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