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.

• PMID: 29203496
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Reference Type

Journal Article | Research Support, Non-U.S. Gov't

Authors

Chrétien AÈ, Gagnon-Arsenault I, Dubé AK, Barbeau X, Després PC, Lamothe C, Dion-Côté AM, Lagüe P, Landry CR

Primary Lit For

Retromer complex

Additional Lit For

Review For