Yeast handbook

The regulatory domain of mouse SPAK (residues 353 to 556) fused to the GAL4 activating domain in pACT2 was originally isolated from a Clontech mouse brain library (Piechotta, et al., 2002 (link)). The clone was re-transformed into PJ69-4A cells (James, et al., 1996 (link))) according to standard yeast handling procedures (Yeast Handbook, Clontech) and plated on –LEU plates. Sense and anti-sense oligonucleotides were purchased from Sigma Genosys. Upon annealing, the oligonucleotides create overhanging 5’ EcoRI and 3’ BamHI sites that are directly used for ligation. The annealed adaptors encode 14 amino acid peptides that includes EF (EcoRI site), QLVG (linker), RFQVT or mutant (PF2 target peptides), and SSK followed by a stop codon. The QLVGRFQVTSSK sequence is original to the SPAK binding site in WNK4 ((Piechotta, et al., 2003 (link); Villa, et al., 2007 (link)). The adaptors are ligated downstream of the Gal4 binding domain in the pGBDUc2 vector. Yeast cells containing the regulatory domain of SPAK in pACT2 were then transformed with individual peptide clones in pGBDUc2 and plated on double dropout -LEU, -URA plates. Yeast clones were then re-streaked on double-dropout plates as controls and triple dropout –LEU, -URA, -His, 2 mM 3-amino-1,2,4-triazole plates.

The plasmids used in this study were constructed by amplifying the glenth coding sequence from giardial genomic DNA and cloned in multicopy URA3 or HIS3 vectors. Expression was driven by the MET25 promoter. DNA manipulations were performed using standard techniques, employing T4 DNA polymerase-mediated ligations in Escherichia coli. Amino acid substitutions were made using the QuikChange XL site-directed mutagenesis kit (Stratagene, La Jolla, CA) according to the manufacturer’s instructions. All restriction enzymes were purchased from New England Biolabs (Ipswich, MA). Yeast was transformed by the Li-Acetate method following the Clontech yeast handbook.