A scalable double-barcode sequencing platform for characterization of dynamic protein-protein interactions

Abstract

Several large-scale efforts have systematically catalogued protein-protein interactions (PPIs) of a cell in a single environment. However, little is known about how the protein interactome changes across environmental perturbations. Current technologies, which assay one PPI at a time, are too low throughput to make it practical to study protein interactome dynamics. Here, we develop a highly parallel protein-protein interaction sequencing (PPiSeq) platform that uses a novel double barcoding system in conjunction with the dihydrofolate reductase protein-fragment complementation assay in Saccharomyces cerevisiae. PPiSeq detects PPIs at a rate that is on par with current assays and, in contrast with current methods, quantitatively scores PPIs with enough accuracy and sensitivity to detect changes across environments. Both PPI scoring and the bulk of strain construction can be performed with cell pools, making the assay scalable and easily reproduced across environments. PPiSeq is therefore a powerful new tool for large-scale investigations of dynamic PPIs.

Mapping DNA damage‐dependent genetic interactions in yeast via party mating and barcode fusion genetics

“…The authors demonstrated the method, showing that a pool corresponding to nine gene pairs could be sequenced to monitor competitive growth of double mutants en masse in different environments (Jaffe et al, 2017). Cre-mediated approaches have been used similarly to map protein-protein interactions (Hastie & Pruitt, 2007;Yachie et al, 2016;Schlecht et al, 2017).…”

Genetics’ Piece of the PI: Inferring the Origin of Complex Traits and Diseases from Proteome‐Wide Protein–Protein Interaction Dynamics

“…It is exciting to anticipate how much more we could get from studies like these if we can simplify the heteromeric screening process in order to practically perform screens with the entire proteome. Some exploratory PCA‐based heteromeric screens have already shown their utility in examining the dynamics of the interaction network, adding valuable edge‐focused information to the node‐focused hd PCA approach . With barcoding of strains and high‐throughput sequencing readouts, we anticipate that joint measurements of genomic variation and in vivo PPI dynamics may become possible in the near future.…”

“…PCAs also allow for a variety of reporters, from those that confer survival to cells under selection to fluorescent reporters that allow for detection of spatiotemporal dynamics of protein complexes following cell perturbations . A particularly simple way to read out survival‐selection PCAs is to integrate sequence barcodes associated with specific genes into cells harboring complementary fragments for a pair of proteins . Importantly, there is no longer the need to physically array individual clones for specific PPI protein pairs, saving large amounts of associated materials, manipulations, and waiting times to perform a screen.…”

A large accessory protein interactome is rewired across environments

“…The library was grown under mild methotrexate selection in 9 environments for 12-18 generations in serial batch culture, diluting 1:8 every ~3 generations, with a bottleneck population size greater than 2 x 10 9 cells (Table S1). Double barcodes were enumerated over 4-5 timepoints by sequencing, and the resulting frequency trajectories (Table S2) were used to estimate the relative fitness (Table S3) of each strain, which is a rough measure of the average PPI abundance over a growth cycle ( Figure 1B) (Levy et al, 2015;Li et al, 2018;Schlecht et al, 2017). We recovered a minimum of two reliable replicate fitness estimates for 1.6 million protein pairs, and downstream analysis was limited to this set.…”