Name: growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae
Uploaded: 2015-02-16T15:00:00
Description: Watch this Scientific Journal Video about Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae at JoVE.com

We thank current and former members of the Rubenstein lab for providing a supportive and enthusiastic research environment. We thank Ryan T. Gibson for assistance in protocol optimization. We thank Mark Hochstrasser (Yale University) and Dieter Wolf (Universit&#228;t Stuttgart) for yeast strains and plasmids. We thank our anonymous reviewers for their help in improving the clarity and utility of this manuscript. This work was supported by a research award from the Ball State University chapter of Sigma Xi to S.G.W., a National Institutes of Health grant (R15 GM111713) to E.M.R., a Ball State University ASPiRE research award to E.M.R, and funds from the Ball State University Provost&#8217;s Office and Department of Biology.

1. Yeast Growth Assay to Identify Candidate Mutants Defective in Protein Degradation
<ol>
	<li>Transform wild-type and mutant yeast cells with a plasmid encoding an unstable protein fused, in frame, to a reporter metabolic enzyme.</li>
	<li>Inoculate transformants in 5 ml of synthetic defined (SD) minimal medium that is selective for cells harboring plasmid molecules. Incubate overnight at 30 &#176;C, rotating.</li>
	<li>Measure the optical density at 600 nm (OD600) of each overnight culture. 
	NOTE: Fo...

The methodology presented here allows for the rapid determination and biochemical confirmation of genetic requirements for protein degradation in yeast cells. These experiments highlight the utility and power of yeast as a model eukaryotic organism (several excellent reviews of yeast biology and compilations of protocols for handling, storing, and manipulating yeast cells (e.g. 41-44) are available for investigators new to the organism). The techniques can readily be applied to investigate the degrada...

Selective protein degradation is essential for eukaryotic life, and altered protein degradation contributes to a number of medical conditions, including several types of cancer, neurodegenerative disease, cardiovascular disease, and cystic fibrosis1-5. The ubiquitin-proteasome system (UPS), which catalyzes selective protein degradation, is an emerging therapeutic target for these conditions6-10. Ubiquitin ligases covalently attach polymers of the 76-amino acid ubiquitin to proteins11. Proteins that have been marked with polyubiquitin chains are recognized and proteolyzed by the ~2.5 megadalton 26S proteasome12. Studies initiated in the model eukaryotic organism Saccharomyces cerevisiae (budding yeast) have been foundational in the elucidation of protein degradation mechanisms in eukaryotic cells. The first demonstrated physiological substrate of the UPS was the yeast transcriptional repressor MAT&#945;213,14, and many highly conserved components of the UPS were first identified or characterized in yeast (e.g. 15-26). Discoveries made in this versatile and genetically tractable model organism are likely to continue to provide important insights into conserved mechanisms of ubiquitin-mediated degradation.
Recognition and degradation of most UPS substrates require the concerted action of multiple proteins. Therefore, an important goal in characterizing the regulated degradation of a given unstable protein is to determine the genetic requirements for proteolysis. Classical approaches (e.g. pulse-chase and cycloheximide-chase experiments27) for monitoring protein degradation in mammalian or yeast cells are laborious and time-consuming. While these types of methodology provide highly sensitive means for detecting protein degradation, they are not suitable for rapid analysis of protein degradation or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the rapid identification of genetic requirements for the degradation of unstable proteins is presented.
In the yeast growth-based method for analyzing protein degradation, an unstable protein of interest (or degradation signal) is fused, in frame, to a protein that is required for yeast growth under specific circumstances. The result is an artificial substrate that may serve as a powerful tool to determine the genetic requirements of protein degradation of the unstable protein of interest. Conveniently, most commonly used laboratory yeast strains harbor a panel of mutations in genes encoding metabolic enzymes involved in the biosynthesis of particular amino acids or nitrogenous bases (e.g. 20,28-30). These enzymes are essential for cellular proliferation in the absence of exogenously provided metabolites in whose synthesis the enzymes participate. Such metabolic enzymes may thus function as growth-based reporters for the degradation of unstable proteins to which they are fused. The genetic requirements for protein degradation can be readily elucidated, since mutations that prevent proteolysis will allow cells harboring the degradation reporter to grow under selective conditions.
A growth advantage is an indirect indication that a particular mutation increases the abundance of the protein of interest. However, direct biochemical analysis is required to confirm that a mutation permits growth through increased protein levels rather than via indirect or artifactual causes. The effect of a mutation on protein abundance may be confirmed by western blot analysis of steady-state protein levels in cells that do and do not harbor the particular mutation. A method for the rapid and efficient extraction of yeast proteins (sequential incubation of yeast cells with sodium hydroxide and sample buffer) in a form suitable for analysis by western blotting is also presented31. Together, these experiments facilitate the rapid identification of candidate regulators of protein degradation.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Desired yeast strains, plasmids, standard medium and buffer components</td>
			<td></td>
			<td></td>
			<td>Yeast strains with desired mutations may be generated in the investigator&#39;s laboratory. Wild-type yeast and a variety of mutants are also commercially available (e.g. from GE Healthcare). Plasmids encoding fusion proteins may be generated in the investigator&#39;s laboratory.</td>
		</tr>
		<tr>
			<td>3-amino-1H-1,2,4-triazole</td>
			<td>Fisher Scientific</td>
			<td>AC264571000</td>
			<td>Competitive inhibitor of His3 enzyme. May be included in medium to increase stringency of growth assay using His3 reporter constructs</td>
		</tr>
		<tr>
			<td>Endoglycosidase H (recombinant form from Streptomyces plicatus)</td>
			<td>Roche</td>
			<td>11088726001</td>
			<td>May be used to assess N-glycosylation of proteins; compatible with SDS and beta-mercaptoethanol concentrations found in 1X Laemmli sample buffer</td>
		</tr>
		<tr>
			<td>Disposable borosilicate glass tubes</td>
			<td>Fisher Scientific</td>
			<td>14-961-32</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180)</td>
			<td>Dot Scientific</td>
			<td>51028068</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)</td>
			<td>New Brunswick</td>
			<td>M1053-0450</td>
			<td>Tube roller is recommended to maintain overnight yeast starter cultures of yeast cells in suspension. A platform shaker or tube roller may be used to maintain larger cultures in suspension.</td>
		</tr>
		<tr>
			<td>New Brunswick TC-7 Roller Drum 120V 50/60 H</td>
			<td>New Brunswick</td>
			<td>M1053-4004</td>
			<td>For use with tube roller</td>
		</tr>
		<tr>
			<td>SmartSpec Plus Spectrophotometer</td>
			<td>Bio-Rad</td>
			<td>170-2525</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Sterile 96-well flat bottom microtest plates with lid individually wrapped</td>
			<td>Sarstedt</td>
			<td>82.1581.001</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Pipetman Neo P8x200N, 20-200 &#956;l</td>
			<td>Gilson</td>
			<td>F14403</td>
			<td>Single-channel and multichannel pipettors are used at various stages of the protocol. While multichannel pipettors reduce the pipetting burden at several steps, single-channel pipettors may be used throughout the entire protocol. Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Pipetman Neo P8x20N, 2-20 &#956;l</td>
			<td>Gilson</td>
			<td>F14401</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Plate imaging system (e.g. 
			Gel Doc XR+ System)</td>
			<td>Bio-Rad</td>
			<td>170-8195</td>
			<td>A variety of systems may be used to image plates, including sophisticated imaging systems, computer scanners, and camera phones.</td>
		</tr>
		<tr>
			<td>Centrifuge 5430</td>
			<td>Eppendorf</td>
			<td>5427 000.216&#160;</td>
			<td>Rotor that is sold with unit holds 1.5 and 2.0 mL microcentrifuge tubes. Rotor may be swapped for one that holds 15 ml and 50 ml conical tubes</td>
		</tr>
		<tr>
			<td>Fixed-Angle Rotor F-35-6-30 with Lid and Adapters for Centrifuge Model 5430/R, 15/50 mL Conical Tubes, 6-Place</td>
			<td>Eppendorf</td>
			<td>F-35-6-30</td>
			<td></td>
		</tr>
		<tr>
			<td>15 ml screen printed screw cap tube 17 x 20 mm conical, polypropylene</td>
			<td>Sarstedt</td>
			<td>62.554.205</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>1.5 ml flex-tube, PCR clean, Natural microcentrifuge tubes</td>
			<td>Eppendorf</td>
			<td>22364120</td>
			<td>Available from a variety of manufacturers</td>
		</tr>
		<tr>
			<td>Analog Dri-Bath Heater</td>
			<td>Fisher Scientific</td>
			<td>1172011AQ</td>
			<td>Boiling water bath with hot plate may also be used to denature proteins</td>
		</tr>
		<tr>
			<td>SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane</td>
			<td></td>
			<td></td>
			<td>Will vary by lab and application</td>
		</tr>
		<tr>
			<td>Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software)</td>
			<td>Li-Cor</td>
			<td>9140-01</td>
			<td>Will vary by lab and application</td>
		</tr>
		<tr>
			<td>EMD Millipore Immobilon PVDF Transfer Membranes</td>
			<td>Fisher Scientific</td>
			<td>IPFL00010</td>
			<td>Will vary by lab and application</td>
		</tr>
		<tr>
			<td>Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))</td>
			<td>Life Technologies</td>
			<td>459250</td>
			<td>Will vary by lab and application</td>
		</tr>
		<tr>
			<td>Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L))</td>
			<td>Life Technologies</td>
			<td>A-21065</td>
			<td>Will vary by lab and application</td>
		</tr>
	</tbody>
</table>

growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

To illustrate this methodology, the His3 enzyme has been fused to the carboxy-terminus of the model endoplasmic reticulum (ER)-associated degradation (ERAD) substrate, Deg1-Sec62 (Figure 2A) to create Deg1-Sec62-His3 (Figure 3). Deg1-Sec62 represents a founding member of a novel class of ERAD substrates that are targeted following persistent, aberrant association with the translocon, the channel primarily responsible for moving proteins across the ER membrane<s...

Watch this Scientific Journal Video about Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae at JoVE.com

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

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