Ez yeast transformation kit

Yeast transformations were performed using the EZ-Yeast transformation kit (MP Biomedicals, USA) as per manufacturer’s recommendations. Briefly, 2 µg each of pGADT7 (AD) and pGBKT7 (BD) fusion constructs were co-transformed in yeast AH109 suspended in 125 µl EZ-Transformation solution along with 5 µl carrier DNA. The mixture was incubated at 42°C for 30 min. The cells were then plated on Synthetic Dropout/-Leucine/-Tryptophan, SD/-Leu/-Trp (SD-LW) plates and incubated at 30°C for 3–4 days. Transformants showing robust growth were then streaked on SD/-Leu/-Trp/-Histidine (SD-LWH), SD/-Leu/-Trp/-His/-Adenine (SD-LWHA) and SD-LWHA + X-α-Gal synthetic dropout solid media plates for selection of transformants displaying protein–protein interactions.

For Y2H library screening, ASK13 was initially sub-cloned into the bait vector pGBKT7BD and transformed into the Y2H Gold strain (Clonetech) using an EZ-Yeast Transformation Kit (MP Biomedicals) according to the manufacturers’ instructions. The pGBKT7BD-ASK13 transformed cells were then mated with a normalized Arabidopsis Y2H cDNA library (purchased from Clonetech) and screening was carried out according to the manufacturer’s guidelines. Selection was performed on SD plates lacking histidine, tryptophan, leucine, and adenine but supplemented with X-α-Gal and aureobasidin A. For Y2H one-to-one interactions, the Gateway-compatible vectors pDEST-GBKT7 and pDEST-GADT7 (from TAIR) were used. Selected F-box, non-F-box, and several other ASK cDNAs were cloned into the pENTR/D-TOPO vector using Gateway cloning technology (Invitrogen) and eventually cloned in the pDEST-GADT7 Y2H vector. The resulting plasmids were co-transformed to the Y2H gold strain and then the transformed cells were selected on SD–Ade–His–Leu–Trp/X-α-Gal/aureobasidin agar media. pGADT7-T- and pGBKT7-Lam-transformed Y2H gold strain was used as a negative control and pGADT7-T- and pGBKT7-53-transformed Y2H gold strain was used as a positive control for interactions.

Y3H analysis was performed as previously described (Chaudhuri et al., 2007 (link), 2009 (link)). NL4-3 Nef or mouse tyrosinase cytosolic tail DNAs were subcloned into the pBridge vector (Clontech, CA) along with rat σ1 or σ2. Rat α and δ subunit DNAs were subcloned into the pGADT7 vector (Clonetech, CA). All the point mutants used in this study were generated by site-directed mutagenesis, using the QuikChange II XL (Agilent technologies, Santa Clara, CA). The canonical dileucine-containing tyrosinase tail construct was included as a positive control for the formation of a functional complex, and the σ1 subunit of AP-1 and the δ subunit of AP-3 were included as negative controls for self-activation. The mutations were verified by DNA sequencing. The Saccharomyces cerevisiae HF7c strain was cotransformed with the indicated pairs of pBridge and pGADT7 constructs, using EZ Yeast Transformation Kit (MP biomedicals, Solon, OH). Double transformants were selected and grown on plates lacking Leu, Trp, and Met (+HIS) for 3 days, then the colonies from each transformant were normalized and plated on + HIS plates and plates lacking Leu, Trp, Met, and HIS (−HIS) with/without 3-AT (3-amino-1,2,4-triazole) for 4 days.

A mating-based split-ubiquitin Y2H approach was used. The bait clones were transformed in the yeast strain NMY51 and prey clones in the NMY61 strain using EZ yeast transformation kit (MP Biomedicals, LLC). To rule out any possible autoactivation, the control pDHB1 and pPR3-N vectors were also transformed into NMY51 and NMY61, respectively. Since the split-ubiquitin system allows for checking the bait expression, the positive (pAI-Alg5) and negative control (pDL2-Alg5) vectors were also transformed into NMY61. Bait clones in NMY51 and prey clones in NMY61 were grown overnight in the corresponding auxotrophic media and 100 µl of each culture was dispensed in a 96 DeepWell PP plate (#260252, Thermo Fisher Scientific) and centrifuged at 700 × g for 5 min. The supernatant was discarded and the pellet was re-suspended in 2.5X YPDA media (pH-5.8). The resuspended culture was allowed to grow overnight at 30 °C. After centrifugation and washing of the pellet with sterile distilled water, it was resuspended in 0.9% NaCl and spotted on double drop-out (SD/-L-W) media. The mated yeast clones were allowed to pass through two rounds of patching on SD/-L-W plates and then used for reporter gene analysis on quadruple drop-out media (SD/-L-W-A-H) and X-gal overlay assay (Supplementary Fig. S1).

The interactions of various combinations between ArF-BAR, ArActin, ArWASP, and ArF-BAR domains in yeast cytoplasm were examined using the split-ubiquitin based DUALhunter system (Dualsystems Biotech). The ORFs were cloned at NotI and AscI restriction sites or by LR clonase II into pGDHB1 and pGPR3-N vectors. The cloned plasmids were co-transformed along with necessary controls into NMY51 strain using the EZ-Yeast transformation kit (MP Biomedicals, USA) and plated on SD/-L/-W plates. The plating of yeast clones on required synthetic media to check protein-protein interactions in yeast was done as described previously [22 (link)]. All the interactions were verified by three independent experiments.