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.