Phosphorylation states at CTD sites influence AtPIP2;1 facilitated cation transport differently in different heterologous expression systems
In the oocyte system, greater Na+ and K+ conductances and intracellular Na+ accumulation were consistently observed for single and double phospho-mimic mutants relative to the other mutants, regardless of which CTD site was mimicking a phosphorylated state. Oocytes expressing AtPIP2;1 S280D, S283D, D/A, A/D and D/D had significantly greater Na+ conductance and accumulation. However, the trends for Na+ accumulation in yeast for the phospho-mimic (S->D) versions were different to that in oocytes. In the B31 yeast system, only the yeast expressing AtPIP2;1 S280A and AtPIP2;1 S283D were observed to have significantly increased net Na+ accumulation compared to the empty vector control (Figure 4). These results indicate that different S280 and S283 phosphorylation states might have distinct effects on facilitating Na+ flux through AtPIP2;1 in yeast. Expression of the AtPIP2;1 double phospho-mimic mutant, D/D, in yeast resulted in the accumulation of similar amounts of Na+ relative to the values for the empty vector control, which differs from the trend in oocytes where there was increased Na+ accumulation observed for D/D injected oocytes (Figure 2e). The fact that AtPIP2;1 S283D sub-cellular localisation was similar to the D/D mutant (Figure 5J) indicates that S280 maybe endogenously phosphorylated by the yeast for the S/D version, and potentially this could be triggered in response to position 283 being a phospho-mimic residue. It was the D/D version that was also associated with particularly clear PM abundance when expressed in the aqy1/aqy2 mutant yeast. We also observed that AtPIP2;1 WT and phospho-mimic mutants differed in K+-associated conductance in oocytes (Figure 2), but we did not observe significant differences in K+ accumulation for these variants when expressed in yeast, following a NaCl treatment (Figure S6). Differences in oocytes relative to yeast cells such as the absence of a vacuole, and associated differences in signalling and regulatory process could result in the different behaviours. Plant aquaporin trafficking to the PM has been reported to be regulated by syntaxin proteins; for example, it has been shown that AtPIP2;7 trafficking depends on SYP61 (Hachez et al., 2014). Yeast also employs a set of SNAREs to drive a series of membrane fusion events (Burri and Lithgow, 2004), which could also potentially interact with AtPIP2;1 to influence the sub-cellular localization and subsequently affect cation transport capacity. Yeast and oocyte cells have distinct sets of endogenous protein kinases, and there may be other phosphorylation sites within AtPIP2;1 that could be differently phosphorylated in the two systems that control ion conductance. One possibility is that another site may be preferentially phosphorylated in yeast which reduces the ion conductance of the S280D mutant. For this mutant and also the S283D there was a large spread in ion conductances in oocytes ranging from near to that of water injected controls up to the maximum ion conductance observed (Figure 3d) which was not observed in yeast (Figure 4). This could indicate that another site is variably phosphorylated in oocytes that reduces ion conductance while it may be more consistently phosphorylated in yeast.