We are thankful for the thoughtful discussions with members of the Ju laboratory and Zhong laboratory, and the financial support from the Wright State University.

NOTE: The protocol described here is designed for screening library plasmids contained in ten 96-well plates but can be scaled up or down accordingly. The protocol needs to be repeated to complete the whole library screening. Usually, screening against 10 plates of library genes each time can be comfortably handled by 1 person.
1. Preparation for 96-well Yeast Transformation
NOTE: This step is done as previously described7,10.
<ol>
	<li>Aliquot 5 &#181;L (about 50&#8211;100 ng) of plasmid DNA from an arrayed plasmid library into each well of a round-bottom 96-well plate. 
	NOTE: An example of such a library would be a yeast gene overexpression library10.</li>
	<li>Inoculate 150 mL of yeast peptone dextrose (YPD) media in a 500 mL flask with a colony (2&#8211;3 mm diameter) of the haploid yeast strain W303&#945;. Incubate them overnight at 30 &#176;C with shaking (200 rpm).</li>
	<li>The following morning, measure the OD600 of the overnight culture and dilute the yeast culture to OD600 = 0.1 in (up to) 2 L of YPD. Incubate it at 30 &#176;C with shaking (200 rpm) for ~5 h until the culture reaches OD600 = 0.4&#8211;0.6.</li>
</ol>
2. Yeast Transformation
<ol>
	<li>Harvest the yeast culture by filling 8 sterile 250 mL centrifuge bottles and centrifuging them at room temperature at 3,000 x g for 10 min. Pour off the supernatant without disrupting the pellet. 
	NOTE: Carry out all centrifugation steps at room temperature.</li>
	<li>Wash the yeast with sterile-distilled H2O. For this, add 100 mL of sterile H2O to each centrifuge bottle and vortex it to resuspend the cell pellet. Combine the washed cells into 2 bottles and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>Wash the cells in each bottle in 100 mL of 0.1M LiOAc/1XTE (100 mM LiOAc; 10 mM Tris, pH 8.0; 1 mM EDTA) and centrifuge them at 3,000 x g for 5 min. Pour off the supernatant.</li>
	<li>While centrifuging, boil 5 mL of salmon sperm DNA (10 mg/mL) at 100 &#176;C using a block heater for 3 min and then cool it on ice.</li>
	<li>Resuspend the cell pellet in 25 mL of 0.1M LiOAc/1XTE in each bottle, combine the resuspended cells and transfer them to a 150 mL flask. Add 5 mL of the pre-cooled salmon sperm DNA and incubate it at 30 &#176;C with shaking (225 rpm) for 30 min.</li>
	<li>Pour the cell mixture into a sterile disposable reagent reservoir and, using a multichannel pipette, transfer 35 &#181;L of the cell mixture to each well of the round-bottom 96-well plate containing the library plasmid DNA. Vortex the 96-well plates using a plate vortexer for 1 min at 1,000 rpm. Incubate the plates for 30 min at 30 &#176;C without shaking. 
	NOTE: Do not stack the plates, so the heat can transfer more efficiently. We have found that vortexing the 96-well plates at 1,000 rpm does not cause liquid to spill out of the wells, but a safe vortex speed should be tested before performing the step.</li>
	<li>In a flask, prepare 200 mL of the transformation buffer containing a final concentration of 40% PEG3350, 10% DMSO, and 0.1M LiOAc. Prepare the transformation buffer immediately before use and mix it thoroughly by shaking.</li>
	<li>Remove 96-well plates from the 30 &#176;C incubator and mix them for 30 s at 1,000 rpm using the plate vortexer. Add 125 &#181;L of the transformation buffer to each well and then vortex the plates for 1 min at 1,000 rpm.</li>
	<li>Incubate the plates at 30 &#176;C for 30 min and then heat shock the yeast by placing the plates in a 42 &#176;C incubator for 15 min. 
	NOTE: Do not stack the plates.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g. Remove the transformation buffer from the wells by inverting the plates over a waste bin and forcefully dumping the buffer from the plates. Quickly blot the inverted plates on a clean paper towel to remove any liquid on the top of the plates.</li>
	<li>Rinse the cells by adding to the plates 200 &#181;L of minimal dropout medium corresponding to the selectable marker on the library plasmid. 
	NOTE: We used a synthetic Ura- medium.</li>
	<li>Centrifuge the plates for 5 min at 3,000 x g and remove the supernatant over a waste bin as in step 2.10.</li>
	<li>Add 160 &#181;L of minimal Ura-medium containing 2% glucose. Vortex the plates for 1 min at 1,000 rpm and incubate them at 30 &#176;C for 48 h without shaking. After 48 h, note that a pellet of transformed yeast is visible at the bottom of each well.</li>
	<li>Using a liquid dispenser, add 100 &#181;L of Ura-media containing glucose into each well of a new set of 96-well plates.</li>
	<li>Vortex the plates containing the transformed yeast (from step 2.13) at 1,000 rpm for 30 s. Using a sterile plastic 96-pin replicator, place the pins into the wells containing the transformed yeast and then inoculate them into the corresponding wells of the new plates filled with media. Incubate the new plates at 30 &#176;C for 24 h. 
	NOTE: If working with 10 plates, the medium can also be dispensed manually using multi-channel pipettes.</li>
	<li>After 24 h, a pellet of yeast should be visible in the bottom of each well containing successfully transformed yeast. To save these yeast strains as glycerol stocks, add 50 &#181;L of 50% glycerol to each well, vortex the plates for 30 s at 1,000 rpm, seal the plates with sealing tape and freeze them at -80 &#176;C. 
	NOTE: The above steps only need to be performed once. Future screenings of different yeast models against the same library disposable reagent reservoir can start from the glycerol stocks and proceed immediately from the steps below.</li>
</ol>
3. Mating Between Cells Containing Library Genes and Query Yeast
<ol>
	<li>To revive the library strains from the glycerol stock, take the 96-well plates out of the -80 &#176;C freezer, remove the sealing tape, and let the yeast thaw at room temperature for approximately 30 min.</li>
	<li>Once the yeast has thawed, use a sterile plastic 96-pin replicator to inoculate the glycerol stocks to 160 &#181;L of fresh Ura-media containing 2% glucose in 96-well plates and incubate them at 30 &#176;C for 24 h. Immediately after the library strains are used, seal the plates with sealing tape, and return them to the -80 &#176;C freezer.</li>
	<li>On the same day the library strains (W303&#945;) are thawed, inoculate the query yeast strain (here, FUS in W303a) to 50 mL of YPD and grow it overnight at 30 &#176;C with shaking at 250 rpm.</li>
	<li>The next morning, pour the query yeast strain to a sterile disposable reagent reservoir, and aliquot 160 &#181;L of the query strain to each well of a 96-well plate using a multichannel pipette.</li>
	<li>Using the liquid dispenser, dispense 160 &#181;L of YPD media into each well of ten 96-well plates. Use these plates later for mating the query yeast strain with the library yeast.</li>
	<li>Briefly vortex the 96-well plate containing the query strain and then use a sterile 96-pin replicator to transfer the query strain to the YPD plates.</li>
	<li>Briefly vortex the library strain plates and, for each library strain plate, use a new sterile 96-pin replicator to transfer the library strains to the YPD plates that have been inoculated with the query strain. 
	NOTE: Cells in glycerol lose their viability after multiple thaw-freeze cycles. Returning the library strains to the long-term storage (-80 &#176;C freezer) as soon as possible will keep the library in good quality, and ready for future use. Properly maintained, the glycerol stock can be frozen and thawed at least 10 times. We also strongly recommend keeping a working copy and backup copies of the glycerol stock. When the working stock does not work, immediately make a new working copy from the backup copy.</li>
	<li>Incubate the YPD plates at 30 &#176;C for 24 h. Note that a pellet of yeast will be visible in the bottom of each well after 24 h.</li>
	<li>Fill 96-well plates with a minimal dropout medium containing 2% raffinose, corresponding to the selectable markers on the plasmid in the query strain as well as on the library plasmid (e.g., here Ura-His-).</li>
	<li>Use a sterile 96-pin replicator to transfer yeast from the YPD mating cultures to the selective media. Incubate the 96-well plates at 30 &#176;C for 48 h; only yeast cells that have mated and formed diploid cells and, therefore, contain both the query plasmid and the library plasmid will be able to grow in this media. After 2 days, observe that a pellet is visible on the bottom of the wells.</li>
</ol>
4. Spotting Assay
<ol>
	<li>After 48 h of growth in the raffinose-containing selective media, spot the yeast on agar plates.
	<ol>
		<li>Prepare 2 sets of Ura-His- dropout plates containing 2% agar using clear polystyrene plates, one containing 2% galactose and the other containing 2% glucose.</li>
		<li>Vortex the 96-well plates for 1 min at 1,000 rpm, then spot the yeast to the Ura-His- dropout plates containing 2% galactose and 2% agar (FUS and library genes are induced) and to the Ura-His- dropout plates containing 2% glucose and 2% agar (FUS and library genes repressed) using a robotic spotting machine, by which the culture in each well is spotted onto the agar plates in quadruplicate (i.e., the culture in 1 well is spotted to 4 spots on the agar plate).</li>
		<li>After spotting, let the agar plates dry, and then place them upside down in a 30 &#176;C incubator. Photograph the agar plates every 24 h to record a faster/more yeast growth in which the toxicity to the query strain is rescued or to record a slower/less growth in which the toxicity to the query strain is exacerbated. Incubate the plates for 4 days. 
		NOTE: The culture in each well can be spotted on agar plates 1-to-1, as shown in a previous transformation-based method10. However, the 1-to-4 spotting here (which can be conveniently set up using the robotic spotting machine) significantly increases the robustness of the assay by reducing the number of false positives. Positive hits are only considered when all 4 colonies from the same well show a similar phenotype.</li>
	</ol>
	</li>
</ol>

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	<li>Dujon, B. A., Louis, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+diversity+and+evolution+in+the+budding+yeasts+(Saccharomycotina).">Genome diversity and evolution in the budding yeasts (Saccharomycotina).</a> Genetics. 206 (2), 717-750 (2017).</li><li>Khurana, V., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modelling+neurodegeneration+in+Saccharomyces+cerevisiae:+why+cook+with+baker's+yeast.">Modelling neurodegeneration in Saccharomyces cerevisiae: why cook with baker's yeast.</a> Nature Reviews Neuroscience. 11 (6), 436-449 (2010).</li><li>Willingham, S., Outeiro, T. F., DeVit, M. J., Lindquist, S. L., Muchowski, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+genes+that+enhance+the+toxicity+of+a+mutant+huntingtin+fragment+or+alpha-synuclein.">Yeast genes that enhance the toxicity of a mutant huntingtin fragment or alpha-synuclein.</a> Science. 302 (5651), 1769-1772 (2003).</li><li>Outeiro, T. F., Lindquist, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Yeast+cells+provide+insight+into+alpha-synuclein+biology+and+pathobiology.">Yeast cells provide insight into alpha-synuclein biology and pathobiology.</a> Science. 302 (5651), 1772-1775 (2003).</li><li>Cooper, A. 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S., McCaffery, J. M., Lindquist, S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+yeast+TDP-43+proteinopathy+model:+Exploring+the+molecular+determinants+of+TDP-43+aggregation+and+cellular+toxicity.">A yeast TDP-43 proteinopathy model: Exploring the molecular determinants of TDP-43 aggregation and cellular toxicity.</a> Proceedings of the National Academy of Sciences of the United States of America. 105 (17), 6439-6444 (2008).</li><li>Fleming, M. S., Gitler, A. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+yeast+plasmid+overexpression+screen.">High-throughput yeast plasmid overexpression screen.</a> Journal of Visualized Experiments. 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The authors have nothing to disclose.

Here, we describe a protocol to perform a plasmid overexpression screen in yeast using mating to introduce the plasmid library into the yeast model. Using this approach, multiple yeast models of neurodegenerative disease protein toxicity can be screened using the same collection of yeast transformed with a plasmid library. The laborious process of transformation only needs to be performed once, after which highly efficient yeast mating is used to introduce the plasmid library into the query strain. This protocol does rely on the use of robotic equipment to dispense media and spot yeast cultures onto agar plates. While the protocol can be performed without the use of&#160;robotic equipment, it will be more time consuming. This method was successfully used to screen for genes that can modify the toxicity of FUS.
We observed&#160;that FUS is slightly less toxic in the diploid yeast background. This is most likely due to&#160;gene copy number and differences in&#160;growth rate of diploid yeast. Unless the phenotype that is being studied is&#160;mating type or ploidy-dependent, the growth phenotype of the toxicity is consistent in haploid and diploid yeast. Because of this, the mating-based method is expected to work widely in many yeast models of various growth phenotypes. Nonetheless, the phenotype of the yeast model should be verified to make sure that it is still present in the diploid background before this screening method is performed. This method can be used to study many different phenotypes in yeast and is not limited to the study of neurodegenerative protein toxicity. In addition, any plasmid library containing yeast expression vectors can be used.
After performing the screen, there are a number of verification assays that will help ensure the identified hits are specific to the yeast model being screened. Enhancers of toxicity should be tested in&#160;yeast without co-expressing the disease protein of interest. Enhancers causing toxicity independent of the disease protein of interest should be eliminated from further study. It is important to consider whether the suppressors of toxicity are affecting&#160;expression of the disease protein by affecting the Gal1 promoter. Any suppressors affecting&#160;expression from the Gal1 promoter should be eliminated from further study.
Yeast strains containing the library plasmid are permanently stored in glycerol stock and can be quickly revived when needed so that the mating-based method may be easily applied to other yeast models in which the same library genes need to be screened against. The efficiency of the mating-based method becomes obvious when the same type of screening is used to remove false positives or when multiple different yeast models need to be studied. We have successfully used this method to screen a yeast model of TDP-43, another protein linked to ALS.

The budding yeast Saccharomyces cerevisiae has been widely used in basic scientific research1 to understand cellular processes directly related to human diseases. Moreover, it has been used as a model organism for studying human disease-associated proteins, such as those linked to the most common neurodegenerative diseases, including Alzheimer&#39;s disease, Parkinson&#39;s disease, Huntington&#39;s disease, and Amyotrophic Lateral Sclerosis (ALS)2. An advantage of the yeast model is the ease with which a genome-wide screen can be performed to identify cellular pathways related to the toxicity of disease-related proteins, thus giving insight into the mechanism of their toxicity. One such screen is called an overexpression library screen, in&#160;which each of the 5,500 yeast genes in an arrayed library is transformed into a yeast model to identify which genes can modify toxicity when overexpressed. This screening method has been successfully applied in the yeast models of multiple neurodegenerative disease-associated proteins, including huntingtin for Huntington&#39;s disease3, &#945;-synuclein for Parkinson&#39;s disease4,5, A&#946; for Alzheimer&#39;s disease6, and FUS and TDP-43 for ALS7,8,9. While it is usually done in a high-throughput manner10, the most labor-intensive step of the screen is individually transforming 5,500 yeast genes from an arrayed library. This step must be performed each time the screening is repeated, and whenever a newly established yeast model needs to be studied. It is important to find a more efficient way to accomplish this task.
Yeast cells can stably exist in both haploid and diploid forms. There are two opposite mating types of haploid cells, mating type a and &#945;. Haploid cells of each mating type produce and secrete their own specific mating pheromone, to which only the opposite mating type cells respond. This allows mating between a and &#945; cells to produce stable diploid cells, a/&#945;. This process is spontaneous and highly efficient11. We can take advantage of this unique life cycle of S. cerevisiae to introduce the plasmid library. More specifically, each gene in the arrayed plasmid library will be transformed into haploid cells of one mating type, i.e., &#945; cell. These cells containing the library genes will then be stored in glycerol stock in an arrayed 96-well format. For each yeast model that needs to be screened, yeast cells containing the library genes can be thawed from the glycerol stock, and the screening can be done through&#160;mating with the yeast model of interest in the opposite mating type, i.e., mating type a. This idea of using mating to bring together two genes into yeast is not new. It has been successfully applied in the high-throughput yeast two-hybrid screening, in which a bait construct (i.e., Gal4-DNA-binding domain fusions) in one mating type is brought together through mating with a prey construct from an arrayed library12. However, this strategy has never been applied in&#160;overexpression library screenings, which have always used traditional transformation methods.
Our laboratory previously established a yeast model of the ALS-associated protein FUS7. Through overexpression library screening using the transformation method we discovered&#160;five yeast genes (ECM32, NAM8, SBP1, SKO1, and VHR1) that rescue toxicity of FUS when overexpressed. These findings were independently confirmed with a similar study by another group8. hUPF1, a human homolog of ECM32, was later shown to suppress toxicity in primary neuronal cells13&#160;and in an animal model of ALS14 as well. Using these five genes as proof of principle, we demonstrate that all five genes similarly rescue FUS toxicity when they are introduced into the FUS yeast model by mating. Since yeast cells containing the library genes can be stored permanently in glycerol stock and revived whenever needed, this mating-based method will remove the time-consuming step of transformation each time the library needs to be screened against. Since mating is highly efficient with no plasmid transformation involved, this strategy also significantly decreases the cost associated with purification and transformation of a large plasmid library. We will successfully apply this method to a library screening against yeast model of FUS.
The procedure for mating-based screening is briefly described in Figure 1. Initially, the arrayed plasmid library is transformed into a haploid yeast strain of mating type &#945; using a high-throughput yeast transformation protocol in which each well of a 96-well plate contains yeast transformed with a specific library plasmid. This collection of transformed yeast is saved as a glycerol stock that can be thawed and revived for use later on. The yeast model of interest, in&#160;this case FUS toxicity, must be generated in a haploid yeast strain with the opposite mating type (mating type a). In a high-throughput manner using sterile 96-pin replicators, the FUS strain and yeast strains containing the plasmid library are transferred to 96-well plates containing rich media and allowed to mate. Following&#160;mating, a small volume from each well of the mating culture is transferred to 96-well plates containing synthetic dropout media in which only diploid yeast containing both the FUS and library genes can grow. A robotic spotting machine is then used to transfer yeast culture from each well onto agar plates where the expression of FUS and the library genes is induced.&#160; Additionally, yeast culture is spotted&#160;to control agar plates where FUS and the library genes are not expressed. Following&#160;growth on&#160;agar plates, genes that rescue or exacerbate FUS toxicity will be identified.

<table><tbody><tr><td>salmon Sperm DNA (SS-DNA)</td><td>Sigma-Aldrich</td><td>&amp;nbsp; D1626</td><td></td></tr><tr><td>YPD broth</td><td>Research Products International (RPI)</td><td>Y20090</td><td></td></tr><tr><td>Granulated Agar</td><td>Fisher Sci</td><td>BP97445</td><td></td></tr><tr><td>D-(+)-Glucose</td><td>Research Products International (RPI)</td><td>G32040</td><td></td></tr><tr><td>D-(+)-Galactose</td><td>Research Products International (RPI)</td><td>G33000</td><td></td></tr><tr><td>D-(+)-Raffinose Pentahydrate</td><td>Research Products International (RPI)</td><td>R20500</td><td></td></tr><tr><td>Ammonium Sulfate</td><td>Fisher Sci</td><td>A702-500</td><td></td></tr><tr><td>Synthetic Ura- drop out medium</td><td>Clontech</td><td>630416</td><td></td></tr><tr><td>Yeast amino acid drop out supplement -Histidine/-Uracil</td><td>Clontech</td><td>630422</td><td></td></tr><tr><td>Yeast Nitrogen Base without Amino Acids and Ammonium Sulfate</td><td>Research Products International (RPI)</td><td>Y20060</td><td></td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>Fisher Sci</td><td>S67496</td><td></td></tr><tr><td>Lithium acetate, anhydrous</td><td>Fisher Sci</td><td>AC268640010</td><td></td></tr><tr><td>Polyethylene Glycol 3350 (PEG-3350)</td><td>Spectrum Chemical&amp;nbsp;</td><td>PO125-12KG</td><td></td></tr><tr><td>96 Pin Replicator&amp;nbsp;</td><td>Scinomix</td><td>SCI-5010-OS</td><td></td></tr><tr><td>Nunc OmniTray</td><td>Thermo Sci</td><td>140156</td><td></td></tr><tr><td>Corning Costar 96 well assay plate, round bottom with lid</td><td>Fisher Sci</td><td>07-200-760</td><td>non-treated, sterile</td></tr><tr><td>Eppendorf&amp;nbsp;Research plus Multichannel Pipette</td><td>Eppendorf</td><td>TI13690052</td><td>30-300ul volume</td></tr><tr><td>Fisherbrand&amp;nbsp;Isotemp Digital Dry Baths/Block Heaters</td><td>Fisher Sci</td><td>88-860-023</td><td></td></tr><tr><td>Eppendorf&amp;nbsp;MixMate</td><td>Eppendorf</td><td>21-379-00</td><td></td></tr><tr><td>Eppendorf 5810R Centrifuge</td><td>Fisher Sci</td><td>05-413-112</td><td></td></tr><tr><td>Avanti J-26 XPI Centrifuge</td><td>Beckman</td><td>393127</td><td></td></tr><tr><td>MultiFlo FX Multi-Mode Dispenser</td><td>BioTek</td><td></td><td></td></tr><tr><td>Rotor HDA&amp;nbsp;&amp;nbsp;</td><td>Singer Instruments</td><td></td><td></td></tr></tbody></table>

mating-based overexpression library screening in yeast

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool commonly used in yeast studies. The expression of a number of neurodegenerative disease-associated proteins in yeast causes cytotoxicity and aggregate formation, recapitulating findings seen in patients with these disorders. Here, we describe a method for screening a yeast model of the Amyotrophic Lateral Sclerosis-associated protein FUS for modifiers of its toxicity. Instead of using transformation, this new screening platform relies on the mating of yeast to introduce an arrayed library of plasmids into the yeast model. The mating method has two clear advantages: first, it is highly efficient; second, the pre-transformed arrayed library of plasmids can be stored for long-term as a glycerol stock, and quickly applied to other screens without the labor-intensive step of transformation into the yeast model each time. We demonstrate how this method can successfully be used to screen for genes that modify the toxicity of FUS.

The ALS-associated protein FUS, an RNA/DNA binding protein, was previously studied in haploid yeast7,8. Genetic screening using the transformation-based method discovered several yeast genes that suppress FUS toxicity. The human homolog of one of the yeast genes was later demonstrated to be effective at suppressing toxicity in a primary neuronal cell and rat model of ALS13. Here, we are using the same yeast model to show that overexpression library screening can be performed by mating as effectively as by transformation.
FUS is toxic to both haploid and diploid yeast cells 
The previous yeast model of FUS and subsequent overexpression library screening was performed in the haploid cell background. For the mating-based method to work, FUS toxicity needs to be demonstrated in diploid yeast. To do this, we mated the FUS yeast model in w303a (mating type a) with w303&#945; transformed with an empty vector (mating type &#945;). As indicated in Figure 2, although&#160;not as strong as in haploid yeast, FUS toxicity is evident&#160;in&#160;diploid yeast.
Suppression genes previously identified work in diploid yeast 
As a proof of principle for the mating-based method, we&#160;tested the five genes previously identified&#160;from the transformation-based method. Mating was used to introduce each of the five genes into the haploid yeast model of FUS (in W303a), and their ability to rescue the toxicity of FUS in the subsequent diploid yeast was tested (W303a/&#945;). As shown in Figure 3, all five genes rescue the toxicity of FUS in diploid yeast, indicating that the mating method was effective.
A pilot screening of 940 genes (arrayed on ten 96-well plates) 
Following the protocols described above, we&#160;applied the mating-based method to an overexpression library screening of 940 genes. Figure 4 displays a picture of one representative plate. As indicated on the right side of the figure (FUS and library genes expressed), FUS was toxic to&#160;diploid yeast. The library gene indicated by the green square&#160;rescued FUS toxicity while that indicated by the red square&#160;enhanced toxicity.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/57978/57978fig1.jpg" /> 
Figure 1: Diagram for screening yeast models of protein toxicity using mating. A plasmid library (under control of the GAL1 promoter which is highly induced in the presence of galactose) is transformed into a haploid yeast strain (MAT&#945;) using a high-throughput transformation protocol. This collection of yeast, transformed with the library plasmids, is stored as a glycerol stock at -80 &#176;C, and is revived when needed to mate with the haploid yeast model of protein toxicity (in our case, one copy of FUS integrated at the&#160;HIS3 locus, GAL1 promoter, MATa). Diploid yeast containing the library plasmid and toxic protein (FUS) are selected and spotted to glucose (FUS and library gene &#39;off&#39;) and galactose (FUS and library gene &#39;on&#39;) agar plates. The growth of the yeast was followed to identify genes that rescue or exacerbate the toxicity of FUS. The green square indicates an example of a suppressor gene that rescues FUS toxicity, and the red square indicates an example of an enhancer gene that exacerbates the FUS toxicity when overexpressed. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/57978/57978fig2.jpg" /> 
Figure 2:&#160;FUS is toxic to both haploid and diploid yeast cells. Haploid yeast (w303 MATa) transformed with pRS303Gal1-FUS were either transformed with an empty plasmid or mated with yeast of the opposite mating type (w303 MAT&#945;) transformed with the same empty plasmid to generate a diploid yeast strain. These yeast strains along with a control strain were then 5x serially diluted (from left to right) and spotted to Ura-His-Glucose medium (left, FUS expression repressed) and Ura-His-Galactose medium (right, FUS expression induced). The picture was taken after 2 days of growth at 30 &#176;C. Nearly identical growth of the haploid and diploid control strain was observed, so only the haploid control strain was shown. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/57978/57978fig3.jpg" /> 
Figure 3: Five yeast genes (ECM32, NAM8, SBP1, SKO1, and VHR1) rescue FUS toxicity through the mating-based method. Plasmids containing previously identified yeast genes that&#160;suppress FUS toxicity were transformed into a haploid yeast strain (w303 MAT&#945;). These yeast were then mated with haploid yeast of the opposite mating type (w303 MATa) transformed with a FUS expression plasmid. Diploid yeast containing FUS and either an empty vector or one of the five suppression genes was&#160;selected and spotted in replicates on agar plates containing glucose (genes &#39;off&#39;) and galactose (genes &#39;on&#39;). (1) shows a control yeast strain transformed with two empty vectors. (2) shows the diploid FUS yeast strain with an empty vector where the expression of FUS is very toxic. (3&#8211;7) show diploid yeast expressing FUS as well as a suppression gene that can rescue FUS toxicity. Each number (1&#8211;7) shows two rows of twelve identical replicates. The picture, representing three independent experiments, was taken after 3 days of growth at 30 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/57978/57978fig4.jpg" /> 
Figure 4: Library screening for genes that rescue or exacerbate FUS toxicity. Haploid yeast containing FUS was mated with haploid yeast containing the library genes. After mating, diploid cells containing both FUS and a library gene were selected and then spotted to glucose (FUS and library genes &#39;off&#39;) and galactose agar plates (FUS and library genes &#39;on&#39;) in quadruplicate. On the galactose plate, in which FUS and the library gene were expressed, most of the yeast were unable to grow well. This indicates that FUS is toxic and most library genes were unable to rescue toxicity. The green square demonstrates an&#160;example of a library gene that suppresses FUS toxicity and allows the yeast to form colonies. The red square indicates an&#160;example of a library gene that exacerbates&#160;FUS toxicity. The plates shown here are representative of 10 plates of library genes that were screened against. The picture was taken after 3 days of growth at 30 &#176;C. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/57978/57978fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

Mating-based Overexpression Library Screening in Yeast

This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Budding yeast has been widely used as a model in studying proteins associated with human diseases. Genome-wide genetic screening is a powerful tool ...

mating-based-overexpression-library-screening-in-yeast

Nanyang Technological University

Research

JoVE Journal

Genetics

7.6K Views.  Wright State University. This article presents a mating-based method to facilitate overexpression screening in budding yeast using an arrayed plasmid library.

Video: Mating-based Overexpression Library Screening in Yeast

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the discovery of high-affinity interactors that are highly enriched in the library of interacting candidates. In a traditional format, the yeast 2-hybrid assay can yield too many colonies to analyze when conducted at low stringency where low affinity interactors might be found. Moreover, without a comprehensive and complete interrogation of the same library against different bait plasmids, a comparative analysis cannot be achieved. Although some of these problems can be addressed using arrayed prey libraries, the cost and infrastructure required to operate such screens can be prohibitive. As an alternative, we have adapted the yeast 2-hybrid assay to simultaneously uncover dozens of transient and static protein interactions within a single screen utilizing a strategy termed DEEPN (Dynamic Enrichment for Evaluation of Protein Networks), which incorporates high-throughput DNA sequencing and computation to follow the evolution of a population of plasmids that encode interacting partners. Here, we describe customized reagents and protocols that allow a DEEPN screen to be executed easily and cost-effectively.

Batch processing of yeast 2-hybrid screens allows for direct comparison of the interaction profiles of multiple bait proteins with a highly complex set of prey fusion proteins. Here, we describe refined methods, new reagents, and how to implement their use for such screens.

Screening for protein-protein interactions using the yeast 2-hybrid assay has long been an effective tool, but its use has largely been limited to the ...

Biochemistry

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely related biochemical pathways. Previous methods such as the Synthetic Genetic Array (SGA) analysis, and random mutagenesis techniques using ultraviolet (UV) or chemicals like ethyl methanesulfonate (EMS) or N-ethyl-N- nitrosourea (ENU), have been widely used but are often costly and laborious. Also, these mutagen-based screening methods are frequently associated with severe side effects on the organism, inducing multiple mutations that add to the complexity of isolating the suppressors. Here, we present a simple and effective protocol to identify suppressor mutations in mutants which confer a growth defect in Schizosaccharomyces pombe. The fitness of cells with a growth deficiency in standard rich liquid media or synthetic liquid media can be monitored for recovery using an automated 96-well plate reader over an extended period. Once a cell acquires a suppressor mutation in the culture, its descendants outcompete those of the parental cells. The recovered cells that have a competitive growth advantage over the parental cells can then be isolated and backcrossed with the parental cells. The suppressor mutations are then identified using whole-genome sequencing. Using this approach, we have successfully isolated multiple suppressors that alleviate the severe growth defects caused by loss of Elf1, an AAA+ family ATPase that is important in nuclear mRNA transport and maintenance of genomic stability. There are currently over 400 genes in S. pombe with mutants conferring a growth defect. As many of these genes are uncharacterized, we propose that our method will hasten the identification of novel functional interactions with this user-friendly, high-throughput approach.

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

We present a simple suppressor screen protocol in fission yeast. This method is efficient, mutagen-free, and&#160; selective&#160; for&#160; mutations that&#160; often&#160; occur at&#160; &#160;a&#160; single&#160; genomic&#160; locus.&#160; The protocol is suitable for isolating suppressors that alleviate growth defects in liquid culture that are caused by a mutation or a drug.

A genetic screen for mutant alleles that suppress phenotypic defects caused by a mutation is a powerful approach to identify genes that belong to closely ...

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and computational approaches. One such method is the yeast one-hybrid (Y1H) assay, in which interactions between TFs and DNA regions are tested in the milieu of the yeast nucleus using reporter genes. Y1H assays involve two components: a &#8216;DNA-bait&#8217; (e.g., promoters, enhancers, silencers, etc.) and a &#8216;TF-prey,&#8217; which can be screened for reporter gene activation. Most published protocols for performing Y1H screens are based on transforming TF-prey libraries or arrays into DNA-bait yeast strains. Here, we describe a pipeline, called enhanced Y1H (eY1H) assays, where TF-DNA interactions are interrogated by mating DNA-bait strains with an arrayed collection of TF-prey strains using a high density array (HDA) robotic platform that allows screening in a 1,536 colony format. This allows for a dramatic increase in throughput (60 DNA-bait sequences against &#62;1,000 TFs takes two weeks per researcher) and reproducibility. We illustrate the different types of expected results by testing human promoter sequences against an array of 1,086 human TFs, as well as examples of issues that can arise during screens and how to troubleshoot them.

Here, we present an enhanced yeast one-hybrid screening protocol to identify the transcription factors (TFs) that can bind to a human DNA region of interest. This method uses a high-throughput screening pipeline that can interrogate the binding of &#62;1,000 TFs in a single experiment.

Identifying the sets of transcription factors (TFs) that regulate each human gene is a daunting task that requires integrating numerous experimental and ...

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

Significant efforts were gathered to generate large-scale comprehensive protein-protein interaction network maps. This is instrumental to understand the pathogen-host relationships and was essentially performed by genetic screenings in yeast two-hybrid systems. The recent improvement of protein-protein interaction detection by a Gaussia luciferase-based fragment complementation assay now offers the opportunity to develop integrative comparative interactomic approaches necessary to rigorously compare interaction profiles of proteins from different pathogen strain variants against a common set of cellular factors.
This paper specifically focuses on the utility of combining two orthogonal methods to generate protein-protein interaction datasets: yeast two-hybrid (Y2H) and a new assay, high-throughput Gaussia princeps protein complementation assay (HT-GPCA) performed in mammalian cells.
A large-scale identification of cellular partners of a pathogen protein is performed by mating-based yeast two-hybrid screenings of cDNA libraries using multiple pathogen strain variants. A subset of interacting partners selected on a high-confidence statistical scoring is further validated in mammalian cells for pair-wise interactions with the whole set of pathogen variants proteins using HT-GPCA. This combination of two complementary methods improves the robustness of the interaction dataset, and allows the performance of a stringent comparative interaction analysis. Such comparative interactomics constitute a reliable and powerful strategy to decipher any pathogen-host interplays.

This article focuses on the identification of high-confident interaction datasets between host and pathogen proteins using a combination of two orthogonal methods: yeast two-hybrid followed by a high-throughput interaction assay in mammalian cells called HT-GPCA.

Significant efforts were gathered to generate  large-scale comprehensive protein-protein interaction network maps. This is  instrumental to understand the ...

Immunology and Infection

High-throughput Yeast Plasmid Overexpression Screen

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, including those with direct relevance to human disease. Because of its short generation time and well-characterized genome, a major experimental advantage of the yeast model system is the ability to perform genetic screens to identify genes and pathways that are involved in a given process. Over the last thirty years such genetic screens have been used to elucidate the cell cycle, secretory pathway, and many more highly conserved aspects of eukaryotic cell biology 1-5. In the last few years, several genomewide libraries of yeast strains and plasmids have been generated 6-10. These collections now allow for the systematic interrogation of gene function using gain- and loss-of-function approaches 11-16. Here we provide a detailed protocol for the use of a high-throughput yeast transformation protocol with a liquid handling robot to perform a plasmid overexpression screen, using an arrayed library of 5,500 yeast plasmids. We have been using these screens to identify genetic modifiers of toxicity associated with the accumulation of aggregation-prone human neurodegenerative disease proteins. The methods presented here are readily adaptable to the study of other cellular phenotypes of interest.

Here we describe a plasmid overexpression screen in Saccharomyces cerevisiae, using an arrayed plasmid library and a high-throughput yeast transformation protocol with a liquid handling robot.

The budding yeast, Saccharomyces cerevisiae, is a powerful model system for defining fundamental mechanisms of many important cellular processes, ...

Biology

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Optimizing Protein Functions Through Yeast Surface Display Techniques

Protein Engineering by Yeast Surface Display

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and versatile tools in the protein engineering field is yeast surface display, where a pool of randomized proteins is expressed on the surface of yeast. The linkage of phenotype (e.g., binding of the yeast-displayed protein to the antigen of interest) and genotype (the plasmid encoding for the protein variant) enables selection of this library for desired properties and subsequent sequencing of enriched variants. By combining magnetic bead selection with flow cytometric sorting, protein variants with enhanced binding to a target antigen can be selected and enriched. Notably, in addition to affinity maturation, binding to a target can also be achieved without any initial binding affinity. Here, we provide a step-by-step protocol that covers all essential parts of a yeast surface display selection campaign and gives examples of typical yeast surface display results. We demonstrate that yeast surface display is a broadly applicable and robust method that can be established in any molecular biology laboratory with access to flow cytometry.

This protocol describes the essential steps for conducting yeast surface display selection campaigns to enrich protein variants binding to an antigen of interest.

Protein engineering enables the improvement of existing functions of a given protein or the generation of novel functions. One of the most widely used and ...

Bioengineering

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived ...

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships with other genes and organization into biological pathways and processes. Although the majority of the genetic screens were initially developed in Saccharomyces cerevisiae, a complementary platform for carrying out these genetic screens has been provided by Schizosaccharomyces pombe. One of the common approaches used to identify genetic interactions is by overexpression of clones from a genome-wide, high-copy-number plasmid library in a loss-of-function mutant, followed by selection of clones that suppress the mutant phenotype.
This paper describes a protocol for carrying out this &#39;multicopy suppression&#39;-based genetic screen in S. pombe. This screen has helped identify multicopy suppressor(s) of the genotoxic stress-sensitive phenotype associated with the absence of the Ell1 transcription elongation factor in S. pombe. The screen was initiated by transformation of the query ell1 null mutant strain with a high-copy-number S. pombe cDNA plasmid library and selecting the suppressors on EMM2 plates containing 4-nitroquinoline 1-oxide (4-NQO), a genotoxic stress-inducing compound. Subsequently, plasmid was isolated from two shortlisted suppressor colonies and digested by restriction enzymes to release the insert DNA. Plasmids releasing an insert DNA fragment were retransformed into the ell1 deletion strain to confirm the ability of these suppressor plasmid clones to restore growth of the ell1 deletion mutant in the presence of 4-NQO and other genotoxic compounds. Those plasmids showing a rescue of the deletion phenotype were sequenced to identify the gene(s) responsible for suppression of the ell1 deletion-associated genotoxic stress-sensitive phenotype.

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe

This work describes a protocol for a multicopy suppressor genetic screen in Schizosaccharomyces pombe. This screen uses a genome-wide plasmid library to identify suppressor clone(s) of a loss-of-function phenotype associated with a query mutant strain. Novel genetic suppressors of the ell1 null mutant were identified using this screen.

Identification of genetic interactions is a powerful tool to decipher the functions of gene(s) by providing insights into their functional relationships ...

Mating-based Overexpression Library Screening in Yeast

Summary

Explore More Videos

Chapters in this video

A Yeast 2-Hybrid Screen in Batch to Compare Protein Interactions

A Deep-sequencing-assisted, Spontaneous Suppressor Screen in the Fission Yeast Schizosaccharomyces pombe

Enhanced Yeast One-hybrid Screens To Identify Transcription Factor Binding To Human DNA Sequences

A Comparative Approach to Characterize the Landscape of Host-Pathogen Protein-Protein Interactions

High-throughput Yeast Plasmid Overexpression Screen

Competitive Genomic Screens of Barcoded Yeast Libraries

Protein Engineering by Yeast Surface Display

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

Genetic Screen for Identification of Multicopy Suppressors in Schizosaccharomyces pombe