Phospho-mimic and deficient AtPIP2;1 mutants had altered Na+ accumulation in S. cerevisiae strain B31
To test whether mimicking AtPIP2;1 C-terminal phosphorylation states influenced cell Na+ accumulation in yeast, the phospho-mimic and deficient mutants were expressed in a Na+ efflux compromised strain B31 (Δena1::HIS3::ena4, Δnha1::LEU2 ). This strain is deficient in Na+ efflux (Bañuelos et al., 1998) enabling greater potential to distinguish any differences in intracellular Na+ accumulation under salt treatment associated with Na+ uptake through plasma membrane localised Na+ transporters. Na+ accumulation in B31 yeast expressing phospho-mimic and deficient AtPIP2;1 mutants was measured after the yeast had been incubated in a 70 mM NaCl uptake buffer for 40 min. The yeast cell Na+ accumulation was measured in samples representing all controls and mutants before and after the uptake assay and no significant differences in yeast cell sodium content were observed in the samples prior to the incubation in the NaCl uptake buffer (Figure S5a).
Yeast expressing AtPIP2;1 WT accumulated greater Na+than the empty vector control and AtPIP2;7 (Figure 4). AtPIP2;7 was used as an additional control as this PIP was previously reported to lack Na+ induced currents when expressed in X. laevis oocytes (Kourghi et al., 2017). Comparison of Na+ accumulation for yeast expressing each of the single and double phospho-mimic/deficient mutants of interest, showed that only yeast expressing AtPIP2;1 S280A and S283D accumulated significantly more Na+ than the empty vector controls (Figure 4). However, mimicking single phosphorylation and de-phosphorylation mutations at S280 and S283 sites influenced Na+ accumulation (Figure 4). Yeast expressing AtPIP2;1 S280A accumulated significantly greater Na+ than S280D. Whereas greater Na+ accumulation was observed for S283D relative to S283A (Figure 4). In the case of A/D and D/A, although yeast expressing both double mutants had Na+contents not significantly different to the empty vector control, A/D caused significantly greater Na+ accumulation than D/A (Figure 4). Interestingly, when both CTD sites were mimicked in either a phosphorylated or de-phosphorylated state, (D/D and A/A), a similar level of Na+ accumulation was observed not significantly different to that of empty vector controls (Figure 4).
AtPIP2;1 WT and CTD mutants were capable of facilitating K+ transport in X. laevis oocytes (Figure 2A and C). To examine whether similar trends occur when the proteins of interest were expressed in yeast, the K+ contents of yeast samples were examined. Prior to the incubation in the NaCl uptake buffer there were no significant differences observed between genotypes (Figure S5b). This was also the case after incubation in the 70 mM NaCl buffer solution for 40 min (Figure S6).