Isolating Interaction-Null/Impaired Mutants Using the Yeast Two-Hybrid Assay

Summary

The interactions of biomolecules, such as protein-protein interactions, are the molecular basis of biological functions. If interaction-null/impaired mutants that specifically lack the relevant interaction can be isolated, they will greatly help to understand the function(s) of this interaction. This article presents an efficient way to isolate interaction-null/impaired mutants.

Abstract

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the role(s) and function(s) of a specific protein-protein interaction is to compare the phenotype of the wild-type (with the relevant protein-protein interaction) and those of mutants that lack the relevant interaction. Therefore, if such mutants can be isolated, they will help to elucidate the related biological processes. The yeast two-hybrid (Y2H) procedure is a powerful approach not only to detect protein-protein interactions but also to isolate interaction-null/impaired mutants. In this article, a protocol is presented to isolate interaction-null/impaired mutants using Y2H technology. First, a mutation library is constructed by combining the polymerase chain reaction and efficient seamless cloning technology, which efficiently excludes the empty vector from the library. Second, interaction-null/impaired mutants are screened by the Y2H assay. Because of a trick in the Y2H vector, undesired mutants, such as those with frameshift and nonsense mutations, are efficiently eliminated from the screening process. This strategy is simple and can, therefore, be applied to any combination of proteins whose interaction can be detected by the two-hybrid system.

Introduction

Interactions between biomolecules are the most basic part of biological phenomena. Protein-protein interactions constitute a significant part of such interactions. Therefore, identification of the interaction partner(s) of a protein of interest is critical to further elucidate the function of the protein/gene of interest. The yeast two-hybrid (Y2H) method is a popular technique to identify protein-protein interactions in vivo¹. In this system, two proteins (X and Y) whose interaction is to be tested are fused to the DNA-binding (DB) domain and transcriptional activation domain (AD), respectively. The DB-X fusion protein binds to a recognition sequence of the DB domain; therefore, when proteins X and Y interact, the AD-Y fusion protein comes into the proximity of the recognition sequence. Consequently, transcription of the reporter gene downstream of the recognition sequence is activated. Therefore, the presence or absence of reporter gene activity can be used to determine the presence or absence of the protein-protein interaction¹.

Once a specific interaction partner of the protein of interest is identified, further analyses should be performed to elucidate the biological function of the interaction. For this purpose, if mutants of the proteins that impair or remove the specific protein-protein interaction can be isolated, they will serve as powerful tools. The Y2H system can be used directly to isolate such mutants by screening 'interaction-negative' clones, starting with the wild-type 'interaction-positive' clone. To accelerate this process, 'reverse' Y2H (rY2H) systems were developed^2,3. In rY2H systems, the host yeast strains harbor counter-selectable marker genes as reporter genes, meaning yeast cells grow only when the AD-Y and DB-X proteins do not interact.

Although both the Y2H and rY2H systems allow the isolation of interaction-negative mutants, the process of isolating the mutants is laborious because not all of the candidates obtained by screening carry the desired type of mutations (usually missense mutations). The most serious issue is that a significant fraction of candidates harbor frameshift or nonsense mutations, and it is necessary to perform western blotting to exclude undesired clones. To overcome this problem, new plasmid vectors have been developed⁴. In these vectors, KanMX, a drug resistance marker, is positioned out-of-frame downstream of the DB domain or AD. The marker gene becomes in-frame with the DB domain or AD only when the gene of interest is inserted. When a random mutation(s) is introduced in the gene of interest, undesirable mutants, such as those with frameshift or nonsense mutations, can be easily eliminated by performing drug resistance selection, and candidates carrying desirable missense mutations can be easily identified with the Y2H screen⁴. This article presents a protocol to isolate interaction-null/impaired mutants of a protein of interest using this strategy.

Protocol

1. Construction of the mutant library

1. Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 µL of the reaction, which is divided into ten aliquots of 5 µL, and amplify the target gene fragment in a PCR machine⁴.
   NOTE: Users should select an appropriate polymerase to efficiently introduce mutations. If the DNA fragment to be amplified is short, then a polymerase with a higher error rate, such as Taq polymerase, which lacks proofreading activity, is needed. If the DNA fragment to be amplified is long, then a higher fidelity polymerase is needed to reduce the number of mutations. Mutations occur every few hundred base pairs after 30 cycles of amplification with regular Taq polymerase⁴. In the Representative results, a different Taq polymerase was used (see Table of Materials), which has higher fidelity than regular Taq polymerase, and the mutation rate was one in 600 base pairs. An example of the PCR mixtures, the primer details, and the reaction conditions are provided in Supplementary File 1.
2. When the PCR is completed, combine all the aliquots of the reaction, confirm that the DNA fragments of interest have been amplified using agarose gel electrophoresis, etc., and purify the DNA⁵.
3. Assemble the DNA fragment generated in step 1.2 with the appropriate Y2H vector using a 'seamless' cloning strategy (Figure 1).
   NOTE: The seamless cloning system, such as Gibson assembly⁶, minimizes contamination of the constructed mutant library by empty vectors generated via self-ligation. A list of Y2H vectors is provided in Table 1. They can be prepared by BamHI digestion before assembly. All plasmids are available from the National BioResource Project - yeast (see Table of Materials).
4. Introduce the reaction mixture generated in step 1.3 into Escherichia coli competent cells prepared according to a highly efficient E. coli transformation protocol (e.g., Inoue et al.⁷). Spread all competent cells on several LB plates (1% tryptone, 0.5% yeast extract, 1% NaCl, and 2% agar) containing appropriate antibiotics (see Table of Materials). At this point, spread a small amount (~1/100 of the total reaction) onto the same selection plate to calculate the library titer. Incubate the plates at 37 °C overnight.
5. Once a large number of E. coli colonies have appeared, scrape all cells from the surface of the plate using a disposable plastic loop. Recover plasmid DNA from these cells using popular methods, such as alkaline lysis⁸. At the same time, count the number of colonies that appear after spreading small aliquots of the transformation mixture on the plate. Using this number, estimate the total number of independent clones in the library.
   NOTE: At this point, pick up a few independent colonies (4-6) that appear on the plate on which the small aliquots of transformation reaction were spread, culture them separately, and isolate plasmids from them to confirm that virtually all clones carry the desired DNA fragments⁵. Many commercial kits are available to isolate plasmids from E. coli. The number of colonies that appear on the plate after spreading small aliquots can be counted manually.
6. Measure the concentration of the library DNA pool. Usually, a few hundred nanograms of library DNA is sufficient to obtain several thousand transformants in the next step.
   NOTE: It is recommended to measure the DNA concentration using a fluorescent dye that binds to DNA because measurement of A₂₆₀ is frequently inaccurate.

2. Transformation of yeast with the mutant library and replica plating

1. Introduce the mutant library into the host yeast strain (TAT-7 (L40-ura3)^9,10 MATa, leu2-3,112, trp1-901, his3-Δ200, ade2-101, gal80Δ, LYS2:(lexAop)₄-HIS3, ura3:(lexAop)₈-lacZ) for the Y2H assay using about 100 ng of DNA. Pre-transform the host cells with the 'bait' plasmid.
   1. After the transformation procedure, suspend yeast cells in sterile water and spread aliquots of different amounts on appropriate plates (usually SC medium lacking leucine and tryptophan: SC-LW [0.67% yeast nitrogen base, 2% glucose, 1.546 g/L SC double drop-out mix -Leu -Trp, and 2% agar, see Table of Materials]). Incubate these plates overnight at 30 °C.
      NOTE: The standard protocol, such as the lithium acetate-PEG method¹¹ with ~5 × 10⁶ logarithmically growing cells, is sufficient for the transformation of yeast. Another method with a high transformation efficiency, such as electroporation, can also be used.
2. The next day, when tiny colonies appear, select the plates with an appropriate number of cells (500-2000) and make replicates from them as follows.
3. Set two sheets of filter paper on a replica block to make several replicas (medium is SC-LW and SC-LW containing kanamycin) (see Table of Materials). Incubate at 30 °C for 1-2 days.
   NOTE: The concentration of kanamycin (G418) is 600 µg/mL. Do not use velvet for replica plating because the resolution of colonies will be lost.
4. Once colonies have grown, compare plates and pick candidates. Perform a Y2H color assay (see step 3) if lacZ is used as the reporter.
   NOTE: For the Y2H reporter, lacZ is usually better at detecting a weak interaction than HIS3.

3. Y2H color assay

1. Place a sheet of filter paper cut in advance to the appropriate size on the replica plate prepared using step 2. Be careful not to allow air bubbles to form between the filter paper and the plate.
   NOTE: Use No. 4A filter paper or Grade 50 filter paper (see Table of Materials). Any SC-LW or SC-LW containing kanamycin plates, which are made by replica plating, can be used for the color assay.
2. When the filter paper on the plate is completely moistened (when the color of the filter paper changes completely), place several sheets of paper towel on top and make it come into contact with the filter paper to remove excess moisture. Remove the paper towel when it absorbs the water and changes color. Repeat this process two or three times.
3. Pick up the edges of the filter paper with tweezers, peel it off the plate quickly, immerse it in liquid nitrogen, and freeze it. If liquid nitrogen is not available, place the filter paper on a plastic tray with the colony side up and immediately place it in a freezer (-20 °C to -80 °C) to freeze completely.
4. Remove the frozen filter paper and place it, with the colony side up, on a dry paper towel to thaw. Immediately after thawing, place the filter paper, with the colony side up, on another filter paper that has been placed on a plastic tray or sealable container and soaked with Z-buffer (60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, and 1 mM MgSO₄, pH 7.0) containing X-gal (5-bromo-5-chloro-3-indolyl-β-D-galactoside) and 2-mercaptoethanol⁹ (see Table of Materials). Be careful not to allow air bubbles to form between the top and bottom filter papers.
5. Cover the plastic tray containing the filter paper and put it into an airtight container or plastic bag. Close the airtight container or plastic bag and incubate it at 37 °C until a blue color appears.
6. When a blue color appears, place the filter paper with the colony side up on about 500 µL of stop solution (1 M Na₂CO₃) placed on a clean plastic tray. Stop solution soaks in quickly; therefore, remove the filter immediately, place it on a fresh paper towel with the colony side up, and allow it to dry.
7. After the paper towel is replaced and the filter is dry enough, use a scanner or other device to capture the data.

4. Recovery and confirmation of candidate clones

1. Select white and Kan^R candidate clones from the replica's original plate (or the replicated plate not used for the color assay). Examples are shown in Figure 1C. Confirm that candidate clones are white and Kan^R by re-streaking them as a small patch on SC-LW medium containing kanamycin and incubating them at 30 °C for 1-2 days. Make at least two sets or make replicates the next day.
   NOTE: White colonies should be selected because pale blue colonies might retain the interaction.
2. Once candidate clones have re-grown well, repeat the color assay with at least one plate generated in step 4.1 as described in step 3 and confirm that they remain white and do not turn blue (Figure 2A).
   NOTE: When re-streaking the candidates, do not carry over large amounts of cells. Too many cells make it impossible to distinguish Kan^R and Kan^S clones.
3. Take clones that are still white and Kan^R generated in step 4.2 from the plate remains and grow them independently in 1 mL of selection medium (SC-L or SC-W, depending on which vector is used for the library construction) overnight at 30 °C.
4. Transfer the culture to a microtube and collect cells by centrifugation (~15,000 × g, for 10-30 seconds at room temperature). Isolate whole DNA from the cell pellet as follows.
5. Suspend the cell pellet in 400 µL of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl (8.0), and 1 mM EDTA) containing 1 µL each of 2-mercaptoethanol and 20 mg/mL Zymolyase 100T (see Table of Materials), and incubate at 37 °C for 30 min to digest cell walls. At this time, mix gently by inverting the tube every 5-10 min.
6. When the cell suspension has become opaque or translucent, add an equal volume of a mixture of phenol-chloroform-isoamyl alcohol (25:24:1) and mix well by vortexing at moderate speed for 10-20 s.
7. Centrifuge the microtubes in a microcentrifuge for 5 min at full speed (~15,000 × g, at room temperature) and recover the aqueous phase containing DNA in a new microtube. Add 0.8 volume of isopropanol and mix well by inverting the tube.
8. Leave the tube at room temperature for 2-3 min and then centrifuge in a microcentrifuge for 4-5 min at full speed (~15,000 × g, at room temperature) to precipitate the DNA.
9. Remove the supernatant and wash the precipitate with about 500 µL of 70% ethanol. Remove ethanol completely and dry the precipitate briefly.
10. Add 20 µL of TE buffer (10 mM Tris-Cl (8.0) and 1 mM EDTA) to the precipitate, mix briefly, and leave the tube overnight at room temperature to dissolve the precipitate.
    NOTE: If users are in a hurry, keep tubes at 37-50 °C. The DNA precipitate will dissolve in a few hours.
11. Once the pellet is completely dissolved, transform E. coli with a small amount of this DNA solution.
    NOTE: About 0.5 µL of DNA solution is sufficient if E. coli with a high transformation efficiency is used.
12. When E. coli colonies appear, pick several independent colonies, culture them, and recover plasmids by standard methods, such as alkaline lysis.
13. Introduce the plasmid DNA obtained in step 4.12 into Y2H host cells harboring the bait plasmid. When transformants appear, check them by the color assay (they should appear white) and western blotting⁴ (they should express a protein of the desired size).
    NOTE: The post-alkaline method¹² is an easy and rapid way to prepare a whole-cell extract from yeast.
14. If the above two criteria are met, determine the mutation site of the clones by nucleotide sequencing⁴.

Results

Recently, it was found that the C-terminal half of Pol2 protein (Pol2-C) interacts with Mcm10. Both proteins are essential for the initiation of DNA replication and, hence, for cell growth in the budding yeast Saccharomyces cerevisiae^13,14,15. To help understand the biological significance of this interaction, Pol2-C mutants that have no/diminished interaction with Mcm10 were isolated using the method described here.

Discussion

This article describes how to isolate interaction-null/impaired mutants using the Y2H assay. Such mutants are powerful tools to analyze the function of a protein of interest. To isolate such mutants, rY2H assays were developed previously by modifying the Y2H host strain^2,3. However, they have not greatly reduced the amount of labor. By contrast, mutants can be isolated with this method without a significant amount of labor. In this method, modification of the Y2H...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.5 M EDTA (8.0)	Nacalai Tesque Inc.	14347-21
10% SDS Solution	Fujifilm Wako Pure Chemical Corp.	313-90275
2-mercaptoethanol	Fujifilm Wako Pure Chemical Corp.	135-07522
2-propanol	Kishida Chemical Co. Ltd.	110-64785
5-Bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal)	Fujifilm Wako Pure Chemical Corp.	021-07852
Agar	Formedium	AGR60
Ampicillin Sodium	Fujifilm Wako Pure Chemical Corp.	68-52-3
Anti-HA-tag mAb-HRP-DirecT	Medical & Biological Laboratories Co. Ltd.	M180-7
DNA from salmon testes	Merck KGaA.	D1626
Ethanol	Merck KGaA.	9-0770-4-4L-J
Filter paper for colony lift (Grade 50)	Whatman, Cytiva	1450-090
Filter paper for colony lift (No.4A)	Advantec Toyo Kaisha, Ltd.	01411090
Filter paper for replicaplating (No.1)	Advantec Toyo Kaisha, Ltd.	00011150
G-418 Sulfate	Fujifilm Wako Pure Chemical Corp.	075-05962
Hydrochloric acid	Kishida Chemical Co. Ltd.	230-37585
KCl	Fujifilm Wako Pure Chemical Corp.	163-03545
Lithium Acetate Dihydrate	Nacalai Tesque Inc.	20604-22
MgSO4•7H2O	Fujifilm Wako Pure Chemical Corp.	131-00405
Na2HPO4•12H2O	Nacalai Tesque Inc.	10039-32-4
NaCl	Nacalai Tesque Inc.	31319-45
NaH2PO4•2H2O	Nacalai Tesque Inc.	31717-25
Paper towel	AS ONE Corp.	7-6200-02
Phenol:Chloroform:Isoamyl Alcohol 25:24:1	Nacalai Tesque Inc.	25970-56
Plasmid DNAs	the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)
Plasmid isolation Kit	Nippon Genetics Co. Ltd.	FG-90502
Polyethylene Glycol #4,000	Nacalai Tesque Inc.	11574-15
SC double drop-out mix -Leu -Trp	Formedium	DSCK172
Seamless cloning kit (In-Fusion assembly )	Takara Bio Inc.	#639648
Skim milk powder	Fujifilm Wako Pure Chemical Corp.	190-12865
Streptomycin Sulfate	Fujifilm Wako Pure Chemical Corp.	3810-74-0
Taq polymerase (GoTaq Green Master Mixes)	Promega Corp.	M7122
TRIS (hydroxymethyl) aminomethane	Formedium	TRIS01
Triton X-100	Nacalai Tesque Inc.	12967-45
Tryptone	ThermoFisher scientific Inc.	211705
Tween 20	Nacalai Tesque Inc.	35624-15
Yeast Extract	ThermoFisher scientific Inc.	212750
Yeast Nitrogen Base (YNB)	Formedium	CYN0210
Zymolyase 100T	Nacalai Tesque Inc.	07665-55