Name: mating-based overexpression library screening in yeast
Uploaded: 2018-07-06T14:00:00
Description: Watch this Scientific Journal Video about Mating-based Overexpression Library Screening in Yeast at JoVE.com

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....

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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 rel...

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.

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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...

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 ...

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