Yeast vector cloning and yeast localisation
Gateway compatible entry clone containing AtPIP2;1 was generated in pENTR1a and used as a template to generate site-directed single and double point phosphomimetic mutations in AtPIP2;1 (Table S1). Additional non-stop codon versions of these genes were PCR amplified from the pENTR1a clones with primers containing attB sites and inserted into pZeo using BP clonase (Invitrogen). The pZeo non-stop-codon gene versions were shuttled into pAG426-GPD-eGFP by LR clonase II reaction (Invitrogen) to create C-terminal GFP fusion for sub-cellular protein localisation. The pAG426-GPD-GFP vector which confers strong constitutive transgene expression in yeast, were obtained from Addgene (plasmid #14150) and originally deposited by Susan Lindquist (Albertiet al., 2007). The pAG426-GPD-GFP AtPIP2;1 wild-type and the single and double AtPIP2;1 S280 and S283 phosphorylation mutant constructs were transformed into the Saccharomyces cerevisiaeaqy1/aqy2 double mutant yeast strain (Matα; leu2::hisG; trp1::hisG, his3::hisG; ura352 aqy1D::KanMX aqy2D::KanMX) using Frozen-EZ Yeast Transformation II kit (Zymo Research). Theaqy1/aqy2 double mutant yeast strain was gifted by Peter Dahl (Hohmann Lab) (Tanghe et al., 2002).
Sub-cellular GFP signal was visualised on a Zeiss LSM780 confocal laser-scanning microscope (Carl Zeiss) operated by Zen Black software and a DIC x40 oil immersion lens. eGFP was excited at 488nm and emission was captured at 495-570nm and RFP was excited at 561nm and emission captured at 580-735, with 24 µm pinhole and master and digital gains identically set for all images and analysis. Between 30 to 160 cells for each of the AtPIP2;1 wild-type and mutant proteins were scored across 3-4 independent experiments with differences in the localisation patterns between the genotypes consistent across sessions. pSM1959 was obtained through Addgene (Susan Michaelis - Addgene plasmid #41837; Metzger et al., 2008).