Genome-wide Protein-protein Interaction Screening by Protein-fragment Complementation Assay (PCA) in Living Cells

Abstract

Proteins are the building blocks, effectors and signal mediators of cellular processes. A protein's function, regulation and localization often depend on its interactions with other proteins. Here, we describe a protocol for the yeast protein-fragment complementation assay (PCA), a powerful method to detect direct and proximal associations between proteins in living cells. The interaction between two proteins, each fused to a dihydrofolate reductase (DHFR) protein fragment, translates into growth of yeast strains in presence of the drug methotrexate (MTX). Differential fitness, resulting from different amounts of reconstituted DHFR enzyme, can be quantified on high-density colony arrays, allowing to differentiate interacting from non-interacting bait-prey pairs. The high-throughput protocol presented here is performed using a robotic platform that parallelizes mating of bait and prey strains carrying complementary DHFR-fragment fusion proteins and the survival assay on MTX. This protocol allows to systematically test for thousands of protein-protein interactions (PPIs) involving bait proteins of interest and offers several advantages over other PPI detection assays, including the study of proteins expressed from their endogenous promoters without the need for modifying protein localization and for the assembly of complex reporter constructs.

The role of structural pleiotropy and regulatory evolution in the retention of heteromers of paralogs

“…The initial denaturation was performed for 5 min at 95°C and was followed by 35 cycles of 30 sec of denaturation at 94°C, 30 sec of annealing at 55°C, 1 min of extension at 72°C and 3 min of a final extension at 72°C. We confirmed 1769 of the 1904 strains from the DHFR collection and 117 strains out of the 143 from (Diss et al, 2017) (Table S5, and Table S7) The missing or non-validated strains were constructed de novo using the standard DHFR strain construction protocol (Michnick et al, 2016;Rochette et al, 2015). The DHFR fragments and associated resistance cassettes were amplified from plasmids pAG25-linker-DHFR-F[1,2]-ADHterm (marked with nourseothricin N-acetyl-transferase) and pAG32-linker-DHFR-F[3]-ADHterm (marked with hygromycin B phosphotransferase) (Tarassov et al, 2008) using oligonucleotides defined in (Table S7).…”

“…The missing or non-validated strains were constructed de novo using the standard DHFR strain construction protocol (Michnick et al, 2016;Rochette et al, 2015). The DHFR fragments and associated resistance cassettes were amplified from plasmids pAG25-linker-DHFR-F[1,2]-ADHterm (marked with nourseothricin N-acetyl-transferase) and pAG32-linker-DHFR-F[3]-ADHterm (marked with hygromycin B phosphotransferase) (Tarassov et al, 2008) using oligonucleotides defined in (Table S9).…”

“…Three DHFR PCA experiments were performed, hereafter referred to as PCA1, PCA2 and PCA3. The configuration of strains on plates and screening was performed using robotically manipulated pin tools (BM5-SC1, S&P Robotics Inc., Toronto, Canada (Rochette et al, 2015)). We first organized haploid strains in 384 colony arrays containing a border of control strains using a cherry-picking 96-pin tool ( Figure S13).…”

A collection of barcoded natural isolates of Saccharomyces paradoxus to study microbial evolutionary ecology

“…The 370 parental strains and 594 barcoded strains of the collection were assembled in two arrays (omnitrays, 86 mm × 128 mm Petri dish) on solid YPD medium (Rochette et al, ). One contained all the SpB and SpD strains while SpC and SpC* strains were on the other second array.…”

“…Genomic DNA was extracted following a protocol modified from Looke, Kristjuhan, and Kristjuhan (2011). Strains from the S. cerevisiae deletion collection were printed onto arrays of 384 colonies on solid yeast peptone dextrose (YPD) with 10 g/L of yeast extract, 20 g/L of tryptone, and 20 g/L of glucose, as outlined in Rochette et al (2015) using a BMC-BC robotic platform (S&P robotics, North York, Canada).…”