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-transformed cells). Incubate overnight at 30 &#176;C, rotating. 
	NOTE: 30 &#176;C is the optimal growth temperature for typical wild-type laboratory yeast strains 38. However, because some mutant yeast strains do not grow optimally at 30 &#176;C and some proteins undergo temperature-dependent degradation, the temperatures used for yeast cell growth and cycloheximide chase should be empirically determined 39.</li>
	<li>Measure the optical density at 600 nm (OD600) of each overnight culture. 
	NOTE: Cells may be in logarithmic or stationary growth phase but should have minimally reached a density that will allow dilution to an OD600 = 0.2 in 15 ml of fresh medium.</li>
	<li>Dilute the overnight cultures to an OD600 value of 0.2 in 15 ml of fresh medium.</li>
	<li>Incubate yeast at 30 &#176;C, shaking until the cells reach mid-logarithmic growth phase (i.e., an OD600 between 0.8 and 1.2).</li>
	<li>During yeast cell growth, perform the following in preparation for the cycloheximide chase procedure:
	<ol>
		<li>Set a heat block that can accommodate 15 ml conical tubes to 30 &#176;C for incubation of cells in the presence of cycloheximide. Add water to the wells of the heat block to allow efficient heat distribution to cultures. Add water to each well such that a 15 ml conical tube will cause the water level to rise to, but not overflow, the lip of the well.</li>
		<li>Set a second heat block that can accommodate 1.5 ml microcentrifuge tubes to 95 &#176;C for protein denaturation following cell lysis.</li>
		<li>Pre-warm fresh growth medium (1.1 ml per time point per culture to be assayed) to 30 &#176;C.</li>
		<li>Add 50 &#181;l 20x Stop Mix to pre-labeled microcentrifuge tubes (one tube per time point per culture to be assayed). Place tubes on ice. 
		CAUTION: Sodium azide, an ingredient in 20x Stop Mix, is toxic via oral ingestion or dermal contact. Follow manufacturer recommendations when preparing, storing, and handling sodium azide. In the event of accidental exposure, consult material safety data sheet provided by manufacturer.</li>
	</ol>
	</li>
	<li>When cells have reached mid-logarithmic growth, collect 2.5 OD600 units of each culture per time point to be assayed (e.g., 7.5 OD600 units to analyze protein abundance at three time points). Centrifuge collected cells in 15 ml conical tubes at 3,000 x g at room temperature for 2 min. 
	NOTE: One OD600 unit is equal to the amount of yeast present in 1 ml culture at an OD600 of 1.0. The volume of culture (in ml) required to harvest X OD600 units (V) may be determined by using the following equation: V = X OD600 units/Measured OD600. For example, to harvest 7.5 OD600 units of yeast cell culture at an OD600 of 1.0, collect 7.5 OD600 units/1.0 = 7.5 ml yeast culture.</li>
	<li>Resuspend each cell pellet in 1 ml of 30 &#176;C (pre-warmed) fresh growth medium per 2.5 OD600 units of cells (e.g., 3 ml for 7.5 OD600 units).</li>
</ol>
2. Cycloheximide Chase
<ol>
	<li>Equilibrate yeast cell suspensions by incubation for 5 min in the 30 &#176;C heat block.</li>
	<li>Prepare a timer to count up from 0:00.</li>
	<li>To begin the cycloheximide chase, press &#34;Start&#34; on the timer. Swiftly, but carefully, perform the following steps:
	<ol>
		<li>Add cycloheximide to a final concentration of 250 &#181;g/ml to the first yeast cell suspension (e.g., add 37.5 &#181;l of 20 mg/ml cycloheximide stock to 3 ml of cell suspension), and vortex briefly to mix. 
		CAUTION: Cycloheximide is a dermal irritant and is toxic via oral ingestion. Follow manufacturer recommendations when preparing, storing, and handling cycloheximide. In the event of accidental exposure, consult material safety data sheet provided by manufacturer.</li>
		<li>Immediately after adding cycloheximide and vortexing, transfer 950 &#181;l (~2.4 OD600 units) of the yeast cell suspension with added cycloheximide to a pre-labeled microcentrifuge tube containing 50 &#181;l ice-cold 20x stop mix. Vortex the microcentrifuge tube, and place on ice until all samples have been collected.</li>
		<li>Return the yeast cell suspension to 30 &#176;C.</li>
	</ol>
	</li>
	<li>Repeat steps 2.3.1 through 2.3.3 for each of the remaining yeast cell suspensions at regular time intervals (e.g., every 30 sec, such that cycloheximide is added to Sample #1 at 0:00, Sample #2 at 0:30, Sample #3 at 1:00, etc.).</li>
	<li>At each subsequent time point, vortex yeast cell suspensions and transfer 950 &#956;l to labeled microcentrifuge tubes containing 50 &#956;l pre-chilled 20x Stop Mix. Vortex and place collected cells on ice. Return 15 ml conical tubes to 30 &#176;C heat block.
	<ol>
		<li>For example, for collection of cells 30 min after cycloheximide addition (assuming 30-sec intervals between cycloheximide addition to yeast cell suspensions at the beginning of the time course), vortex and remove 950 &#956;l of cell suspension from Sample #1 at 30:00. Repeat for Sample #2 at 30:30, and so on. 
		NOTE: To prevent settling of yeast, vortex cell suspensions in 15 ml conical tubes approximately every 5 min throughout the course of the chase. Alternatively, yeast cell suspensions may be maintained in a continuously agitating water bath for the duration of the cycloheximide chase experiment.</li>
	</ol>
	</li>
	<li>When all samples have been collected, pellet collected cells by centrifugation at 6,500 x g at room temperature for 30 sec. Remove the supernatant by pipetting or aspiration. Cells are now ready for alkaline lysis. Alternatively, snap-freeze pelleted cells in liquid nitrogen and store at -80 &#176;C.</li>
</ol>
3. Post-alkaline Protein Extraction (Modified from 16,40)
<ol>
	<li>Add 100 &#181;l of distilled water to each cell pellet. Resuspend by pipetting up and down or vortexing.</li>
	<li>Add 100 &#181;l&#160;of 0.2 M NaOH to each sample. Mix by pipetting up and down or vortexing.</li>
	<li>Incubate suspended cells at room temperature for 5 min. At this stage, yeast cells have not been lysed, and proteins have not been released 40.</li>
	<li>Pellet cells by centrifugation at 18,000 x g at room temperature for 30 sec. Remove supernatant by pipetting or aspiration.</li>
	<li>To lyse cells, add 100 &#181;l of Laemmli sample buffer to each cell pellet. Resuspend by pipetting up and down or vortexing. 
	NOTE: Sequential incubation of cells with NaOH and Laemmli sample buffer releases proteins in a form compatible with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-glycine running buffer system.</li>
	<li>Incubate at 95 &#176;C for 5 min to fully denature proteins. 
	NOTE: Incubation at 95 &#176;C may not be suitable for analysis of proteins that are prone to aggregation (e.g., proteins with several transmembrane segments). These proteins may become insoluble when incubated at elevated temperatures 41. Thus, for the analysis of such proteins, lysates should be incubated at lower temperatures (e.g., 37 &#176;C - 70 &#176;C) for 10 - 30 min, as empirically determined.</li>
	<li>Centrifuge lysates at 18,000 x g at room temperature for 1 min to pellet insoluble material. The supernatant (solubilized extracted protein) is ready for separation by SDS-PAGE and subsequent western blot analysis. Alternatively, store lysates at -20 &#176;C.</li>
</ol>
4. Representative SDS-PAGE and Western Blotting Procedure (Adapted from 16)
<ol>
	<li>Load protein molecular weight standards and empirically determined volume of lysates onto an SDS-PAGE gel. 
	NOTE: Choose the percentage of acrylamide and bis-acrylamide in the SDS-PAGE gel based on the molecular weight of the protein of interest. In general, lower percentage gels are better suited for resolving higher molecular weight proteins.</li>
	<li>Run gel at 200 V until the dye front has reached the bottom edge of the gel.</li>
	<li>Transfer proteins from the gel to polyvinylidene fluoride (PVDF) membrane by wet transfer at 20 V for 60 - 90 min at 4 &#176;C.</li>
	<li>Block membrane by incubating in Tris-Buffered Saline (TBS) containing 5% skim milk, rocking, at room temperature for 1 hr or at 4 &#176;C overnight. 
	NOTE: To prevent microbial growth in the presence of a membrane incubated overnight, it is recommended to include sodium azide in the blocking solution at a final concentration of 0.02%.</li>
	<li>Incubate membrane with primary antibody specific for protein of interest in TBS with 0.1% Tween-20 (TBS/T) and 1% skim milk, rocking, at room temperature for 1 hr.</li>
	<li>Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.</li>
	<li>Incubate membrane with appropriate fluorophore-conjugated secondary antibody in TBS/T with 1% skim milk at room temperature for 1 hr, rocking. 
	NOTE: Fluorophores are light-sensitive. Therefore, dilutions of antibodies conjugated to fluorophores should be prepared in the dark, and incubation of membranes in the presence of antibodies conjugated to fluorophores and subsequent wash steps should occur in lightproof (e.g., foil-wrapped) containers.</li>
	<li>Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.</li>
	<li>Image membrane using LI-COR Odyssey CLx and Image Studio software (or comparable imaging equipment and software), following manufacturer recommendations.</li>
	<li>After acquiring membrane image, incubate the membrane with a primary antibody specific for a loading control protein in TBS/T with 1% skim milk at room temperature for 1 hr, rocking.</li>
	<li>Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.</li>
	<li>Incubate membrane with appropriate fluorophore-conjugated secondary antibody in TBS/T with 1% skim milk at room temperature for 1 hr, rocking.</li>
	<li>Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.</li>
	<li>Image membrane as in step 4.9.</li>
</ol>

The authors have nothing to disclose.

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">
	<tbody>
		<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 ...

cycloheximide-chase-analysis-protein-degradation-saccharomyces

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28.7K Views.  Ball State University. 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).

Video: Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Dissection of Saccharomyces Cerevisiae Asci

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae exists in either a haploid or diploid state. The ability to combine alleles from two haploids and the ability to introduce modifications to the genome requires the production and dissection of asci. Asci production from haploid cells begins with the mating of two yeast haploid strains with compatible mating types to produce a diploid strain. This can be accomplished in a number of ways either on solid medium or in liquid. It is advantageous to select for the diploids in medium that selectively promotes their growth compared to either of the haploid strains. The diploids are then allowed to sporulate on nutrient-poor medium to form asci, a bundle of four haploid daughter cells resulting from meiotic reproduction of the diploid. A mixture of vegetative cells and asci is then treated with the enzyme zymolyase to digest away the membrane sac surrounding the ascospores of the asci. Using micromanipulation with a microneedle under a dissection microscope one can pick up individual asci and separate and relocate the four ascopores. Dissected asci are grown for several days and tested for the markers or alleles of interest by replica plating onto appropriate selective media.

Dissection of Saccharomyces Cerevisiae Asci

Micromanipulation of yeast cells is needed for meiotic genetic analysis or to select diploid zygotes. These micromanipulations are carried out using the microneedle of a dissection microscope. The microneedle is used to relocate cells and is controlled by a micromanipulator which are available with various degrees of automation.

Yeast is a highly tractable model system that is used to study many different cellular processes. The common laboratory strain Saccharomyces cerevisiae ...

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground using a cryolysis procedure in a ball mill grinder and the resulting grindate brought into solution by urea solubilization. This procedure allows for the lysis of the cells in a metabolically inactive state, preserving phosphorylation and preventing reorientation of the phosphoproteome during cell lysis.  Following reduction, alkylation, trypsin digestion of the proteins, the samples are desalted on C18 columns and the sample complexity reduced by fractionation using hydrophilic interaction chromatography (HILIC). HILIC columns preferentially retain hydrophilic molecules which is well suited for phosphoproteomics.  Phosphorylated peptides tend to elute later in the chromatographic profile than the non phosphorylated counterparts. After fractionation, phosphopeptides are enriched using immobilized metal chromatography, which relies on charge-based affinities for phosphopeptide enrichment. At the end of this procedure the samples are ready to be quantitatively analyzed by mass spectrometry.

Quantitative Phosphoproteomics in Fatty Acid Stimulated Saccharomyces cerevisiae

Description of a quantitative phosphorylation procedure using cryolysis, urea solubilziation, HILIC fractionation and IMAC enrichment of phosphorylated peptides.

This protocol describes the growth and stimulation, with the fatty acid oleate, of isotopically heavy and light S. cerevisiae cells. Cells are ground ...

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent. In the experiment, yeast is grown for 48 hours in 1/2X YPD broth containing 3X glucose. The culture is split into a control and treated group. The experiment culture is treated with 0.5 mM H2O2 in Hanks Buffered Saline (HBSS) for 1 hour. The control culture is treated with HBSS only. Total RNA is extracted from both cultures and is converted to a biotin-labeled cRNA product through a multistep process. The final synthesis product is taken back to the UVM Microarray Core Facility and hybridized to the Affymetrix yeast GeneChips. The resulting gene expression data are uploaded into bioinformatics data analysis software.

Microarray Analysis for Saccharomyces cerevisiae

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen peroxide (H2O2), an oxidizing agent.

In this protocol, gene expression in yeast (Saccharomyces cerevisiae) is changed after exposure to oxidative stress induced by the addition of hydrogen ...

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride channel, that when mutated, can give rise to cystic fibrosis in humans.There is therefore considerable interest in this protein, but efforts to study its structure and activity have been hampered by the difficulty of expressing and purifying sufficient amounts of the protein1-3. Like many 'difficult' eukaryotic membrane proteins, expression in a fast-growing organism is desirable, but challenging, and in the yeast S. cerevisiae, so far low amounts were obtained and rapid degradation of the recombinant protein was observed 4-9. Proteins involved in the processing of recombinant CFTR in yeast have been described6-9 .In this report we describe a methodology for expression of CFTR in yeast and its purification in significant amounts. The protocol describes how the earlier proteolysis problems can be overcome and how expression levels of CFTR can be greatly improved by modifying the cell growth conditions and by controlling the induction conditions, in particular the time period prior to cell harvesting. The reagants associated with this protocol (murine CFTR-expressing yeast cells or yeast plasmids) will be distributed via the US Cystic Fibrosis Foundation, which has sponsored the research. An article describing the design and synthesis of the CFTR construct employed in this report will be published separately (Urbatsch, I.; Thibodeau, P. et al., unpublished). In this article we will explain our method beginning with the transformation of the yeast cells with the CFTR construct - containing yeast plasmid (Fig. 1). The construct has a green fluorescent protein (GFP) sequence fused to CFTR at its C-terminus and follows the system developed by Drew et al. (2008)10. The GFP allows the expression and purification of CFTR to be followed relatively easily. The JoVE visualized protocol finishes after the preparation of microsomes from the yeast cells, although we include some suggestions for purification of the protein from the microsomes. Readers may wish to add their own modifications to the microsome purification procedure, dependent on the final experiments to be carried out with the protein and the local equipment available to them. The yeast-expressed CFTR protein can be partially purified using metal ion affinity chromatography, using an intrinsic polyhistidine purification tag. Subsequent size-exclusion chromatography yields a protein that appears to be &gt;90% pure, as judged by SDS-PAGE and Coomassie-staining of the gel.

Expression and Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein in Saccharomyces cerevisiae

Attempts to express the cystic fibrosis transmembrane conductance regulator (CFTR) in Saccharomyces cerevisiae have, until now, yielded relatively low amounts of protein. This protocol and the associated reagents distributed via the Cystic Fibrosis Foundation should allow the preparation of milligram amounts of this 'difficult' eukaryotic membrane protein.

The  cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride  channel, that when mutated, can give rise to cystic fibrosis in  ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must be maintained to avoid deleterious consequences 1. External or internal factors that disrupt this balance can lead to protein aggregation, toxicity and cell death. In humans this is a major contributing factor to the symptoms associated with neurodegenerative disorders such as Huntington's, Parkinson's, and Alzheimer's diseases 10. It is therefore essential that the proteins involved in maintenance of proteostasis be identified in order to develop treatments for these debilitating diseases. This article describes techniques for monitoring in vivo protein folding at near-real time resolution using the model protein firefly luciferase fused to green fluorescent protein (FFL-GFP). FFL-GFP is a unique model chimeric protein as the FFL moiety is extremely sensitive to stress-induced misfolding and aggregation, which inactivates the enzyme 12. Luciferase activity is monitored using an enzymatic assay, and the GFP moiety provides a method of visualizing soluble or aggregated FFL using automated microscopy. These coupled methods incorporate two parallel and technically independent approaches to analyze both refolding and functional reactivation of an enzyme after stress. Activity recovery can be directly correlated with kinetics of disaggregation and re-solubilization to better understand how protein quality control factors such as protein chaperones collaborate to perform these functions. In addition, gene deletions or mutations can be used to test contributions of specific proteins or protein subunits to this process. In this article we examine the contributions of the protein disaggregase Hsp104 13, known to partner with the Hsp40/70/nucleotide exchange factor (NEF) refolding system 5, to protein refolding to validate this approach.

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

This article describes the use of a firefly luciferase-GFP fusion protein to investigate in vivo protein folding in Saccharomyces cerevisiae. Using this reagent, refolding of a model heat-denatured protein can be monitored simultaneously by fluorescence microscopy and an enzymatic assay to probe the roles of proteostasis network components in protein quality control.

Proteostasis, defined as the combined processes of protein folding/biogenesis, refolding/repair, and degradation, is a delicate cellular balance that must ...

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that currently limits the average life expectancy of sufferers to &#60;40 years of age. The development of novel drug molecules to restore the activity of CFTR is an important goal in the treatment CF, and the isolation of functionally active CFTR is a useful step towards achieving this goal.
We describe two methods for the purification of CFTR from a eukaryotic heterologous expression system, S. cerevisiae. Like prokaryotic systems, S. cerevisiae can be rapidly grown in the lab at low cost, but can also traffic and posttranslationally modify large membrane proteins. The selection of detergents for solubilization and purification is a critical step in the purification of any membrane protein. Having screened for the solubility of CFTR in several detergents, we have chosen two contrasting detergents for use in the purification that allow the final CFTR preparation to be tailored to the subsequently planned experiments.
In this method, we provide comparison of the purification of CFTR in dodecyl-&#946;-D-maltoside (DDM) and 1-tetradecanoyl-sn-glycero-3-phospho-(1&#39;-rac-glycerol) (LPG-14). Protein purified in DDM by this method shows ATPase activity in functional assays. Protein purified in LPG-14 shows high purity and yield, can be employed to study post-translational modifications, and can be used for structural methods such as small-angle X-ray scattering and electron microscopy. However it displays significantly lower ATPase activity.

Purification of the Cystic Fibrosis Transmembrane Conductance Regulator Protein Expressed in Saccharomyces cerevisiae

Heterologous expression and purification of the cystic fibrosis transmembrane conductance regulator (CFTR) are significant challenges and limiting factors in the development of drug therapies for cystic fibrosis. This protocol describes two methods for the isolation of milligram quantities of CFTR suitable for functional and structural studies.&#160;

Defects in the cystic fibrosis transmembrane conductance regulator (CFTR) protein cause cystic fibrosis (CF), an autosomal recessive disease that ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. Saccharomyces cerevisiae, or budding yeast, is a model eukaryote for which a complete collection of ~6,000 gene deletion mutants and hypomorphic essential gene mutants are commercially available. These collections of mutants can be used to systematically detect chemical-gene interactions, i.e. genes necessary to tolerate a chemical. This information, in turn, reports on the likely mode of action of the compound. Here we describe a protocol for the rapid identification of chemical-genetic interactions in budding yeast. We demonstrate the method using the chemotherapeutic agent 5-fluorouracil (5-FU), which has a well-defined mechanism of action. Our results show that the nuclear TRAMP RNA exosome and DNA repair enzymes are needed for proliferation in the presence of 5-FU, which is consistent with previous microarray based bar-coding chemical genetic approaches and the knowledge that 5-FU adversely affects both RNA and DNA metabolism. The required validation protocols of these high-throughput screens are also described.

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Here we present a cost-effective method for defining chemical-genetic interactions in budding yeast. The approach is built on fundamental techniques in yeast molecular biology and is well suited for the mechanistic interrogation of small to medium collections of chemicals and other media environments.