(A) Scheme of rPCA, a systematic method for detecting trans-acting mutations that dissociate a specific PPI in <i>S. cerevisiae</i>.

<p><i>Step 1</i>. The <i>MAT</i>α query strain carries two complementary fragments of mDHFR (F[1,2], and F[3]), fused at the C-terminus of the proteins of interest (X and Y), and linked to dominant selectable markers, NatMX and HygB respectively (X::mDHFR F[1,2]-NatMX, and Y::mDHFR F[3]-HygB), which confer resistance to the antibiotics nourseothricin (ClonNAT), and HygromycinB (HygB). In addition, these strains also contain the MFA1pr-<i>HIS3</i>, <i>can1</i>Δ and <i>lyp1</i>Δ SGA haploid selection markers (HSM). The query strain is crossed to an ordered array of <i>MAT</i><b>a</b> viable deletion mutants (<i>zzz</i>Δ), each carrying a gene deletion mutation linked to a <i>kanMX</i> marker, which confers resistance to the antibiotic Geneticin (G418) (illustrated here), or <i>MAT</i><b>a</b> libraries of temperature sensitive alleles of most essential genes. <i>Step 2</i>. The growth of resultant zygotes is selected on medium containing ClonNAT and G418. <i>Step 3</i>. The heterozygous diploids are transferred to medium with reduced levels of carbon and nitrogen to induce sporulation and the formation of haploid meiotic spore progeny. <i>Step 4</i>. Spores are transferred to a haploid selection medium, i.e., synthetic medium lacking histidine, which allows for selective germination of <i>MAT</i><b>a</b> meiotic progeny (since only these cells express the MFA1pr-<i>HIS3</i> reporter), and containing canavanine and thialysine, which allows for selective germination of meiotic progeny that carry the <i>can1</i>Δ and <i>lyp1</i>Δ HSMs. <i>Step 5</i>. The <i>MAT</i><b>a</b> meiotic progeny are then transferred to haploid selection medium containing G418 (for growth of meiotic progeny that carry the gene deletion mutation), and HygB and clonNAT, which select for the fusion proteins. Medium lacking MTX (−MTX) (<i>left</i>), is used as a control, and provides an indication of mutants that affect growth rate <i>per-se</i> under normal conditions (indicated by a dashed black arrow). The haploids that were selected for further analysis showed impaired growth on the experimental MTX-containing medium (+MTX) (<i>right</i>), when compared to the control array (indicated by black arrows). <b>(B) Example of colonies that were selected for further analysis.</b> Representative images of colonies obtained 2 days after pinning of a single 1536-density array plate from step-5 (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003838#pgen-1003838-g001" target="_blank">Figure 1A</a>). Example of a mutant that affects growth rate <i>per-se</i> under normal conditions (circled in yellow), and a mutant that specifically affects growth in the presence of MTX, and was selected for further analysis (circled in red). The formula shows the calculation that was used for the scoring of each colony.