Extended Linkers Improve the Detection of Protein-protein Interactions (PPIs) by Dihydrofolate Reductase Protein-fragment Complementation Assay (DHFR PCA) in Living Cells

Andrée-Ève Chrétien,Isabelle Gagnon-Arsenault,Alexandre K Dubé,Xavier Barbeau,Philippe C Després,Claudine Lamothe,Anne-Marie Dion-Côté,Patrick Lagüe,Christian R Landry

doi:10.1074/mcp.tir117.000385

Andrée-Ève Chrétien, Isabelle Gagnon-Arsenault + Show 7 more

Open Access

https://doi.org/10.1074/mcp.tir117.000385

Copy DOI

Abstract

Understanding the function of cellular systems requires describing how proteins assemble with each other into transient and stable complexes and to determine their spatial relationships. Among the tools available to perform these analyses on a large scale is Protein-fragment Complementation Assay based on the dihydrofolate reductase (DHFR PCA). Here we test how longer linkers between the fusion proteins and the reporter fragments affect the performance of this assay. We investigate the architecture of the RNA polymerases, the proteasome and the conserved oligomeric Golgi (COG) complexes in living cells and performed large-scale screens with these extended linkers. We show that longer linkers significantly improve the detection of protein-protein interactions and allow to measure interactions further in space than the standard ones. We identify new interactions, for instance between the retromer complex and proteins related to autophagy and endocytosis. Longer linkers thus contribute an enhanced additional tool to the existing toolsets for the detection and measurements of protein-protein interactions and protein proximity in living cells.

Highlights

Much of our knowledge of cell biology, including on protein interaction networks and protein localization, derives from reporter proteins that require the use of short peptides to link proteins and protein fragments together (44)
We show that DHFR PCA, with a modest increase in linker size from 41 Å to 82 Å, can be used to detect PPIs in these specific conditions with an increased signal-to-noise ratio and with an enhanced ability to detect PPIs among distant proteins, including PPIs among complexes and subcomplexes within large complexes
Because only a single longer linker is generally sufficient to detect new PPIs, the current strains from the DHFR PCA collection could directly be used as preys while requiring only the construction of baits with different linker sizes