The overnight yeast cultures were diluted to 0.05 at OD600nm before further growing in liquid media containing 2% galactose (W/V) to induce the gene expression in B31 yeast for 18 h. To test the Na+influx, yeast culture were first centrifuged at 2,500 x g for 3 min and the supernatant was removed. Yeast cells were then gently resuspended in Na+ uptake buffer (70 mM NaCl, 10 mM MES, 10 mM EGTA, pH 5.6) and incubated at 30 °C for 40 min before being harvested through a 0.45 µm Millipore filter (Merck). Yeast pellets collected by Millipore filter were washed three times using ice-cold washing buffer (20 mM MgCl2 and D-mannitol to adjust the osmolarity similar to Na+ uptake buffer). To reduce the potential for uncontrolled ion fluxes across yeast membrane during wash, a running vacuum was connected to the Millipore filter to quickly remove the supernatant and washing buffer. Harvested yeast samples were dried on the Millipore filter at 70 °C for two days and were digested in 1% HNO3 acid at same temperature for another 2 days. Digested yeast sample was vortexed and centrifuged briefly at 13,000 x g and the supernatant was diluted with MilliQ water. Yeast Na+ and K+ contents were measured using Atomic Absorption Spectrophotometer (AAS; Shimadzu AA-7000) according to manufacturer’s instructions.

Results