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

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 ba...

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Bibliographic Details
Published in:Molecular & cellular proteomics 2018-02, Vol.17 (2), p.373-383
Main Authors: Chrétien, Andrée-Ève, Gagnon-Arsenault, Isabelle, Dubé, Alexandre K, Barbeau, Xavier, Després, Philippe C, Lamothe, Claudine, Dion-Côté, Anne-Marie, Lagüe, Patrick, Landry, Christian R
Format: Article
Language:English
Subjects:
Assaying
Autophagy
Binding sites
Cells (biology)
Dihydrofolate reductase
Endocytosis
Fragmentation
Golgi apparatus
Phagocytosis
Proteasomes
Protein interaction
Proteins
Ribonucleic acid
RNA
Screens
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Summary: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.
ISSN:1535-9476
1535-9484
DOI:10.1074/mcp.RA117.000385