Abstract
We present a technological advancement for the estimation of the affinities of Protein-Protein Interactions (PPIs) in living cells. A novel set of vectors is introduced that enables a quantitative yeast two-hybrid system based on fluorescent fusion proteins. The vectors allow simultaneous quantification of the reaction partners (Bait and Prey) and the reporter at the single-cell level by flow cytometry. We validate the applicability of this system on PPIs with different affinities. After only two hours of reaction, expression of the reporter can easily be detected even for the weakest PPI. Through a simple gating analysis, it is possible to select only cells with identical expression levels of the reaction partners. As a result of this standardization of expression levels, the mean reporter levels directly reflects the affinities of the studied PPIs. With a set of PPIs with known affinities, it is straightforward to construct an affinity ladder that permits rapid classification of PPIs with thus far unknown affinities. Conventional software can be used for this analysis. To permit automated high-throughput analysis, we provide a graphical user interface for the Python-based FlowCytometryTools package.
- Abbreviations
- PPIs
- Protein-Protein interactions
- qY2H
- quantitative yeast two-hybrid
- BD-Bait
- DNA Binding Domain fused to the Bait
- AD-Prey
- Activation Domain fused to the Prey
Introduction
Protein-protein interactions (PPIs) are essential for many functions in living cells, including communication of signals, modulation of enzyme activity, active transportation, or stabilization of the cell structure by the cytoskeleton (1–4). Resolving the complex cellular network of PPIs remains one of the major challenges in proteomics (5). Thus, the quest for reliable methods that identify PPIs and quantify their strength is unbroken.
The yeast two-hybrid technique (Y2H) is a commonly used approach to probe the interaction between proteins (6–8). In contrast to biochemical in-vitro methods (such as mass spectrometry, ITC or SPR) that require purified proteins, Y2H is based on a genetic assay. It relies on the in-cellula expression of fusions of the two proteins of interest, usually named Bait and Prey. Upon physical interaction of Bait and Prey, a functional transcription factor is reconstituted that drives the expression of a reporter gene (e.g., β-galactosidase). As a consequence, a read-out is observed (e.g., color, fluorescence, or growth) that permits high-throughput screens.
Y2H has been extensively used in the past decades to decipher PPI networks (9, 10). With growing experience, the scientific community became aware of the limitations of this approach. Standard Y2H is prone to false positive/negative results (8). For example, the absence of a detectable read-out may reflect insufficient expression of the Bait and/or Prey. More laborious Western blottings can be performed to verify the expression (11). Furthermore, Y2H provides often only a qualitative result. With X-gal-based Y2H (12), for instance, the measured read-out (color) cannot be assumed to be proportional to the reporter level, i.e., β-galactosidase activity, but exceptions exist (13, 14). Also, the extent of β-galactosidase activity does not necessarily reflect the extent of interaction between Bait and Prey (due to varying expression levels of Bait and Prey fusions).
Several groups tried to overcome the qualitative limitations of the two-hybrid system in yeast and other organisms. Extensive overviews can be found in the literature, for example Ref. (8). Many applied methods could rank PPIs according to their affinity using the quantified read-out, examples are included in Refs. (13–17). It should be noted, however, that mainly mutants were compared. Similar expression levels for the Bait and Prey fusions can be assumed for such mutational studies. Comparing proteins from different families often breaks the correlation (11). It speaks to the need of quantifying not only the read-out but the Bait/Prey fusions as well. Thus far, only low-throughput methods exist that address this necessity. For example, by measuring the fraction of co-localized fluorescent “Bait” and “Prey” fusions in human cells by high-resolution microscopy (18) it was possible to optimize the affinity of an inhibitor (19). Another approach used a fluorescent antibody to quantify the amount of retained Prey by the Bait associated to the periplasm (20). Following this idea, different yeast surface two-hybrid approaches emerged (17, 21) using antibodies or purified proteins.
Here we present a novel set of Y2H vectors that enable the quantification of the reaction partners (Bait, Prey) and the reporter at the single-cell level without the need of any antibodies or purified proteins (Fig. 1). Three different fluorescent tags serve as sensors to probe the cellular expression levels by flow cytometry. The vectors have been validated on protein-protein interactions with varying affinities (see Table 1) to encompass the sensitivity of the novel quantitative Y2H (qY2H) system.
Experimental Procedures
Experimental design
Starting from the original plasmids pLexA and pB42AD (22), several new cloning sites were introduced that permit convenient sub-cloning into the expression cassettes via homologous recombination in yeast (see Suppl. Fig. S1 for detailed vector maps). Thus, the newly designed vectors facilitate the construction of novel fusions proteins with tailored functionalities. Here we generated cassettes that code for BD-Bait, AD-Prey and reporter fusions with several new features as shown in Fig. 1. We copied the HA tag (that was originally only in the AD-Prey expression cassette) to the BD-Bait cassette to enable the simultaneous quantification of expressed BD-Bait and AD-Prey fusions by Western Blotting. In addition, we added red and green fluorescent tags (Tag RFP and yEGFP) to the BD-Bait and AD-Prey cassettes, respectively. Furthermore, the original reporter (β-galactosidase) was replaced by a tandem of the Tag BFP in the pSH18-34 vector (22). The three fluorescent tags emit at considerably different wavelength ranges so that their individual expression levels can be simultaneously monitored at the single-cell level by flow cytometry (Fig. 1B). Also, a spacer sequence was added between the fluorescent tags and the Prey/Bait to avoid steric hindrance in the expressed fusions.
The new constructs were tested on two well-studied protein-protein interactions (23–29): the association between the bacterial ribonuclease Barnase and its inhibitor Barstar, as well as between the human GTPase HRas and the Ras-binding domain of CRaf. Different mutants were applied (Table 1) that span a wide range of affinities (known from independent in-vitro experiments). By exchanging Bait and Prey for a given couple it is possible to probe the corresponding PPI in two different orientations. We cross-tested all proteins of Table 1 as BD-Bait and AD-Prey fusions with proper negative controls, i.e., empty AD-EGFP and BD-RFP fusions, respectively, to eliminate orientations that lead to false positive interactions (see Material & Methods).
For a given couple, the two orientations may also lead to different read-outs (15). Table 1 lists the couples in the orientation with the stronger reporter level (when the cellular contents of BD-Bait and AD-Prey are standardized, see below). This orientation is considered as the molecular configuration with the higher accessibility of the PPI binding interface (15). Or in other words, this orientation may feature the smaller steric hindrance due to the fused fluorescent BD and AD and probably resembles more closely the situation of free (not fused) proteins. We focus therefore in the following sections on the orientation given in Table 1.
Haploid cells were transformed with either Prey or Bait plasmids; in the latter case we used haploid cells that were previously transformed with the reporter plasmid. Transformed yeast cells were mated and amplified to generate diploid colonies for the desired Bait/Prey-couples. Selection and amplification of the diploids occurred in glucose medium which represses the expression of the AD-Prey fusion (under the control of the GAL1 promoter). Transfer of the diploid cells into Galactose/Raffinose medium induced the expression of the AD-Prey fusion and enabled the expression of the reporter.
After fixation, the samples were submitted to flow cytometry measurements to monitor the fluorescence intensities at the single cell level for three different channels matching the emission ranges of the fluorescent BD-Bait, AD-Prey and reporter fusions; hereafter these channels are named Tag-RFP-H, yEGFP-H and Tag-BFP-H, respectively.
We analyzed the expression level of the fluorescent proteins either for the entire cell population (to which refer as “global” hereafter) or for subpopulations using interval gatings. The entire procedure starting from the transformation up to the flow cytometry measurement was repeated at least three times for each protein-protein interaction.
Plasmid creation
In order to generate the pSB_1Bait plasmid, the pLexA (22) vector was linearized using EcoRI and SalI (Thermo Scientific) to remove all DNA between the LexA cDNA and the ADH terminator. The Barstar WT coding sequence was ordered for synthesis to Eurofin Genomics as part of a new expression cassette. At the 5’ end we added the sequences of the HA-Tag and our MCS-spacer (EcoRI, AscI, and XhoI). At the 3’ end, after the stop codon of Barstar, we inserted one XhoI site, created 3 stop codons (1 per ORF) and regenerated the SalI site. The upstream (LexA) and downstream (Terminator) 30bp required for homologous recombination in yeasts (30, 31) were also added. This new optimized expression cassette was amplified by PCR (Phusion DNA polymerase, Thermo Scientific), using the primers primSB_0001 and 2 (see Suppl. Table S1), and inserted in the previously linearized pLexA vector. As a result, we obtained the pSB_1Bait_Barstar. The coding sequence for Tag-RFP was subsequently introduced in the EcoRI site through PCR from pTag_RFP-Actin (Evrogen), using the primers primSB_0003 and 0004, combined with homologous recombination in yeasts to obtain the pSB_1Bait_RFP-Barstar plasmid. The pSB_1Bait_RFP-Empty and pSB_1Bait-Empty vectors were generated by digesting the pSB_1Bait_RFP-Barstar and pSB_1Bait_Barstar, respectively, with XhoI (Thermo Scientific), followed by self-ligation. The coding sequences of the mutants of Barstar, and Ras_G12V_C186A were ordered to Eurofin Genomics, amplified (primSB_0018 and 0019) and introduced in the pSB_1Bait_RFP-Empty linearized with XhoI by homologous recombination.
To create the pSB_1Prey vector, the pB42AD plasmid (22) was linearized using EcoRI and XhoI. The sequence coding for the non-toxic Barnase mutant H102A was ordered from Operon MWG. At the 5’ end we inserted the same MCS-spacer sequence as in the pSB_1Bait vector to allow easy transfer from one plasmid to the other. At the 3’ end, we inserted one XhoI site, created 3 stop codons and one NcoI restriction site. The upstream (HA-Tag) and downstream (Terminator) 30bp required for homologous recombination in yeasts were also introduced. This new expression cassette was then amplified by PCR (primSB_0010 and 0011) and inserted in the pB42AD by homologous recombination in yeast to obtain the pSB_1Prey_Barnase-H102A vector. The coding sequence of the yEGFP was amplified from the pGY-LexA-GFP_KanMX (kindly provided by Dr Gaël Yvert) using the primers primSB_0012 and 0013, and then introduced in the EcoRI sit of our MCS as previously to generate the pSB_1Prey_yEGFP-Barnase-H102A vector. The pSB_1Prey-Empty and pB_1Prey_yEGFP-Empty were created by removing the coding sequence of Barnase H102A with XhoI and performing a self-ligation. The coding sequences of the other Preys (CRaf RBD WT, and CRaf RBD A85K) were ordered to Eurofin Genomics, amplified by PCR (primSB_0020 and 21) and introduced in the pSB_1Prey_yEGFP-Empty linearized with XhoI by homologous recombination.
To create the reporter plasmid, the pSH18-34 (22) was digested using the unique SalI (In the modified Gal1 promoter) and RsrII (downstream to the β-Galactosidase coding sequence) restriction sites. We subsequently reconstructed the expression cassette using four PCR products:
1) The Gal1 promoter delta Gal4 with 8 operator LexA and the Kozack sequence with a new downstream MCS (AscI, NheI) (primSB_0076 and 0077).
2) The Gal1 Nterm sequence (I10-C20), originally expressed by the pSH18-34, is used as spacer (primSB-0078 and 0079) between the two copies of the Tag BFP.
3) The coding sequence of the Tag-BFP (from pTag_BFP-Actin, Evrogen) borded with 2 XhoI sites, (primSB_0084 and 0085).
4) The terminator sequence (primSB_0080 and 0081).
These 4 amplicons were then used to perform directly a gap repair in yeasts. Thus, we obtained the pSB_3RO plasmid. A second copy of the Tag-BFP (primSB_0120 and 121) was inserted in our new NheI site (Thermo Scientific), by homologous recombination to generate the pBFP2 plasmid. This final vector allows the expression of a dimer of Tag-BFP as reporter of the yeast two hybrid reaction.
Western blot
Total protein extracts were obtained from 6 OD590nm exponentially growing diploids yeasts as previously described (32) into 60µl of sample buffer. Ten microliters were used for SDS-Page analysis on Bolt™ 4-12% Bis-Tris Plus Gels (Thermo Scientific). Electrophoresis separation was performed in NuPAGE™ MOPS SDS Running Buffer (Thermo Scientific). Proteins were then transferred on a Nitrocellulose Membrane 0.45µm (Biorad), using a Trans-Blot® Turbo™ Transfer System (Biorad) for 14 minutes, at 1A and 25V. The membrane was subsequently blocked 1 hour at room temperature in PBS + tween 0.2% (v/v) supplemented with 5% (w/v) low-fat milk powder. HA tagged proteins were labeled overnight at 4°C with the mouse HA.11 Clone 16B12 Monoclonal Antibody (Eurogentec) 1/2000 in PBS + tween 0.2% (v/v) + 10 mg/ml BSA (Albumin bovine fraction V, Euromedex). The membrane was then washed four times seven minutes in blocking buffer at room temperature. The membrane was then incubated for one hour at room temperature in presence of a sheep anti-mouse whole IgG HRP (GE Healthcare) secondary antibody diluted 1/5000 in blocking buffer. The excess of antibody was removed with two washing steps of five minutes in PBSt at room temperature. Labelled proteins were then revealed with Super Signal West Pico chemiluminescent substrate (Thermo Scientific) using a Biorad Chemidoc apparatus, following instructions provided by the suppliers.
qY2H in liquid phase
Chemo-competent EGY42 (MATa; trp1, his3, ura3, leu2) and TB50 (MATα; trp1, his3, ura3, leu2, rme1) yeasts were generated as previously described (33).
Competent EGY42a yeasts were transformed with 1µg of pBFP2 and grown on selective SD-U medium. Chemo-competent EGY42a pBFP2 yeasts were then generated and transformed with 1µg of Bait vectors. Haploid Bait yeast strains were then selected on SD-UH medium. Competent TB50α yeasts were transformed with 1µg of Prey vector. Haploid Prey yeast strains were selected on SD-W medium. Matrix mating assay were performed for one night with 50µl of Bait and Prey strains (each) resuspended in YPAD medium at 0.1 OD at 30°C. The next morning YPAD medium was removed and the yeast diploids were harvested and amplified in 1ml of SD- UHW for 3 days at 30°C.
The qY2H assay was performed in pre-heated (30°C) and oxygenated SGR-UHW supplemented with Galactose 0.25% (Euromedex) and Raffinose 1% (Sigma-Aldrich) to induce the expression of the Prey proteins. To ensure we obtained an excessive number of cells (about 107) for the analysis, a culture of 100 ml was inseminated with 600 µl of saturated diploids per couple of interest. It turned out that for a typical analysis a number of 106 cells is adequate, so that actually 10 times smaller cultures and insemination volumes can be used. The yeasts were incubated for 2h at 30°C without shaking, and then harvested after a centrifugation step of 10min at 1000g. The yeast were resuspended in 1ml PBS (Dominique Dutscher), centrifuged 1min at 13000 rpm, and washed again with 1ml of PBS. The yeasts were resuspended in 500µl of PBS 4% PFA (Sigma-Aldrich, Catalog n°P6148) and incubated for 10min at 4°C. The fixation reaction was blocked by 2 washing steps with 1ml PBS, and one incubation of 15min at 4°C in 500µl of PBS 0.1M Glycine (Euromedex). Finally, the yeasts were washed twice in PBS, and stored in 1ml of PBS at 4°C for not longer than 24 hours.
Flow cytometry and data analyses
The expression levels of BD-Bait, AD-Prey and reporter were acquired in linear scale using a MacsQuantVYB flow cytometer (the settings are presented in Suppl. Table S2). To ensure homogeneous sampling of the yeasts cells in suspension, we used the strong mixing mode. With the apparatus at our disposal, this mode generates at very early acquisition times a small population of particles with abnormal characteristics for yeast cells (a high red fluorescence intensity, even for non-fluorescent samples). We suspect these are micro-bubbles. To rigorously eliminate this population, we skipped the first 20 000 events of all samples files in the subsequent analysis. The flow-cytometry files were analyzed using the FlowCytometryTools package for python (http://eyurtsev.github.io/FlowCytometryTools). When a hlog-transformation (34) was applied, the following settings were used: b = 2000, r = 10000, and d = 5.42.
Our python scripts (with a graphical user interface and user guide for the automated generation of the qY2H affinity ladder) can be downloaded here: http://github.com/LBMC/qY2H-Affinity-Ladder. The flow cytometry files of the experiment shown in Fig. 4 can be downloaded from http://flowrepository.org under accession number FR-FCM-ZYUL.
Results
qY2H enables the monitoring of the expression level of the reaction partners after 2h
Two hours after induction of the Y2H reaction, the expression of all BD-Bait and AD-Prey fusions can be easily detected. Fig. 2A displays their fluorescence intensities for a subset of couples. The fluorescence intensity typically spans several orders of magnitude higher than the negative controls. However, expression problems can be seen for BD-HRas (Fig. 2C). Independent quantification by Western Blotting (Fig. 3) shows that the expression level of BD-HRas is indeed impaired. In fact, BD-HRas cannot be detected in the Western blot. Flow cytometry, on the other hand, indicates a slight shift in the probability distribution for BD-HRas expressing cells (with respect to the negative control, see also Fig. 4A).
Thus, flow cytometry gives an immediate indication on eventual expression problems of the Bait and Prey fusions during acquisition. This contrasts with standard Y2H experiments where the expression level of the BD-Bait and AD-Prey fusions is usually unknown at the time of reporter detection. More laborious Western Blots are usually required to gain this information.
Reporter level is correlated with the expression level of the reaction partners
Even for the weakest Bait-Prey interaction the reporter can already be detected two hours after induction (Fig. 2); it is clearly above the level of the negative control (panel B). Moreover, we observe that the reporter level (i.e., blue fluorescence intensity) is correlated with the green and red fluorescence intensity: more reaction partners (i.e., higher amount of interacting BD-Bait and AD-Prey fusions) yielded more product (reporter). This obvious correlation has consequences for the extraction of quantitative information on the strength of PPIs as we demonstrate later on. In the case of the Bait/Prey-couple BD-B112/AD-Barnase H102A (Fig. 2F) this correlation is basically only observed for the red fluorescence (BD-Bait). The B112 acid blob acts as an activation domain (35, 36) so that this specific BD-Bait fusion is a functional transcription factor by itself that does not depend on the AD-Prey fusion.
Standardization of BD-Bait and AD-Prey levels is required to gain information on binding strength
In all repetitions of the experiment, the reporter level of the global cell population roughly reflects the magnitude of the (in-vitro) affinity. As shown in Fig. 4A for a single experiment and a small subset of PPIs, the Bait-Prey couples with high affinity (Kd ∼ pM) could be easily distinguished from medium-affinity couples (Kd ∼ nM) based on their mean reporter level. It confirms results of previous studies that the global Y2H read-out correlates with in-vitro affinity (13–17). In addition, our approach discloses the influence of the expression levels of the reaction partners, i.e., the BD-Bait and AD-Prey fusions. Their levels may vary significantly between the studied couples (Fig. 2 & 3) and to a smaller extent between different experiments. These variations complicate the discrimination of Bait-Prey couples based on their affinities. Fig. 4A presents a particularly illustrative experiment: The couple BD-HRas / AD-CRaf displays a 20 times lower in-vitro Kd-value than the couple BD-Barstar D39A / AD-Barnase H102A. Yet the mean reporter level (Fig. 4A, right column) is lower for the former couple than for the latter couple; the opposite would have been expected according to the in-vitro affinities. We note, however, that the former couple exhibits a significant lower expression level of the BD-Bait fusion (Fig. 4A, left column) than the latter. Thus, can quantitative information on the strength of the interaction (i.e., relative affinities) be reliably extracted from such an experiment?
To address this question, we tried to correct for differences in the expression level by sub-selecting (gating) only cells that display a red and green fluorescence intensity within a certain narrow interval (see Fig. 4B & C). And indeed, when standardizing the red fluorescence intensity (i.e., gating for cells with similar BD-Bait expression level), the mean reporter level reflects the in-vitro affinities for the two couples BD-HRas / AD-CRaf and BD-Barstar D39A / AD-Barnase H102A (Fig. 4B). Similarly, the couple BD-Barstar Y29F / AD-Barnase H102A has the lowest AD-Prey expression level and displays a weaker reporter level than expected. Standardization of the AD-Prey level corrects the reporter levels of the studied couples according to their reported in-vitro affinities (Fig. 4C). Changing the location of the gating intervals leads to the same conclusions (see Suppl. Fig. S2). Nevertheless, suggestions can be made to choose the optimal the gating intervals (see “Recommendations”).
Affinity ladder permits rapid classification of PPIs based on their strength
Often the goal is to rank PPIs based on their affinity or to obtain an upper and lower bound for the dissociation constant. With the above gating approach, an affinity ladder can easily be generated with a set of PPIs with known dissociation constants (Fig. 5). Standard software of flow cytometers can be used to perform the required gatings and calculate mean fluorescence intensities. We provide a graphical user interface to automate the generation of the affinity ladder (see Experimental Procedures).
The generated affinity ladder can then be used for a rapid visual classification of PPIs with thus far unknown affinities within a given range (here from nano- to picomolar). This is demonstrated at the example of the mutation D35A of Barstar. Thus far, no in-vitro affinity data is available for the interaction of this mutant with Barnase H102A. With the affinity ladder of Fig. 5 we can rank the affinity between those of HRas/CRaf 122 nM) and HRas/CRafA85K (11 nM).
Our experiment indicates that the Barstar mutant D35A exhibits a significantly higher affinity for Barnase H102A than the Barstar mutant D39A (420 nM). To validate this observation, we performed independent alchemical free-energy calculations (see Supp. Mat.). Through the use of a thermodynamic cycle (Suppl. Fig. S3A) we calculated the difference in binding free energy between the mutants Barstar D35A and Barstar D39A. We obtained a value of −1.9 ± 0.3 kcal/mol which indicates that the dissociation constant of the mutant D35A is about 20 times lower than that of the mutant D39A. Thus, we can estimate a dissociation constant of about 20 nM for the mutant D35A in agreement with the qY2H experiment.
Discussion
Quantitative features of the tri-fluorescent yeast two-hybrid system
The tri-fluorescent qY2H system offers the novelty of identifying expression correlations for the genes involved in the actual Y2H reaction. For true positive interactions the read-out is correlated with both reaction partners. For false positive interactions where the Bait acts as an AD (e.g., B112), the read-out is basically only correlated with one reaction partner. This characteristic correlation patterns can serve as additional criteria to discriminate such false positive interactions from true positives. It complements the use of proper controls (i.e., empty Bait and Prey plasmids) routinely applied in Y2H assays.
In the past, significant effort has been spent to render the Y2H read-out quantitative and thereby gain quantitative information on the strength of interactions (see cited literature in the Introduction). Our study clearly demonstrates that the quantification of the reaction partners is important, too. We have shown that variations in the expression levels of BD-Bait and AD-Prey can lead to reporter levels that are not ordered according to the underlying PPI affinities. Through a simple gating process, it is, however, possible to standardize the expression levels of BD-Bait and AD-Prey and thereby overcome this difficulty.
In-cellula is not in-vitro
The observed agreement between in-cellula reporter levels and in-vitro affinities (Fig. 4B) cannot be presumed a priori. The in-vitro experiment measures the affinity between the interactors alone (or with tags) whereas the qY2H system relies on fusions proteins (Fig. 1). If the fused domains influence the interaction between the Bait and Prey, e.g., by blocking the binding interface, the resulting in-cellula reporter level would be impaired and most likely not correlate with the in-vitro affinity.
In-vitro experiments measure the affinity under well-defined buffer-controlled equilibrium conditions. In contrast, our in-cellula experiments take place in non-equilibrium microvessels (37) where the interaction partners can interact with the endogenous complex solution of biomolecules. This may lead to effectively smaller concentrations of the reaction partners. Also, post-translational modification(s) could impact the interactions.
With prior knowledge about the Bait or Prey the amino-acid sequence can be optimized to take into account some effects, like specific sub-cellular localization. An illustrative example is the protein HRas, which is usually found to be associated to the cytoplasmic membrane through its C-terminal anchor. Mutation of C186A abolishes the anchor function (38) and the protein can be used for the in-cellula titrations.
Recommendations
Beside potential sequence optimizations (as proposed above), we recommend the following precautions to be taken for the measurement with the qY2H system:
1) As in any Y2H screen, BD-Bait and AD-Prey constructs should be tested against negative controls, i.e., an empty AD and BD construct, respectively, to identify potential false positive interactions. Also, a mating of yeasts expressing only empty (but fluorescent) AD and BD constructs is recommended; it serves to remove the background of the system.
2) The PPIs should be tested in both orientations, i.e., with the proteins switched between the Bait and Prey vectors, to identify the orientation with the lower background and with the higher reporter level (for standardized levels of reaction partners).
3) We recommend to pre-transform BD-Bait-expressing haploids with the read-out plasmid; it increases the read-out; two subsequent transfections are more efficient than a single double transfection. Use only freshly transformed yeast cells for the qY2H experiment. Storing diploids yeast cells for a week in the refrigerator decreases the level of AD-Prey and read-out by a factor two to five.
4) For the construction of the affinity ladder, the gating interval for the red fluorescence intensity (BD-Bait) was positioned at the lowest possible location to avoid saturation effects, i.e., it was set just above the 95-% threshold of the negative control. The gating intervals of the green fluorescence intensity was set to a medium range value to reach the desired sensitivity but to avoid saturation and protein burden effects (39). The width of each interval gate should be about 20-30% of the value of its lower border.
5) If the gating intervals are not directly applied at acquisition time on the flow cytometer, at least 106 cells should be acquired for analysis. This number is sufficient to reach a converged ladder after gating (see Suppl. Fig. S4). For this number of cells cultures of 10ml are sufficient.
Conclusion
The newly constructed vectors provide access to a novel quantitative Y2H system with fluorescent tags for the reaction partners (BD-Bait, AD-Prey) and the reporter. The established protocol is rapid, sensitive and highly reproducible. It permits easy detection of expression problems of the reaction partners. Using flow cytometry, the expression levels of the reaction partners can be monitored cell by cell simultaneously with the level of the reporter. The single-cell data can be exploited to identify correlation patterns as indicators of physical interactions.
The qY2H method presented in this work offers also an approach to quantitative data on the strength of protein-protein interactions in living cells. In this context, we have demonstrated the importance of quantifying the product and the reaction partners of the Y2H reaction: standardization is critical to correct for differences in expression levels between couples. Using a straightforward gating analysis, an affinity ladder can be easily generated that permits rapid classification of PPIs according to their affinity. We would like to emphasize, however, that these in-cellula affinities are effective quantities that depend on the cell’s complex microenvironment; and this environment may change as a function of the yeast strain and the experimental conditions (temperature, medium, etc).
All steps of the protocol have been optimized in liquid phase that can be easily automated for the use of microplates and integrated within robotic pipelines. It sets the stage for high-throughput quantitative Y2H screenings of PPIs using cross-mating approaches (40, 41) with libraries of yeast clones. As an outlook, quantitative PPI networks can be created by attributing weights to the PPI edges according to their in-cellula reporter levels. It contrasts standard Y2H screens that yield networks with only binary information (YES or NO). The topology of force-directed networks may help identifying key pathways within the network, and how these paths change as a function of environmental conditions (stress, metabolism, etc). Thus, we anticipate that high-throughput qY2H data would boost the modelling of interactomes and thereby advance significantly systems biology.
Data Availability
Our python scripts (with a graphical user interface and user guide for the automated generation of the qY2H affinity ladder) can be downloaded here: http://github.com/LBMC/qY2H-Affinity-Ladder.
The flow cytometry files of the experiment shown in Fig. 4 can be downloaded from http://flowrepository.org under accession number FR-FCM-ZYUL. Other experimental data can be provided upon request.
Acknowledgements
This project was supported by a grant from the Fond Recherche of the ENS de Lyon. We are grateful to the Pôle Scientifique de Modélisation Numérique (Lyon, France) for computer time, and the SFR Biosciences Gerland-Lyon Sud (UMS344/US8) for the access to the MacsQuantVYB flow cytometer. We thank Dr Francesca Palladino and Matthieu Caron for technical support on the Western blots and Dr Gaël Yvert for helpful comment on the manuscript.