Name: growth assays to assess polyglutamine toxicity in yeast
Uploaded: 2012-03-05T12:00:00
Description: Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Work in the Duennwald laboratory is supported by grants from the American Federation for Aging Research (AFAR), the Hereditary Disease Foundation (HDF) and the William Wood Foundation.

1. Expression of Toxic PolyQ-expansion Proteins in Yeast
A systematic analysis has established the precise amino acid sequence of a polyQ-expansion protein that is required to produce toxicity in yeast 7. This toxic polyQ-expansion protein contains an amino-terminal FLAG-tag followed by 17 amino acids from the original sequence of the huntingtin protein, a polyQ region, and a carboxy-terminal fusion to a fluorescent protein (either GFP or CFP, see Figure 1 a). The expression of protei...

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This manuscript outlines three complementary experimental approaches to measure polyQ-toxicity in the model organism yeast based on reduced growth of yeast cells expressing toxic polyQ-expansion proteins. Work in yeast has offered profound insights into the basic cellular and molecular mechanisms of protein misfolding and its ensuing toxicity, including the misfolding and toxicity of polyQ-expansion proteins 9,11,12. Experiments based on the protocols presented here have already helped to identify and characte...

<table border="1">
	<tbody>
		<tr>
			<td>Name of instrument</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Frogger (6x8 pins)</td>
			<td>V&amp;P Scientific, San Diego</td>
			<td>VP 407 AH</td>
		</tr>
		<tr>
			<td>BioscreenC</td>
			<td>Growthcurves USA</td>
			<td>5101370</td>
		</tr>
		<tr>
			<td>100-well Honeycomb plates</td>
			<td>Growthcurves USA</td>
			<td>9602550</td>
		</tr>
	</tbody>
</table>

growth assays to assess polyglutamine toxicity in yeast

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's disease, and Huntington's disease 1. Huntington's disease (HD) is caused by the abnormal expansion of a polyglutamine (polyQ) region within the protein huntingtin. The polyQ-expanded huntingtin protein attains an aberrant conformation (i.e. it misfolds) and causes cellular toxicity 2. At least eight further neurodegenerative diseases are caused by polyQ-expansions, including the Spinocerebellar Ataxias and Kennedy&#x2019;s disease 3.
The model organism yeast has facilitated significant insights into the cellular and molecular basis of polyQ-toxicity, including the impact of intra- and inter-molecular factors of polyQ-toxicity, and the identification of cellular pathways that are impaired in cells expressing polyQ-expansion proteins 3-8. Importantly, many aspects of polyQ-toxicity that were found in yeast were reproduced in other experimental systems and to some extent in samples from HD patients, thus demonstrating the significance of the yeast model for the discovery of basic mechanisms underpinning polyQ-toxicity. 
A direct and relatively simple way to determine polyQ-toxicity in yeast is to measure growth defects of yeast cells expressing polyQ-expansion proteins. This manuscript describes three complementary experimental approaches to determine polyQ-toxicity in yeast by measuring the growth of yeast cells expressing polyQ-expansion proteins. The first two experimental approaches monitor yeast growth on plates, the third approach monitors the growth of liquid yeast cultures using the BioscreenC instrument. 
Furthermore, this manuscript describes experimental difficulties that can occur when handling yeast polyQ models and outlines strategies that will help to avoid or minimize these difficulties. The protocols described here can be used to identify and to characterize genetic pathways and small molecules that modulate polyQ-toxicity. Moreover, the described assays may serve as templates for accurate analyses of the toxicity caused by other disease-associated misfolded proteins in yeast models.

Watch this Scientific Journal Video about Growth Assays to Assess Polyglutamine Toxicity in Yeast at JoVE.com

Growth Assays to Assess Polyglutamine Toxicity in Yeast

This manuscript describes three complementary protocols for assessing the toxicity of polyglutamine (polyQ)-expansion proteins in the yeast Saccharomyces cerevisiae. These protocols can easily be modified to monitor the toxicity of other misfolded proteins in yeast.

Protein misfolding is associated with many human diseases, particularly neurodegenerative diseases, such as Alzheimer&#x2019;s disease, Parkinson's ...

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