Phosphorylation state of S280 and S283 influences AtPIP2;1
localisation in yeast
Mimicking changes in C-terminal phosphorylation states of AtPIP2;1 not
only altered Na+ and H2O conductance
in X. laevis oocytes (Figure 2 and 3), but also influenced the
sub-cellular localisation of AtPIP2;1 in S.cerevisiaeaqy1/aqy2 double mutant yeast strain (Figure 5). Sub-cellular
localisation tendencies of the AtPIP2;1 phospho-mutants were
monitored in yeast using both N- or C-terminal GFP fusions. Fusion of
AtPIP2;1 wild-type or AtPIP2;1 phospho-mutants to GFP, redirected GFP
from a diffuse cytosolic pattern (Figure 5A) to a predominantly sharp
ring around the cell perimeter coinciding with the plasma membrane (PM;
Figure 5C-I). Weaker GFP signal associated with the tonoplast of the
vacuole was also frequently observed. In addition, a proportion of cells
showed internal and patchy perimeter GFP signal, matching the
localisation pattern of the SEC63::RFP endoplasmic reticulum (ER) marker
(Figure 5B). The detectable frequency and intensity of the ER
co-localisation differed between the AtPIP2;1 wild-type and some of the
AtPIP2;1 280/283 phospho-mimic mutants (Figure 5J). A difference was
observed for the AtPIP2;1 S280D mutant, which had frequently occurring
intense GFP signal co-localising to the ER (Figure 5D, E and J),
relative to the trend observed for the AtPIP2;1 S283D and AtPIP2;1 D/D
mutants, which had distinct GFP signal around the perimeter consistent
with PM localisation, and less frequent or intense ER co-localisation
(Figure 5F, G, H and J). The tendency of the AtPIP2;1 S280A and A/D
mutants to co-localise to the ER was more likely than for wild-type
(Figure 5I and J). GFP localisation patterns for the phospho-deficient
AtPIP2;1 S283A and A/A mutants along with the D/A mutant were equivalent
to that of wild-type AtPIP2;1 (Figure 5J). There was no discernible
difference in the localisation patterning whether the GFP was fused to
the N- or C- terminal (data not shown).
Comparisons between the phospho-mutants reveal co-ordinated effects of
positions S280 and S283 in determining sub-cellular localisation. The
more prominent PM targeting of the AtPIP2;1 D/D mutant in comparison to
the A/A, D/A, or A/D mutants, indicates that mimicking of
phosphorylation of both S280 and S283 was required to promote more
distinctly PM localisation (Figure 5J). The distinct ER co-localisation
of AtPIP2;1 S280D was not observed in either of the two other S280D
phospho-mimic mutants (D/A or D/D) (Figure 5J), indicating that a serine
at position 283 could be specifically required in combination with the
phospho-mimic aspartic acid at position 280 to achieve the distinct ER
co-localisation.
Discussion
AtPIP2;1 is able to facilitate water and monovalent cation transport
activity in vivo , and this function is influenced by the
phosphorylation states of CTD residues S280 and S283 (Figures 1-4). InX. laevis oocytes, S280 and S283 phosphorylation status
influenced the transport function of AtPIP2;1, such that relatively
greater water transport occurred when these sites mimicked an
un-phosphorylated state and greater ion transport occurred when they
mimicked a phosphorylated state (Figure 2 and 3). A
phosphorylation-dependent inverse relationship was observed for
AtPIP2;1-facilitated water transport relative to ion transport (Figure 3
and Figure S3, 4), where there was approximately ten-fold changes in
both permeabilities (Figure S3). Yeast expressing the different AtPIP2;1
S280 and S283 mutation combinations accumulated different amounts of
intracellular Na+ following incubation in a buffer
containing 70 mM NaCl (Figure 4). S280 and S283 phosphorylation status
also influences the distribution and abundance of AtPIP2;1 protein
localising between the ER and PM in yeast (Figure 5).