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

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. 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. Protein-protein interactions (PPIs) 1The abbreviations used are: PPIs, Protein-protein interactions. 1The abbreviations used are: PPIs, Protein-protein interactions. are central to all cellular functions and are largely responsible for translating genotypes into phenotypes (1.Vidal M. Cusick M.E. Barabasi A.L. Interactome networks and human disease.Cell. 2011; 144: 986-998Abstract Full Text Full Text PDF PubMed Scopus (1181) Google Scholar). 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For the budding yeast data stored in BioGRID (2017) (26.Chatr-Aryamontri A. Oughtred R. Boucher L. Rust J. Chang C. Kolas N.K. O'Donnell L. Oster S. Theesfeld C. Sellam A. Stark C. Breitkreutz B.J. Dolinski K. Tyers M. The BioGRID interaction database: 2017 update.Nucleic Acids Res. 2017; 45: D369-D379Crossref PubMed Scopus (677) Google Scholar), AP-MS has contributed more than twice as much (n = 58455) in terms of PPIs than PCA and Y2H combined (n = 6577 and 15973 respectively). Overall, these methods are complementary because on the one hand, they tell us which proteins assemble into complexes in the cell and on the other hand, how proteins may be physically located relative to one another (20.Benschop J.J. Brabers N. van Leenen D. Bakker L.V. van Deutekom H.W. van Berkum N.L. Apweiler E. Lijnzaad P. Holstege F.C. Kemmeren P. A consensus of core protein complex compositions for Saccharomyces cerevisiae.Mol. 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In the first large-scale study performed using the dihydrofolate reductase (DHFR) PCA in yeast, it was shown that distance constraint determined by linker length could affect the ability to detect PPIs (7.Tarassov K. Messier V. Landry C.R. Radinovic S. Serna Molina M.M. Shames I. Malitskaya Y. Vogel J. Bussey H. Michnick S.W. An in vivo map of the yeast protein interactome.Science. 2008; 320: 1465-1470Crossref PubMed Scopus (579) Google Scholar). For the RNA polymerase (RNApol) II complex and several other protein complexes, PPIs were 3.5 times more likely to be detected if the C-termini were within less than 82 Å of each other. In addition, an earlier study in mammalian cells showed that increasing linker length of the PCA reporter allows to detect configuration changes in a dimeric membrane receptor (34.Remy I. Wilson I.A. Michnick S.W. Erythropoietin receptor activation by a ligand-induced conformation change.Science. 1999; 283: 990-993Crossref PubMed Scopus (536) Google Scholar). Together, these results suggest that linkers of variable sizes could improve the detection of PPIs and even be used as a ruler to infer, albeit roughly, distances between proteins and this, in living cells. However, a recent study on luminescence PCA showed that the relationship between the linker size and the signal-to-noise ratio of PCA assays is not trivial (35.Dixon A.S. Schwinn M.K. Hall M.P. Zimmerman K. Otto P. Lubben T.H. Butler B.L. Binkowski B.F. Machleidt T. Kirkland T.A. Wood M.G. Eggers C.T. Encell L.P. Wood K.V. NanoLuc complementation reporter optimized for accurate measurement of protein interactions in cells.ACS Chem. Biol. 2016; 11: 400-408Crossref PubMed Scopus (553) Google Scholar). Although reducing linker size from 15 to 10 amino acid long reduces signal-to-noise ratio by 40%, further reduction has little effect until the linker is completely eliminated. Therefore, the effect of linker size requires to be tested in a quantitative manner if it is to be implemented in large-scale screen and this, possibly for each specific assay. Here we test these effects on the ability to detect PPIs by PCA in vivo using the yeast DHFR PCA. We examine how linker size influences our ability to detect PPIs in large-scale screens and in small-scale experiments by testing PPIs within complexes whose structures are known. We show that extended linkers improve the signal-to-noise ratio in these experiments and thus, helps to detect previously unreported PPIs. They also allow detecting PPIs among proteins within the same complex but that are more distant based on the location of their C-termini. All resources and reagents used in this study are described in details in supplemental Table S1. Yeast strains used in this study were constructed (as described below) or are from the Yeast Protein Interactome Collection (7.Tarassov K. Messier V. Landry C.R. Radinovic S. Serna Molina M.M. Shames I. Malitskaya Y. Vogel J. Bussey H. Michnick S.W. An in vivo map of the yeast protein interactome.Science. 2008; 320: 1465-1470Crossref PubMed Scopus (579) Google Scholar) (Table S2A). They all derive from the BY4741 (MATa his3Δ leu2Δ met15Δ ura3Δ) and BY4742 (MATα his3Δ leu2Δ lys2Δ ura3Δ) backgrounds. Cells were grown on YPD medium (1% yeast extract, 2% tryptone, 2% glucose, and 2% agar (for solid medium)) containing 100 μg/ml nourseothricin (clonNAT) and/or 250 μg/ml hygromycin B (HygB) for transformations and diploid selection. For the DHFR PCA experiment, cells were grown on MTX medium (0.67% yeast nitrogen base without amino acids and without ammonium sulfate, 2% glucose, 2.5% noble agar, drop-out without adenine, methionine and lysine, and 200 μg/ml methotrexate (MTX) diluted in DMSO). Escherichia coli MC1061 was used for all DNA cloning and propagation steps. Cells were grown on 2YT medium (1% yeast extract, 1.6% tryptone, 0.2% glucose, 0.5% NaCl, and 2% agar (for solid medium)) supplemented with 100 μg/ml ampicillin (Amp). Plasmids pAG25-linker-DHFR-F[1,2]-ADHterm and pAG32-linker-DHFR-F[3]-ADHterm were used as templates to create new plasmids containing DHFR fragments fused to a linker of varying size. Both original plasmids contained the sequence coding for two of the of the for the and two for the were between the linker and the DHFR in plasmids and The new were of to the same the from pAG25-linker-DHFR-F[1,2]-ADHterm with the and DNA fragments were and in the containing and The fragments were by (Table with and The pAG25-linker-DHFR-F[1,2]-ADHterm was with and The fragment to the without the was by The fragments and plasmids were by cloning L. of DNA to several Methods. 2009; 6: PubMed Scopus Google Scholar) with an ratio of were in E. coli and were on were and by with and and The plasmids were used as a to the and fragments were from and Table The DHFR fragment was from pAG32-linker-DHFR-F[3]-ADHterm Table All were with and pAG32-linker-DHFR-F[3]-ADHterm was with and The fragment to the without the was on The were performed as described for the with an (DHFR ratio of were constructed in BY4741 and BY4742 for the DHFR and DHFR (Table S2A). All were performed at the of fragments with the (for DHFR or (for DHFR for to and were by from their with specific to the gene to be fused with the DHFR fragments Table BY4741 and BY4742 cells were with the standard and selection was performed on (DHFR or (DHFR and for all strains DHFR fragment Protein was performed for several strains with proteins fused with the and by on proteins for which the proteins were of cells were in 200 of containing μg/ml μg/ml and μg/ml were and cells were using a for of were and were in a new Protein to of cells were on or and and on a membrane using a in at were with by M. N. J. or (as a in 0.2% at 10 in 0.2% were with or in 0.2% of 10 in 0.2% were performed and on was detected using were performed with For the PCA experiment, of 15 proteins fused to that are of proteins fused to the were to the that they were to the same complexes as the (20.Benschop J.J. Brabers N. van Leenen D. Bakker L.V. van Deutekom H.W. van Berkum N.L. Apweiler E. Lijnzaad P. Holstege F.C. Kemmeren P. A consensus of core protein complex compositions for Saccharomyces cerevisiae.Mol. Cell. 2010; 38: 916-928Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) or that they were PPI of one of based on data in BioGRID in (26.Chatr-Aryamontri A. Oughtred R. Boucher L. Rust J. Chang C. Kolas N.K. O'Donnell L. Oster S. Theesfeld C. Sellam A. Stark C. Breitkreutz B.J. Dolinski K. Tyers M. The BioGRID interaction database: 2017 update.Nucleic Acids Res. 2017; 45: D369-D379Crossref PubMed Scopus (677) Google Scholar). A of strains to proteins in the or the was also in the of as was in two on each so each was were to location the of the PCA experiment, we that are of large complexes Pore and complexes of complexes complex and retromer or that are not of a known stable complex were fused to the and and were the DHFR of (n = after for data and that with was but in an for a of the PPIs For the experiment, we performed a of the and the consensus protein complexes by (20.Benschop J.J. Brabers N. van Leenen D. Bakker L.V. van Deutekom H.W. van Berkum N.L. Apweiler E. Lijnzaad P. Holstege F.C. Kemmeren P. A consensus of core protein complex compositions for Saccharomyces cerevisiae.Mol. Cell. 2010; 38: 916-928Abstract Full Text Full Text PDF PubMed Scopus (60) Google Scholar) to central and proteins of the the II and the and the complexes were because they in size (n = II (n = (n = and proteins (n = proteasome (n = and complex (n = and PPIs among protein of these complexes have shown to be at by DHFR PCA (7.Tarassov K. Messier V. Landry C.R. Radinovic S. Serna Molina M.M. Shames I. Malitskaya Y. Vogel J. Bussey H. Michnick S.W. An in vivo map of the yeast protein interactome.Science. 2008; 320: 1465-1470Crossref PubMed Scopus (579) Google Scholar) based on data available in (26.Chatr-Aryamontri A. Oughtred R. Boucher L. Rust J. Chang C. Kolas N.K. O'Donnell L. Oster S. Theesfeld C. Sellam A. Stark C. Breitkreutz B.J. Dolinski K. Tyers M. The BioGRID interaction database: 2017 update.Nucleic Acids Res. 2017; 45: D369-D379Crossref PubMed Scopus (677) Google Scholar). In addition, there are structures available for the and proteasome complexes, it to our results with known protein complex We constructed and of the strains in and and in for the and proteasome and for the In strains proteins fused to and/or were a of of the proteins at first are with and in at one of the proteins. cells were used as different of cells were all strains and were so that in a of strains all of linker sizes could be tested for a specific of was in for the and complexes, and in for the proteasome The were on the was to a of a a interaction to effects on the of the were first from 10 in (for or (for that were on YPD and at for Cells were on a with a (or a