Y. Tanaka performed the technical improvement of Y2H. This work is supported by JSPS KAKENHI Grant Number JP22K06336 and the Institute for Fermentation, Osaka.

1. Construction of the mutant library
<ol>
	<li>Set up the polymerase chain reaction (PCR) (an example is shown below). Generally, prepare 50 &#181;L of the reaction, which is divided into ten aliquots of 5 &#181;L, 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.</li>
	<li>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.</li>
	<li>Assemble the DNA fragment generated in step 1.2 with the appropriate Y2H vector using a &#39;seamless&#39; 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).</li>
	<li>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 &#176;C overnight.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
2. Transformation of yeast with the mutant library and replica plating
<ol>
	<li>Introduce the mutant library into the host yeast strain (TAT-7 (L40-ura3)9,10 MATa, leu2-3,112, trp1-901, his3-&#916;200, ade2-101, gal80&#916;, 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 &#39;bait&#39; plasmid.
	<ol>
		<li>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 &#176;C. 
		NOTE: The standard protocol, such as the lithium acetate-PEG method11 with ~5 &#215; 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.</li>
	</ol>
	</li>
	<li>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.</li>
	<li>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 &#176;C for 1-2 days. 
	NOTE: The concentration of kanamycin (G418) is 600 &#181;g/mL. Do not use velvet for replica plating because the resolution of colonies will be lost.</li>
	<li>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.</li>
</ol>
3. Y2H color assay
<ol>
	<li>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.</li>
	<li>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.</li>
	<li>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 &#176;C to -80 &#176;C) to freeze completely.</li>
	<li>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-&#946;-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.</li>
	<li>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 &#176;C until a blue color appears.</li>
	<li>When a blue color appears, place the filter paper with the colony side up on about 500 &#181;L 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.</li>
	<li>After the paper towel is replaced and the filter is dry enough, use a scanner or other device to capture the data.</li>
</ol>
4. Recovery and confirmation of candidate clones
<ol>
	<li>Select white and KanR candidate clones from the replica&#39;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 KanR by re-streaking them as a small patch on SC-LW medium containing kanamycin and incubating them at 30 &#176;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.</li>
	<li>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 KanR and KanS clones.</li>
	<li>Take clones that are still white and KanR 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 &#176;C.</li>
	<li>Transfer the culture to a microtube and collect cells by centrifugation (~15,000 &#215; g, for 10-30 seconds&#160;at room temperature). Isolate whole DNA from the cell pellet as follows.</li>
	<li>Suspend the cell pellet in 400 &#181;L of lysis buffer (2% Triton X-100, 1% SDS, 100 mM NaCl, 10 mM Tris-Cl (8.0), and 1 mM EDTA) containing 1 &#181;L each of 2-mercaptoethanol and 20 mg/mL Zymolyase 100T (see Table of Materials), and incubate at 37 &#176;C for 30 min to digest cell walls. At this time, mix gently by inverting the tube every 5-10 min.</li>
	<li>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.</li>
	<li>Centrifuge the microtubes in a microcentrifuge for 5 min at full speed (~15,000 &#215; g,&#160;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.</li>
	<li>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 &#215; g, at room temperature) to precipitate the DNA.</li>
	<li>Remove the supernatant and wash the precipitate with about 500 &#181;L of 70% ethanol. Remove ethanol completely and dry the precipitate briefly.</li>
	<li>Add 20 &#181;L 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 &#176;C. The DNA precipitate will dissolve in a few hours.</li>
	<li>Once the pellet is completely dissolved, transform E. coli with a small amount of this DNA solution. 
	NOTE: About 0.5 &#181;L of DNA solution is sufficient if E. coli with a high transformation efficiency is used.</li>
	<li>When E. coli colonies appear, pick several independent colonies, culture them, and recover plasmids by standard methods, such as alkaline lysis.</li>
	<li>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.</li>
	<li>If the above two criteria are met, determine the mutation site of the clones by nucleotide sequencing4.</li>
</ol>

Biomolecular Interaction Studies Using Yeast Two-Hybrid Technique 

<ol>
	<li>Fields, S., Song, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+genetic+system+to+detect+protein-protein+interactions.">A novel genetic system to detect protein-protein interactions.</a> Nature. 340 (6230), 245-246 (1989).</li><li>Leanna, C. A., Hannink, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+reverse+two-hybrid+system:+A+genetic+scheme+for+selection+against+specific+protein/protein+interactions.">The reverse two-hybrid system: A genetic scheme for selection against specific protein/protein interactions.</a> Nucleic Acids Res. 24 (17), 3341-3347 (1996).</li><li>Vidal, M., Brachmann, R. K., Fattaey, A., Harlow, E., Boeke, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reverse+two-hybrid+and+one-hybrid+systems+to+detect+dissociation+of+protein-protein+and+DNA-protein+interactions.">Reverse two-hybrid and one-hybrid systems to detect dissociation of protein-protein and DNA-protein interactions.</a> Proc Natl Acad Sci U S A. 93 (19), 10315-10320 (1996).</li><li>Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+efficient+method+for+the+isolation+of+interaction-null/impaired+mutants+using+the+yeast+two-hybrid+technique.">An efficient method for the isolation of interaction-null/impaired mutants using the yeast two-hybrid technique.</a> Genes Cells. 24 (12), 781-788 (2019).</li><li>Green, M. R., Sambrook, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Molecular cloning: A laboratory manual. 4th edn. , (2012).</li><li>Gibson, D. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enzymatic+assembly+of+DNA+molecules+up+to+several+hundred+kilobases.">Enzymatic assembly of DNA molecules up to several hundred kilobases.</a> Nat Methods. 6 (5), 343-345 (2009).</li><li>Inoue, H., Nojima, H., Okayama, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+efficiency+transformation+of+Escherichia+coli+with+plasmids.">High efficiency transformation of Escherichia coli with plasmids.</a> Gene. 96 (1), 23-28 (1990).</li><li>Birnboim, H. C., Doly, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+alkaline+extraction+procedure+for+screening+recombinant+plasmid+DNA.">A rapid alkaline extraction procedure for screening recombinant plasmid DNA.</a> Nucleic Acids Res. 7 (6), 1513-1523 (1979).</li><li>Bartel, P. L., Fields, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analyzing+protein-protein+interactions+using+two-hybrid+system.">Analyzing protein-protein interactions using two-hybrid system.</a> Meth Enzymol. 254, 241-263 (1995).</li><li>Hollenberg, S. M., Sternglanz, R., Cheng, P. F., Weintraub, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+new+family+of+tissue-specific+basic+helix-loop-helix+proteins+with+a+two-hybrid+system.">Identification of a new family of tissue-specific basic helix-loop-helix proteins with a two-hybrid system.</a> Mol Cell Biol. 15 (7), 3813-3822 (1995).</li><li>Gietz, R. D., Woods, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transformation+of+yeast+by+lithium+acetate/single-stranded+carrier+DNA/polyethylene+glycol+method.">Transformation of yeast by lithium acetate/single-stranded carrier DNA/polyethylene glycol method.</a> Meth Enzymol. 350, 87-96 (2002).</li><li>Kushnirov, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+reliable+protein+extraction+from+yeast.">Rapid and reliable protein extraction from yeast.</a> Yeast. 16 (9), 857-860 (2000).</li><li>Van Deursen, F., Sengupta, S., De Piccoli, G., Sanchez-Diaz, A., Labib, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+associates+with+the+loaded+DNA+helicase+at+replication+origins+and+defines+a+novel+step+in+its+activation.">Mcm10 associates with the loaded DNA helicase at replication origins and defines a novel step in its activation.</a> EMBO J. 31 (9), 2195-2206 (2012).</li><li>Watase, G., Takisawa, H., Kanemaki, M. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mcm10+plays+a+role+in+functioning+of+the+eukaryotic+replicative+DNA+helicase,+cdc45-mcm-gins.">Mcm10 plays a role in functioning of the eukaryotic replicative DNA helicase, cdc45-mcm-gins.</a> Current Biology: CB. 22 (4), 343-349 (2012).</li><li>Miyazawa-Onami, M., Araki, H., Tanaka, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-initiation+complex+assembly+functions+as+a+molecular+switch+that+splits+the+mcm2-7+double+hexamer.">Pre-initiation complex assembly functions as a molecular switch that splits the mcm2-7 double hexamer.</a> EMBO Rep. 18 (10), 1752-1761 (2017).</li></ol>

The authors declare that they have no conflict of interest.

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 strain2,3. 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...

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 developed2,3. 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.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.5 M EDTA (8.0)</td>
			<td>Nacalai Tesque Inc.</td>
			<td>14347-21</td>
			<td></td>
		</tr>
		<tr>
			<td>10% SDS Solution</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>313-90275</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>135-07522</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>110-64785</td>
			<td></td>
		</tr>
		<tr>
			<td>5-Bromo-4-chloro-3-indolyl-&#946;-D-galactopyranoside (X-Gal)</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>021-07852</td>
			<td></td>
		</tr>
		<tr>
			<td>Agar</td>
			<td>Formedium</td>
			<td>AGR60</td>
			<td></td>
		</tr>
		<tr>
			<td>Ampicillin Sodium</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>68-52-3</td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-HA-tag mAb-HRP-DirecT&#160;&#160;</td>
			<td>Medical &#38; Biological Laboratories Co. Ltd.</td>
			<td>M180-7</td>
			<td></td>
		</tr>
		<tr>
			<td>DNA from salmon testes</td>
			<td>Merck KGaA.</td>
			<td>D1626</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Merck KGaA.</td>
			<td>9-0770-4-4L-J</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (Grade 50)</td>
			<td>Whatman, Cytiva</td>
			<td>1450-090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for colony lift (No.4A)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>01411090</td>
			<td></td>
		</tr>
		<tr>
			<td>Filter paper for replicaplating (No.1)</td>
			<td>Advantec Toyo Kaisha, Ltd.</td>
			<td>00011150</td>
			<td></td>
		</tr>
		<tr>
			<td>G-418 Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>075-05962</td>
			<td></td>
		</tr>
		<tr>
			<td>Hydrochloric acid</td>
			<td>Kishida Chemical Co. Ltd.</td>
			<td>230-37585</td>
			<td></td>
		</tr>
		<tr>
			<td>KCl</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>163-03545</td>
			<td></td>
		</tr>
		<tr>
			<td>Lithium Acetate Dihydrate</td>
			<td>Nacalai Tesque Inc.</td>
			<td>20604-22</td>
			<td></td>
		</tr>
		<tr>
			<td>MgSO4&#8226;7H2O</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>131-00405</td>
			<td></td>
		</tr>
		<tr>
			<td>Na2HPO4&#8226;12H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>10039-32-4</td>
			<td></td>
		</tr>
		<tr>
			<td>NaCl</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31319-45</td>
			<td></td>
		</tr>
		<tr>
			<td>NaH2PO4&#8226;2H2O</td>
			<td>Nacalai Tesque Inc.</td>
			<td>31717-25</td>
			<td></td>
		</tr>
		<tr>
			<td>Paper towel</td>
			<td>AS ONE Corp.</td>
			<td>7-6200-02</td>
			<td></td>
		</tr>
		<tr>
			<td>Phenol:Chloroform:Isoamyl Alcohol 25:24:1</td>
			<td>Nacalai Tesque Inc.</td>
			<td>25970-56</td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid DNAs</td>
			<td>the National BioResource Project - yeast (https://yeast.nig.ac.jp/yeast/top.xhtml)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Plasmid isolation Kit</td>
			<td>Nippon Genetics Co. Ltd.</td>
			<td>FG-90502</td>
			<td></td>
		</tr>
		<tr>
			<td>Polyethylene Glycol #4,000</td>
			<td>Nacalai Tesque Inc.</td>
			<td>11574-15</td>
			<td></td>
		</tr>
		<tr>
			<td>SC double drop-out mix -Leu -Trp</td>
			<td>Formedium</td>
			<td>DSCK172</td>
			<td></td>
		</tr>
		<tr>
			<td>Seamless cloning kit (In-Fusion assembly )</td>
			<td>Takara Bio Inc.</td>
			<td>#639648</td>
			<td></td>
		</tr>
		<tr>
			<td>Skim milk powder</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>190-12865</td>
			<td></td>
		</tr>
		<tr>
			<td>Streptomycin Sulfate</td>
			<td>Fujifilm Wako Pure Chemical Corp.</td>
			<td>3810-74-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Taq polymerase (GoTaq Green Master Mixes)</td>
			<td>Promega Corp.</td>
			<td>M7122</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIS (hydroxymethyl) aminomethane</td>
			<td>Formedium</td>
			<td>TRIS01</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Nacalai Tesque Inc.</td>
			<td>12967-45</td>
			<td></td>
		</tr>
		<tr>
			<td>Tryptone</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>211705</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween 20</td>
			<td>Nacalai Tesque Inc.</td>
			<td>35624-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Extract</td>
			<td>ThermoFisher scientific Inc.</td>
			<td>212750</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast Nitrogen Base (YNB)</td>
			<td>Formedium</td>
			<td>CYN0210</td>
			<td></td>
		</tr>
		<tr>
			<td>Zymolyase 100T</td>
			<td>Nacalai Tesque Inc.</td>
			<td>07665-55</td>
			<td></td>
		</tr>
	</tbody>
</table>

isolating interaction-null&#47;impaired mutants using the yeast two-hybrid assay

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.

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 cerevisiae13,14,15. 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.</p...

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Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

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.

Isolating Interaction-Null&#47;Impaired Mutants Using the Yeast Two-Hybrid Assay

Protein-protein interactions are one of the most basic processes that underlie biological phenomena. One of the simplest and best ways to understand the ...

We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a comparison of the wild-type and mutant is a direct and effective way. Yeast two-hybrid assay, being simple detection and will enhance the process of isolating interaction-null mutants.This will contribute to advancement of research in the field.

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345 Views.  Kochi University of Technology. We are interested in understanding the regulatory mechanisms of the cell cycle in Eukaryotes, especially the relationship between DNA replication, stable germ maintenance, and cell proliferation. The progression of the cell cycle is regulated by cyclin-dependent kinases, or CDKs, in Eukaryotes. This is the case for the initiation of DNA replication.We have identified a way, on substrates of CDKs, to initiate DNA replication. To understand the function of the protein of your interest, a...

Video: Author Spotlight: Enhanced Isolation of Interaction-Null Mutants in Yeast

Measuring Replicative Life Span in the Budding Yeast

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding yeast Saccharomyces cerevisiae has been used extensively to study the biology of aging, and several determinants of yeast longevity have been shown to be conserved in multicellular eukaryotes, including worms, flies, and mice 1. Due to the lack of easily quantified age-associated phenotypes, aging in yeast has been assayed almost exclusively by measuring the life span of cells in different contexts, with two different life span paradigms in common usage 2. Chronological life span refers to the length of time that a mother cell can survive in a non-dividing, quiescence-like state, and is proposed to serve as a model for aging of post-mitotic cells in multicellular eukaryotes. Replicative life span, in contrast, refers the number of daughter cells produced by a mother cell prior to senescence, and is thought to provide a model of aging in mitotically active cells. Here we present a generalized protocol for measuring the replicative life span of budding yeast mother cells. The goal of the replicative life span assay is to determine how many times each mother cell buds. The mother and daughter cells can be easily differentiated by an experienced researcher using a standard light microscope (total magnification 160X), such as the Zeiss Axioscope 40 or another comparable model. Physical separation of daughter cells from mother cells is achieved using a manual micromanipulator equipped with a fiber-optic needle. Typical laboratory yeast strains produce 20-30 daughter cells per mother and one life span experiment requires 2-3 weeks.

In this article we present a general protocol for measuring the replicative life span of yeast mother cells.

Aging is a degenerative process characterized by a progressive deterioration of cellular components and organelles resulting in mortality. The budding ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Split-Ubiquitin Based Membrane Yeast Two-Hybrid (MYTH) System: A Powerful Tool For Identifying Protein-Protein Interactions

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput identification of protein-protein interactions (PPI) for full-length transmembrane proteins. To this end, our lab developed the split-ubiquitin based Membrane Yeast Two-Hybrid (MYTH) system. This technology allows for the sensitive detection of transient and stable protein interactions using Saccharomyces cerevisiae as a host organism. MYTH takes advantage of the observation that ubiquitin can be separated into two stable moieties: the C-terminal half of yeast ubiquitin (Cub) and the N-terminal half of the ubiquitin moiety (Nub). In MYTH, this principle is adapted for use as a 'sensor' of protein-protein interactions. Briefly, the integral membrane bait protein is fused to Cub which is linked to an artificial transcription factor. Prey proteins, either in individual or library format, are fused to the Nub moiety. Protein interaction between the bait and prey leads to reconstitution of the ubiquitin moieties, forming a full-length 'pseudo-ubiquitin' molecule. This molecule is in turn recognized by cytosolic deubiquitinating enzymes, resulting in cleavage of the transcription factor, and subsequent induction of reporter gene expression. The system is highly adaptable, and is particularly well-suited to high-throughput screening. It has been successfully employed to investigate interactions using integral membrane proteins from both yeast and other organisms.

Split-Ubiquitin Based Membrane Yeast Two-Hybrid MYTH System: A Powerful Tool For Identifying Protein-Protein Interactions

MYTH allows the sensitive detection of transient and stable interactions between proteins that are expressed in the model organism Saccharomyces cerevisiae. It has been successfully applied to study exogenous and yeast integral membrane proteins in order to identify their interacting partners in a high throughput manner.

The fundamental biological and clinical importance of integral membrane proteins prompted the development of a yeast-based system for the high-throughput ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Modified Yeast-Two-Hybrid System to Identify Proteins Interacting with the Growth Factor Progranulin

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role in a variety of physiologic and disease processes, including early embryogenesis, wound healing 1, inflammation 2, 3, and host defense 4. PGRN also functions as a neurotrophic factor 5, and mutations in the PGRN gene resulting in partial loss of the PGRN protein cause frontotemporal dementia 6, 7. Our recent studies have led to the isolation of PGRN as an important regulator of cartilage development and degradation 8-11. Although PGRN, discovered nearly two decades ago, plays crucial roles in multiple physiological and pathological conditions, efforts to exploit the actions of PGRN and understand the mechanisms involved have been significantly hampered by our inability to identify its binding receptor(s). To address this issue, we developed a modified yeast two-hybrid (MY2H) approach based on the most commonly used GAL4 based 2-hybrid system. Compared with the conventional yeast two-hybrid screen, MY2H dramatically shortens the screen process and reduces the number of false positive clones. In addition, this approach is reproducible and reliable, and we have successfully employed this system in isolating the binding proteins of various baits, including ion channel 12, extracellular matrix protein 10, 13, and growth factor14. In this paper, we describe this MY2H experimental procedure in detail using PGRN as an example that led to the identification of TNFR2 as the first known PGRN-associated receptor 14, 15.

We have modified the conventional yeast two-hybrid screening, an effective genetic tool in identifying protein interaction. This modification markedly shortens the process, reduces the workload, and most importantly, reduces the number of false positives. In addition, this approach is reproducible and reliable.

Progranulin (PGRN), also known as granulin epithelin precursor (GEP), is a 593-amino-acid autocrine growth factor. PGRN is known to play a critical role ...

Isolating Primary Melanocyte-like Cells from the Mouse Heart

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice. To investigate the electrical and biological properties of cardiac melanocytes we developed a procedure to isolate them from mouse hearts that we derived from those designed to isolate neonatal murine cardiomyocytes. In order to obtain healthier cardiac melanocytes suitable for more extensive patch clamp or biochemical studies, we developed a refined procedure for isolating and plating cardiac melanocytes based on those originally designed to isolate cutaneous melanocytes. The refined procedure is demonstrated in this review and produces larger numbers of healthy melanocyte-like cells that can be plated as a pure population or with cardiomyocytes.

In this protocol, we identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial arrhythmia triggers in mice.&#160;

We identified a novel population of melanocyte-like cells (also known as cardiac melanocytes) in the hearts of mice and humans that contribute to atrial ...

Reverse Yeast Two-hybrid System to Identify Mammalian Nuclear Receptor Residues that Interact with Ligands and/or Antagonists

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking metabolism and inflammation (e.g.&#160;hepatic steatosis)1,2. There has been much progress in the identification of agonist ligands for PXR, however, there are limited descriptions of drug-like antagonists and their binding sites on PXR3,4,5. A critical barrier has been the inability to efficiently purify full-length protein for structural studies with antagonists despite the fact that PXR was cloned and characterized in 1998. Our laboratory developed a novel high throughput yeast based two-hybrid assay to define an antagonist, ketoconazole&#39;s, binding residues on PXR6. Our method involves creating mutational libraries that would rescue the effect of single mutations on the AF-2 surface of PXR expected to interact with ketoconazole. Rescue or &#34;gain-of-function&#34; second mutations can be made such that conclusions regarding the genetic interaction of ketoconazole and the surface residue(s) on PXR are feasible. Thus, we developed a high throughput two-hybrid yeast screen of PXR mutants interacting with its coactivator, SRC-1. Using this approach, in which the yeast was modified to accommodate the study of the antifungal drug, ketoconazole, we could demonstrate specific mutations on PXR enriched in clones unable to bind to ketoconazole. By reverse logic, we conclude that the original residues are direct interaction residues with ketoconazole. This assay represents a novel, tractable genetic assay to screen for antagonist binding sites on nuclear receptor surfaces. This assay could be applied to any drug regardless of its cytotoxic potential to yeast as well as to cellular protein(s) that cannot be studied using standard structural biology or proteomic based methods. Potential pitfalls include interpretation of data (complementary methods useful), reliance on single Y2H method, expertise in handling yeast or performing yeast two-hybrid assays, and assay optimization.

Ketoconazole binds to and antagonizes Pregnane X Receptor (PXR) activation. Yeast high throughput screens of PXR mutants define a unique region for ketoconazole binding. This yeast-based genetic method discovers novel nuclear receptor interactions with ligands that associate with surface binding sites.

As a critical regulator of drug metabolism and inflammation, Pregnane X Receptor (PXR), plays an important role in disease pathophysiology linking ...

Isolating Potentiated Hsp104 Variants Using Yeast Proteinopathy Models

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic lateral sclerosis, and &#945;-synuclein, implicated in Parkinson&#8217;s disease, are toxic and form cytoplasmic aggregates in yeast. These features recapitulate protein pathologies observed in patients with these disorders. Thus, yeast are an ideal platform for isolating toxicity suppressors from libraries of protein variants. We are interested in applying protein disaggregases to eliminate misfolded toxic protein conformers. Specifically, we are engineering Hsp104, a hexameric AAA+ protein from yeast that is uniquely capable of solubilizing both disordered aggregates and amyloid and returning the proteins to their native conformations. While Hsp104 is highly conserved in eukaryotes and eubacteria, it has no known metazoan homologue. Hsp104 has only limited ability to eliminate disordered aggregates and amyloid fibers implicated in human disease. Thus, we aim to engineer Hsp104 variants to reverse the protein misfolding implicated in neurodegenerative disorders. We have developed methods to screen large libraries of Hsp104 variants for suppression of proteotoxicity in yeast. As yeast are prone to spontaneous nonspecific suppression of toxicity, a two-step screening process has been developed to eliminate false positives. Using these methods, we have identified a series of potentiated Hsp104 variants that potently suppress the toxicity and aggregation of TDP-43, FUS, and &#945;-synuclein. Here, we describe this optimized protocol, which could be adapted to screen libraries constructed using any protein backbone for suppression of toxicity of any protein that is toxic in yeast.

Yeast proteinopathy models are valuable tools to assess the toxicity and aggregation of proteins implicated in disease. Here, we present methods for screening Hsp104 variant libraries for toxicity suppressors. This protocol could be adapted to screen any protein library for toxicity suppressors of any protein that is toxic in yeast.

Many protein-misfolding disorders can be modeled in the budding yeast Saccharomyces cerevisiae. Proteins such as TDP-43 and FUS, implicated in amyotrophic ...

Using Microfluidic Devices to Measure Lifespan and Cellular Phenotypes in Single Budding Yeast Cells

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects on the lifespan across species. However, the molecular causes of aging and death remain elusive. To gain a systematic understanding of the molecular mechanisms underlying yeast aging, we need high-throughput methods to measure lifespan and to quantify various cellular and molecular phenotypes in single cells. Previously, we developed microfluidic devices to track budding yeast mother cells throughout their lifespan while flushing away newborn daughter cells. This article presents a method for preparing microfluidic chips and for setting up microfluidic experiments. Multiple channels can be used to simultaneously track cells under different conditions or from different yeast strains. A typical setup can track hundreds of cells per channel and allow for high-resolution microscope imaging throughout the lifespan of the cells. Our method also allows detailed characterization of the lifespan, molecular markers, cell morphology, and the cell cycle dynamics of single cells. In addition, our microfluidic device is able to trap a significant amount of fresh mother cells that can be identified by downstream image analysis, making it possible to measure the lifespan with higher accuracy.

This article presents a protocol optimized for the production of microfluidic chips and the setup of microfluidic experiments to measure the lifespan and cellular phenotypes of single yeast cells.

Budding yeast Saccharomyces cerevisiae is an important model organism in aging research. Genetic studies have revealed many genes with conserved effects ...