Y2146

The detailed protocol of the Y2H assay are as described in Jani et al. (2015) (link). Briefly, the Y2HGold yeast strain (Clontech) was transformed with different bait and prey plasmids as indicated in the figure (Table S4) by a lithium acetate transformation protocol. The yeast transformants were selected on minimal medium plates supplemented with complete amino acid mix (Y0750, Sigma-Aldrich), lacking leucine and tryptophan (referred to here as +His medium). Next, transformants were grown to exponential phase, serially diluted and then spotted on +His, −His (Y2146 –Sigma-Aldrich) and −His [+2 or 10 mM 3AT (3-Amino-1,2,4-triazole)] plates. Plates were incubated for 3–5 days at 30°C and then imaged under white light in a Bio-Rad Molecular Imager. The yeast transformants that grew on –His (+3AT) were considered as demonstrating a positive interaction between bait and prey proteins.

The yeast strain Y2HGold (Clontech) was maintained on YPD (yeast extract, peptone, dextrose) plates. Transformation of different bait and prey plasmids (supplementary material Table S1) in the Y2HGold was performed by a modified lithium acetate procedure as described in the Yeast Two-Hybrid System book (Golemis and Brent, 1997 ). The yeast transformants were selected on minimal medium plates supplemented with yeast synthetic drop-out amino acid mix (Y0750 from Sigma-Aldrich) lacking leucine and tryptophan (referred to here as +His or +Histidine medium). For the reporter assay, transformants were grown to an absorbance of 0.4–0.5 at 600 nm and then spotted on plates that were +His, −His (−His medium, Y2146 from Sigma-Aldrich) and −His containing 2 mM 3AT after serial diluting the cultures by tenfold in sterile water. Plates were grown at 30°C for 3–5 days and then imaged under white light in a Bio-Rad Molecular Imager. Note that the STX13 (WT and Y3F) constructs showed autoactivation on −His, but not on −His (3AT) reporter plates (supplementary material Fig. S3B). Thus, growth of yeast transformants on −His (3AT) reporter plates was considered as criteria for protein–protein interaction.

The coding sequences of the 21 putative interactors and 26 calmodulin/calmodulin-like CaM genes were cloned into pGADT7 (Clontech) vector and PdIQD10 into pGBKT7 (Clontech) vector using In-Fusion^® Advantage PCR Cloning Kit (Clontech). The primers used for cloning are listed in Table 2. The generated constructs were transformed into yeast Y2H Gold (Clontech) competent cells using Fast^TM Yeast Transformation (G-Biosciences Cat. #GZ-1) kit. Transformed yeast cells were plated on Yeast Synthetic Drop-out (SD) media lacking Leucine and Tryptophan amino acids (Sigma–Aldrich #Y0750) to select the positive transformants for both plasmids. At least two clones were individually tested either on SD media lacking histidine, leucine, and tryptophan (Sigma–Aldrich #Y2146) or on SD media lacking histidine, leucine, tryptophan and adenine (Sigma–Aldrich #Y2021) in three different transformation experiments to determine the interaction result.