Name: cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae
Uploaded: 2016-04-18T15:00:00
Description: Watch this Scientific Journal Video about Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae at JoVE.com

The authors thank current and former members of the Rubenstein lab for providing a supportive and enthusiastic research environment. The authors thank Mark Hochstrasser (Yale University) for sharing reagents and expertise. E.M.R. thanks Stefan Kreft (University of Konstanz) and Jennifer Bruns (University of Pittsburgh) for sharing invaluable expertise in kinetic analysis of proteins. This work was supported by a National Institutes of Health grant (R15 GM111713) to E.M.R., a Ball State University ASPiRE research award to E.M.R, a research award from the Ball State University chapter of Sigma Xi to S.M.E., and funds from the Ball State University Provost's Office and Department of Biology.

1. Growth and Harvest of Yeast Cells
<ol>
	<li>If not analyzing degradation kinetics of an endogenous yeast protein, transform desired yeast strain(s) with a plasmid encoding the protein of interest. Reliable methods for yeast transformation have been previously described 37.</li>
	<li>Inoculate yeast in 5 ml of appropriate medium (e.g., selective synthetic defined (SD) medium for plasmid maintenance of transformed cells or non-selective yeast extract-peptone-dextrose (YPD) medium for non-transforme...

In this paper, a method for analyzing protein degradation kinetics is presented. This technique can be readily applied to a range of proteins degraded by a variety of mechanisms. It is important to note that cycloheximide chase experiments report on degradation kinetics of the steady state pool of a given protein. Other techniques may be used to analyze the degradation kinetics of specific populations of proteins. For example, the degradative fate of nascent polypeptides can be tracked by pulse chase analysis 55</su...

Proteins perform crucial functions in virtually every cellular process. Many physiological processes require the presence of a specific protein (or proteins) for a defined period of time or under particular circumstances. Organisms therefore monitor and regulate protein abundance to meet cellular needs 1. For example, cyclins (proteins that control cell division) are present at specific phases of the cell cycle, and the loss of regulated cyclin levels has been associated with malignant tumor formation 2. In addition to regulating protein levels to meet cellular needs, cells employ degradative quality control mechanisms to eliminate misfolded, unassembled, or otherwise aberrant protein molecules 3. Control of protein abundance involves regulation of both macromolecular synthesis (transcription and translation) and degradation (RNA decay and proteolysis). Impaired or excessive protein degradation contributes to multiple pathologies, including cancer, cystic fibrosis, neurodegenerative conditions, and cardiovascular disorders 4-8. Proteolytic mechanisms therefore represent promising therapeutic targets for a range of illnesses 9-12.
Analysis of proteins at a single time point (e.g., by western blot 13, flow cytometry 14, or fluorescence microscopy 15) provides a snapshot of steady state protein abundance without revealing the relative contributions of synthesis or degradation. Similarly, growth-based reporter assays reflect steady state protein levels over an extended time period without discriminating between the influences of synthesis and degradation 15-20. It is possible to infer the contribution of degradative processes to steady state protein levels by comparing abundance before and after inhibiting specific components of the degradative mechanism (e.g., by pharmacologically inactivating the proteasome 21 or knocking out a gene hypothesized to be required for degradation 13). A change in steady state protein levels after inhibiting degradative pathways provides strong evidence for the contribution of proteolysis to the control of protein abundance 13. However, such an analysis still does not provide information regarding the kinetics of protein turnover. Cycloheximide chase followed by western blotting overcomes these weaknesses by allowing researchers to visualize protein degradation over time 22-24. Further, because protein detection following cycloheximide chase is typically performed by western blotting, radioactive isotopes and lengthy immunoprecipitation steps are not required for cycloheximide chase, unlike many commonly used pulse chase techniques, which are also performed to visualize protein degradation25.
Cycloheximide was first identified as a compound with anti-fungal properties produced by the gram-positive bacterium Streptomyces griseus 26,27. It is a cell-permeable molecule that specifically inhibits eukaryotic cytosolic (but not organellar) translation by impairing ribosomal translocation 28-31. In a cycloheximide chase experiment, cycloheximide is added to cells, and aliquots of cells are collected immediately and at specific time points following addition of the compound 22. Cells are lysed, and protein abundance at each time point is analyzed, typically by western blot. Decreases in protein abundance following the addition of cycloheximide can be confidently attributed to protein degradation. An unstable protein will decrease in abundance over time, while a relatively stable protein will exhibit little change in abundance.
Mechanisms of selective protein degradation have been highly conserved throughout Eukarya. Much of what is known about protein degradation was first learned in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast) 25,32-36. Studies with yeast are likely to continue providing novel and important insights into protein degradation. A method for cycloheximide chase in yeast cells followed by western blot analysis of protein abundance is presented here.

<table border="0" cellpadding="0" cellspacing="0" width="607">
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		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Desired yeast strains, plasmids, standard medium and buffer components</td>
			<td style="width:105px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Disposable borosilicate glass tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">14-961-32</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Temperature-regulated incubator (e.g. Heratherm Incubator Model IMH180)</td>
			<td style="width:105px;">Dot Scientific</td>
			<td style="width:119px;">51028068</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:216px;">New Brunswick Interchangeable Drum for 18 mm tubes (tube roller)</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:119px;">M1053-0450</td>
			<td style="width:167px;">A tube roller is recommended to maintain overnight&#160; starter cultures of yeast cells in suspension. Alternatively, if a tube roller is unavailable, a platform shaker in a temperature-controlled incubator may be used for overnight starter cultures. A platform shaker or tube roller may be used to maintain larger cultures in suspension.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">New Brunswick TC-7 Roller Drum 120V 50/60 H</td>
			<td style="width:105px;">New Brunswick</td>
			<td style="width:119px;">M1053-4004</td>
			<td>For use with tube roller</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">SmartSpec Plus Spectrophotometer</td>
			<td style="width:105px;">Bio-Rad</td>
			<td>170-2525</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">Centrifuge 5430</td>
			<td style="width:105px;">Eppendorf</td>
			<td>5427 000.216&#160;</td>
			<td style="width:167px;">Rotor that is sold with unit holds 1.5- and 2.0-ml microcentrifuge tubes. Rotor may be swapped for one that holds 15- and 50-ml conical tubes</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">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 style="width:105px;">Eppendorf</td>
			<td>F-35-6-30</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">15-ml screen printed screw cap tube 17 x 20 mm conical, polypropylene</td>
			<td style="width:105px;">Sarstedt</td>
			<td style="width:119px;">62.554.205</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1.5-ml flex-tube, PCR clean, natural microcentrifuge tubes</td>
			<td style="width:105px;">Eppendorf</td>
			<td style="width:119px;">22364120</td>
			<td style="width:167px;">Available from a variety of manufacturers</td>
		</tr>
		<tr height="357">
			<td height="357" style="height:357px;width:216px;">Analog Dri-Bath Heaters</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">1172011AQ</td>
			<td style="width:167px;">It is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 &#176;C water bath may be used for incubation of cells in the presence of cycloheximide. Boiling water bath with hot plate may altertnatively be used to denature proteins.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">Heating block for 12 x 15-ml conical tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td>11-473-70</td>
			<td style="width:167px;">For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Heating block for 20 x 1.5-ml conical tubes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td>11-718-9Q</td>
			<td style="width:167px;">For use with Dri-Bath Heater to boil lysates for protein denaturation.</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:216px;">SDS-PAGE running and transfer apparatuses, power supplies, and imaging equipment or darkrooms for SDS-PAGE and transfer to membrane</td>
			<td style="width:105px;"></td>
			<td style="width:119px;"></td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Western blot imaging system (e.g. Li-Cor Odyssey CLx scanner and Image Studio Software)</td>
			<td style="width:105px;">Li-Cor</td>
			<td>9140-01</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">EMD Millipore Immobilon PVDF Transfer Membranes</td>
			<td style="width:105px;">Fisher Scientific</td>
			<td style="width:119px;">IPFL00010</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Primary antibodies (e.g. Phosphoglycerate Kinase (Pgk1) Monoclonal antibody, mouse (clone 22C5D8))</td>
			<td style="width:105px;">Life Technologies</td>
			<td style="width:119px;">459250</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Secondary antibodies (e.g. Alexa-Fluor 680 Rabbit Anti-Mouse IgG (H+L))</td>
			<td>Life Technologies</td>
			<td>A-21065</td>
			<td style="width:167px;">Will vary by lab and application</td>
		</tr>
	</tbody>
</table>

cycloheximide chase analysis of protein degradation in saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein synthesis and protein degradation. Many assays reporting on protein abundance (e.g., single-time point western blotting, flow cytometry, fluorescence microscopy, or growth-based reporter assays) do not allow discrimination of the relative effects of translation and proteolysis on protein levels. This article describes the use of cycloheximide chase followed by western blotting to specifically analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast). In this procedure, yeast cells are incubated in the presence of the translational inhibitor cycloheximide. Aliquots of cells are collected immediately after and at specific time points following addition of cycloheximide. Cells are lysed, and the lysates are separated by polyacrylamide gel electrophoresis for western blot analysis of protein abundance at each time point. The cycloheximide chase procedure permits visualization of the degradation kinetics of the steady state population of a variety of cellular proteins. The procedure may be used to investigate the genetic requirements for and environmental influences on protein degradation.

To illustrate cycloheximide chase methodology, the stability of Deg1-Sec62 (Figure 1), a model yeast endoplasmic reticulum (ER)-associated degradation (ERAD) substrate, was analyzed 42-44. In ERAD, quality control ubiquitin ligase enzymes covalently attach chains of the small protein ubiquitin to aberrant proteins localized to the ER membrane. Such polyubiquitylated proteins are subsequently removed from the ER and degraded by the proteasome, a large, ...

Watch this Scientific Journal Video about Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae at JoVE.com

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Protein abundance reflects the rates of both protein synthesis and protein degradation. This article describes the use of cycloheximide chase followed by western blotting to analyze protein degradation in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast).

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Regulation of protein abundance is crucial to virtually every cellular process. Protein abundance reflects the integration of the rates of protein ...

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