Matchmaker two hybrid system 3

For the yeast-two-hybrid assays, we used yeast strain AH109 and Matchmaker Two-Hybrid System 3 from Clontech. Briefly, full-length or truncated cDNA was subcloned into pGADT7 or pGBKT7 vector using specific restriction enzyme sites or Gibson assembly. To test the interaction of defined protein partners, pGADT7 and pGBKT7 plasmids containing one of the two protein partners were cotransformed into yeasts. Transformed yeasts were selected by growing on two-marker dropout medium (SD/-Leu/-Trp). These two markers select for the presence of both plasmids. Colonies from the previous dropout medium were transferred to a four-marker dropout medium (SD/-Ade/-His/-Leu/-Trp) with X-α-Gal. Only colonies growing on four-marker plates were considered as evidence for a positive interaction.

For yeast two-hybrid assay, the MATCHMAKER Two-Hybrid System 3 (CLONTECH Laboratories, K1612-1) was used. Bait plasmids and prey plasmids were constructed by inserting cDNAs into a modified pGBKT7 (CLONTECH Laboratories, K1612-B) or pGADT7 (CLONTECH Laboratories, K1612-A) vector, respectively. Bait and prey plasmids were co-transformed into the strain AH109 (CLONTECH Laboratories, K1612-1), and transformants were selected on the double dropout medium (SD/−Leu/−Trp). The activation of the HIS3 reporter gene was assessed on the triple dropout medium (SD/−His/−Leu/−Trp), and the activation of the HIS3 and ADE2 reporter genes was assessed on quadruple dropout medium (SD/−Ade/−His/−Leu/−Trp).

The pACT2-mouse cDNA library in Escherichia coli (Clontech cat# ML4008AH/cat# 638841) was pre-transformed in the yeast AH109 strain (more than 10⁷ independent clones). The full-length FAIM-L and the 22 additional amino acids at the N-terminal FAIM isoform (FAIM-L) bait proteins were subcloned into pGBKT7 vector and transformed in Y187 yeast strain. The two-hybrid selection was performed by mating, following the matchmaker two-hybrid system 3 protocol (cat# K1612-1 Clontech). Positive colonies were selected in drop out medium lacking leucine, tryptophan, and histidine and containing 20 mM aminotriazole. Colonies were analyzed by polymerase chain reaction (PCR). cDNA was sequenced and transformed in E. coli (WB Cat# OP50), and interactions of bait and prey were confirmed by back transformation in yeast.

The Matchmaker Two-Hybrid system 3 (Clontech, USA) was used according to the manufacturer's instructions. cDNA of genes of interest for two hybrid tests was amplified with primers listed in Table S3 spanning the coding region from start to stop codon as annotated by the N. crassa database (http://www.broadinstitute.org/annotation/genome/neurospora/MultiHome.html) and cloned either into the pGADT7 vector containing the GAL4 activation domain or into pGBKT7 containing the DNA-binding domain. Because the pGBKT7-POD-6 fusion protein was determined to be self-activating, we generated two fragments containing the N-terminal kinase domain (aa 1–412) and C-terminal non-catalytic domain of POD-6 (aa 415–929). Fusion proteins were (co-) expressed in S. cerevisiae AH109 and potential interactions determined by growth tests on SD medium lacking the amino acids adenine, histidine, leucine and tryptophan or on SD medium supplemented with 10 mM 3-amino-1,2,4-triazole and lacking leucine, tryptophane and histidine.

The B9/C2 domain of human MKS1 (amino acids 144–470; Figure 4a) was cloned into the Gal4 vector pB27 and screened against a human fetal brain RP1 prey cDNA library. Yeast-2-hybrid screens were performed by Hybrigenics SA as described previously (Dawe et al., 2009 (link)). Confirmatory ‘1-to-1’ pairwise assays for selected interactants were performed with the MatchMaker Two-Hybrid System 3 (Clontech Inc).

Yeast transformation was performed using S. cerevisiae Direct Transformation Kit (Wako, Japan). Yeast two-hybrid assays were performed using MATCHMAKER Two-Hybrid System3 (Clontech) as previously described (Kayano et al., 2018 (link)). Briefly, yeast strain AH109 was transformed with prey and bait vectors, pGADT7 and pGBKT7 (Supplementary Table S2) and transformants were isolated on SD medium lacking leucine and tryptophan. Transformants were then cultured on SD medium lacking leucine, tryptophan, histidine and adenine (-LTHA), and growth on the latter indicates an interaction between bait and prey. Co-immunoprecipitation assay was performed as described by Xu et al. (2015) (link) using monoclonal antibodies against GFP (mFX75 012-22541, Wako) and FLAG (anti-DYKDDDDK 018-22381, Wako).