Name: growth-based determination and biochemical confirmation of genetic requirements for protein degradation in saccharomyces cerevisiae
Uploaded: 2015-02-16T15:00:00.000+00:00
Duration: 10 min 57 s
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: Following overnight incubation, cells in culture may be in either logarithmic or stationary growth phase but should have reached a minimal OD600 of 0.2. Very slow-growing yeast strains may require incubation times longer than one night, or inoculation of a greater number of cells, as determined empirically.</li>
	<li>Prepare six-fold serial dilutions of transformed yeast cells in a sterile 96-well plate, beginning with cells diluted to an OD600 of 0.2. Place each yeast transformant to be assayed in a different row in the 96-well plate.
	<ol>
		<li>For each transformant, calculate the volume of overnight culture required to dilute cells to an OD600 of 0.2 in a final volume of 200 &#181;l. Add this volume of overnight culture to the corresponding well in Column 1. Add the appropriate amount of sterile water to bring the volume to 200 &#181;l.</li>
		<li>For each row of yeast, add 125 &#181;l of sterile water to the wells in Columns 2, 3, and 4. 
		NOTE: Individually wrapped sterile 96-well plates may be packaged with sterile lids. The lids may be used as reservoirs for the sterile water that is distributed in this step. This allows simultaneous transfer of sterile water to all wells in a given column with a multichannel pipettor.</li>
		<li>Mix the contents of the first column (yeast diluted to an OD600 of 0.2) by pipetting up and down with a multichannel pipettor.</li>
		<li>Transfer 25 &#181;l of yeast from Column 1 to Column 2, using a multichannel pipettor. Mix by pipetting up and down. Transfer 25 &#181;l from Column 2 to Column 3, and 25 &#181;l from Column 3 to Column 4 (mixing well at each step).</li>
	</ol>
	</li>
	<li>Mix each sample with a multichannel pipettor. Proceeding from most dilute to least dilute columns of yeast, pipette 4 &#181;l of each sample onto two plates containing the appropriate selective medium. Use one plate with medium that maintains plasmid selection (this plate serves as a yeast spotting and growth control). Use a second plate with medium that selects for plasmid maintenance and expression of the unstable protein fused to the reporter enzyme. Because yeast settle rapidly, mix cells by pipetting up and down at regular intervals. 
	NOTE: Drier plates will more readily absorb liquid than freshly prepared plates and are therefore recommended for these experiments. Damp plates may be dried by incubation at room temperature in low humidity for 1 &#8211; 2 days or shorter incubations in a laminar flow hood. Plates may dry unevenly if the laminar air flow is parallel to the bench. Use of a template makes it easier to spot yeast cells at regular distances. Two sample templates are provided in Figure 1. These may be printed, cut out, and affixed to the inside of a Petri dish lid.</li>
	<li>Allow plates to dry on the bench top.</li>
	<li>Incubate plates at 30 &#176;C for 2 &#8211; 6 days.</li>
	<li>Photograph each plate after incubation.</li>
</ol>
<img alt="Figure 1" src="/files/ftp_upload/52428/52428fig1large.jpg" width="650" /> 
Figure 1. Templates for spotting yeast cells onto 100-mm agar plates. These templates may be used to facilitate spotting yeast at regular distances with a multichannel pipettor. Templates may be printed, cut out, and affixed to the inside of a Petri dish lid. Place Petri dish with growth medium inside lid with template affixed. Templates are marked with a notch to track orientation. It is recommended that plates used in growth assays be similarly marked with a notch to track orientation. Templates for spotting four (A) or five (B) serial dilutions of yeast cells are provided. <a href="https://www.jove.com/files/ftp_upload/52428/150126 Fig 1.pdf" target="_blank">Please click here to view a printable version of this figure with 100-mm templates.</a>
2. Biochemical Confirmation of Yeast Growth Assay
<ol>
	<li>Growth of Yeast Cells and Post-Alkaline Protein Extraction (modified from 31)
	<ol>
		<li>Transform wild-type and mutant yeast cells with a plasmid encoding the unstable protein.</li>
		<li>Inoculate transformants in 5 ml of SD medium that is selective for cells harboring plasmid molecules. Incubate overnight at 30 &#176;C, rotating.</li>
		<li>Measure the OD600 of each overnight culture. 
		NOTE: Following overnight incubation, cells may be in either logarithmic or stationary growth phase but should have reached an OD600 that will permit dilution to an OD600 of 0.2 in 10 ml fresh selective medium (step 2.1.4). Very slow-growing yeast strains may require incubation times longer than one night, or inoculation of a greater number of cells, as determined empirically.</li>
		<li>Dilute yeast cells to an OD600 of 0.2 in 10 ml fresh selective medium.</li>
		<li>Continue to incubate cells at 30 &#176;C, rotating or shaking, until cultures reach an OD600 between 0.8 and 1.2 (i.e. are in mid-logarithmic growth). 
		NOTE: If the unstable protein of interest is under the control of a regulatable promoter, the optimal timing of induction of protein expression and cell harvest may vary according to previous studies or empirical observations.</li>
		<li>Collect 2.5 OD600 units of culture in a 15-ml conical tube by centrifugation at 5,000 x g for 5 min at room temperature. Remove supernatant by pipetting or aspiration. 
		NOTE: One OD600 unit is defined as the amount of yeast present in 1 ml of culture at OD600 of 1.0. The volume of culture (in ml) required to harvest 2.5 OD600 units (V) can be determined using the following equation: V = 2.5 OD600 units / Measured OD600</li>
		<li>Resuspend cells in 1 ml distilled water. Transfer suspended cells to a microcentrifuge tube.</li>
		<li>Pellet cells by centrifugation at 6,500 x g for 30 sec at room temperature. Remove supernatant by pipetting or aspiration.</li>
		<li>Resuspend cells in 100 &#181;l distilled water by pipetting up and down or vortexing, and add 100 &#181;l 0.2 M NaOH. Mix by pipetting up and down. Incubate samples for 5 min at room temperature.</li>
		<li>Pellet cells (most of which have not yet released proteins and are still viable) by centrifugation at 18,000 x g for 5 min. Remove supernatant by pipetting or aspiration.</li>
		<li>Resuspend pellet in 50 &#8211; 100 &#181;l 1x Laemmli sample buffer, which will lyse cells, by pipetting up and down or vortexing. 
		NOTE: Removal of the alkaline supernatant following centrifugation and subsequent resuspension of cells in Laemmli sample buffer extracts proteins at a pH compatible with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-glycine running buffer system and western blotting.</li>
		<li>To fully denature proteins, incubate lysates at 95 &#176;C for 5 min. 
		NOTE: Aggregation-prone proteins (e.g. proteins with several transmembrane segments) may become insoluble when incubated at 95 &#176;C. Therefore, lysates should be incubated at lower temperatures (e.g. 37 &#176;C &#8211; 70 &#176;C) for 10 &#8211; 30 min, as empirically determined, for the analysis of such proteins.</li>
		<li>Cool lysates by placing on ice for 5 min.</li>
		<li>Centrifuge lysates at 18,000 x g for 1 min at room temperature to pellet insoluble material. Separate the supernatant (solubilized extracted protein) by SDS-PAGE prior to subsequent western blot analysis (section 2.2). Alternatively, store lysates at -20 &#176;C.</li>
	</ol>
	</li>
	<li>Representative Western Blotting Protocol
	<ol>
		<li>Load empirically determined volume of lysates in an SDS-PAGE gel.</li>
		<li>Run gel at 200 V until dye front has reached the bottom of the gel.</li>
		<li>Transfer proteins from gel to polyvinylidene fluoride (PVDF) membrane by wet transfer at 20 V for 60 &#8211; 90 min at 4 &#176;C.</li>
		<li>Block membrane by incubating in 5% skim milk in Tris-Buffered Saline (TBS), rocking, for 1 hr at room temperature or overnight at 4 &#176;C.</li>
		<li>Decant blocking solution.</li>
		<li>Incubate membrane with primary antibody specific for protein of interest (or epitope tag thereof) in 1% skim milk in TBS with 0.1% Tween-20 (TBS/T) for 1 hr at room temperature, rocking.</li>
		<li>Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.</li>
		<li>Incubate membrane with appropriate fluorophore-conjugated secondary antibody in 1% skim milk in TBS/T for 1 hr at room temperature, rocking. 
		NOTE: Because fluorophores are light-sensitive, dilutions of fluorophore-conjugated antibodies should be prepared in the dark. Additionally, incubation of membranes in the presence of fluorophore-conjugated antibodies should occur in lightproof containers. This can be accomplished by wrapping incubation trays in aluminum foil.</li>
		<li>Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.</li>
		<li>Acquire image of membrane using Li-Cor Odyssey CLx and Image Studio software (or comparable imaging equipment and software), according to manufacturer recommendations.</li>
		<li>After imaging membrane, incubate the membrane with a primary antibody specific for a loading control protein in 1% skim milk in TBS/T for 1 hr at room temperature, rocking.</li>
		<li>Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.</li>
		<li>Incubate membrane with appropriate fluorophore-conjugated secondary antibody in 1% skim milk in TBS/T for 1 hr at room temperature, rocking.</li>
		<li>Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.</li>
		<li>Acquire image of membrane using Li-Cor Odyssey CLx and Image Studio software (or comparable imaging equipment and software), according to manufacturer recommendations.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

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><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&#039;s laboratory. Wild-type yeast and a variety of mutants are also commercially available (&lt;em&gt;e.g.&lt;/em&gt; from GE Healthcare). Plasmids encoding fusion proteins may be generated in the investigator&#039;s laboratory.</td></tr><tr><td>3-amino-1&lt;em&gt;H&lt;/em&gt;-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 &lt;em&gt;Streptomyces plicatus&lt;/em&gt;)</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 (&lt;em&gt;e.g.&lt;/em&gt; 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 P8x20N, 2-20 &amp;mu;l</td><td>Gilson</td><td>F14401</td><td>Available from a variety of manufacturers&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&amp;nbsp;&lt;br /&gt;&lt;br /&gt;&amp;nbsp;</td></tr><tr><td colspan="4">[header]</td></tr><tr><td>Pipetman Neo P8x200N, 20-200 &amp;mu;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>Centrifuge 5430</td><td>Eppendorf</td><td>5427 000.216</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>Plate imaging system (&lt;em&gt;e.g.&lt;/em&gt; 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>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 (&lt;em&gt;e.g.&lt;/em&gt; 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 (&lt;em&gt;e.g.&lt;/em&gt; 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 (&lt;em&gt;e.g.&lt;/em&gt; 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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Biology

9.7K Views.  Ball State University. 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.

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

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins remains elusive. Here, we describe a robust and effective method for determining the suborganellar localization of yeast mitochondrial proteins, which is considered a fundamental step during mitochondrial protein function elucidation. This method involves an initial step that consists of obtaining highly pure intact mitochondria. These mitochondrial preparations are then subjected to a subfractionation protocol consisting of hypotonic shock (swelling) and incubation with proteinase K (protease). During swelling, the outer mitochondrial membrane is selectively disrupted, allowing the proteinase K to digest proteins of the intermembrane space compartment. In parallel, to obtain information about the topology of membrane proteins, the mitochondrial preparations are initially sonicated, and then subjected to alkaline extraction with sodium carbonate. Finally, after centrifugation, the pellet and supernatant fractions from these different treatments are analyzed by SDS-PAGE and western blot. The submitochondrial localization as well as the membrane topology of the protein of interest is obtained by comparing its western blot profile with known standards.

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Despite recent advances, many yeast mitochondrial proteins still remain with their functions completely unknown. This protocol provides a simple and reliable method to determine the submitochondrial localization of proteins, which has been fundamental for the elucidation of their molecular functions.

Despite recent advances in the characterization of yeast mitochondrial proteome, the submitochondrial localization of a significant number of proteins ...

Biochemistry

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting them for degradation. This system is exploited in many non-plant eukaryotes, such that a target protein, tagged with the AID motif, is degraded upon auxin addition. The level of TIR1 expression is critical; excessive expression leads to degradation of the AID-tagged protein even in the absence of auxin, whereas low expression leads to slow depletion. A &#946;-estradiol-inducible AID system was created, with expression of TIR1 under the control of a &#946;-estradiol inducible promoter. The level of TIR1 is tunable by changing the time of incubation with &#946;-estradiol before auxin addition. This protocol describes how to rapidly deplete a target protein using the AID system. The appropriate &#946;-estradiol incubation time depends on the abundance of the target protein. Therefore, efficient depletion depends on optimal timing that also minimizes auxin-independent depletion.

Here, we present a protocol to effectively and specifically deplete a protein of interest in the yeast Saccharomyces cerevisiae using the &#946;-est AID system.

The plant auxin binding receptor, TIR1, recognizes proteins containing a specific auxin-inducible degron (AID) motif in the presence of auxin, targeting ...

Immunology and Infection

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 ...

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.

Determining the mode of action of bioactive chemicals is of interest to a broad range of academic, pharmaceutical, and industrial scientists. ...

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular function of many proteins. PTMs of kinetochore proteins that associate with centromeric DNA mediate faithful chromosome segregation to maintain genome stability. Biochemical approaches such as mass spectrometry and western blot analysis are most commonly used for identification of PTMs. Here, a protein purification method is described that allows the detection of both sumoylation and ubiquitination of the kinetochore proteins, Ndc10 and Ndc80, in Saccharomyces cerevisiae. A strain that expresses polyhistidine-Flag-tagged Smt3 (HF-Smt3) and Myc-tagged Ndc10 or Ndc80 was constructed and used for our studies. For detection of sumoylation, we devised a protocol to affinity purify His-tagged sumoylated proteins by using nickel beads and used western blot analysis with anti-Myc antibody to detect sumoylated Ndc10 and Ndc80. For detection of ubiquitination, we devised a protocol for immunoprecipitation of Myc-tagged proteins and used western blot analysis with anti-Ub antibody to show that Ndc10 and Ndc80 are ubiquitinated. Our results show that epitope tagged-protein of interest in the His-Flag tagged Smt3 strain facilitates the detection of multiple PTMs. Future studies should allow exploitation of this technique to identify and characterize protein interactions that are dependent on a specific PTM.

This manuscript describes the detection of sumoylation and ubiquitination of kinetochore proteins, Ndc10 and Ndc80, in the budding yeast Saccharomyces cerevisiae.

Post-translational Modifications (PTMs), such as phosphorylation, methylation, acetylation, ubiquitination, and sumoylation, regulate the cellular ...

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that involves the construction, cloning and expression of mutant libraries, coupled to high frequency homologous DNA recombination in vivo. Here, we present a protocol to create and screen mutant libraries in yeast based on the example of a fungal aryl-alcohol oxidase (AAO) to enhance its total activity. Two protein segments were subjected to focused-directed evolution by random mutagenesis and in vivo DNA recombination. Overhangs of ~50 bp flanking each segment allowed the correct reassembly of the AAO-fusion gene in a linearized vector giving rise to a full autonomously replicating plasmid. Mutant libraries enriched with functional AAO variants were screened in S. cerevisiae supernatants with a sensitive high-throughput assay based on the Fenton reaction. The general process of library construction in S. cerevisiae described here can be readily applied to evolve many other eukaryotic genes, avoiding extra PCR reactions, in vitro DNA recombination and ligation steps.

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

We present a detailed protocol to construct and screen mutant libraries for directed evolution campaigns in Saccharomyces cerevisiae.

Directed evolution in Saccharomyces cerevisiae offers many attractive advantages when designing enzymes for biotechnological applications, a process that ...

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.

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).

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

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

The encoding of biological information that is accessible to future generations is generally achieved via changes to the DNA sequence. Long-lived inheritance encoded in protein conformation (rather than sequence) has long been viewed as paradigm-shifting but rare. The best characterized examples of such epigenetic elements are prions, which possess a self-assembling behavior that can drive the heritable manifestation of new phenotypes. Many archetypal prions display a striking N/Q-rich sequence bias and assemble into an amyloid fold. These unusual features have informed most screening efforts to identify new prion proteins. However, at least three known prions (including the founding prion, PrPSc) do not harbor these biochemical characteristics. We therefore developed an alternative method to probe the scope of protein-based inheritance based on a property of mass action: the transient overexpression of prion proteins increases the frequency at which they acquire a self-templating conformation. This paper describes a method for analyzing the capacity of the yeast ORFeome to elicit protein-based inheritance. Using this strategy, we previously found that &gt;1% of yeast proteins could fuel the emergence of biological traits that were long-lived, stable, and arose more frequently than genetic mutation. This approach can be employed in high throughput across entire ORFeomes or as a targeted screening paradigm for specific genetic networks or environmental stimuli. Just as forward genetic screens define numerous developmental and signaling pathways, these techniques provide a methodology to investigate the influence of protein-based inheritance in biological processes.

High-throughput Screening for Protein-based Inheritance in S. cerevisiae

This protocol describes a high-throughput methodology to functionally screen for protein-based inheritance in S. cerevisiae.

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

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Identification of protein complexes with quantitative proteomics in S. cerevisiae

Assessment of Submitochondrial Protein Localization in Budding Yeast Saccharomyces cerevisiae

Tuning Degradation to Achieve Specific and Efficient Protein Depletion

Coupled Assays for Monitoring Protein Refolding in Saccharomyces cerevisiae

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Rapid Identification of Chemical Genetic Interactions in Saccharomyces cerevisiae

Protein Purification Technique that Allows Detection of Sumoylation and Ubiquitination of Budding Yeast Kinetochore Proteins Ndc10 and Ndc80

Directed Evolution Method in Saccharomyces cerevisiae: Mutant Library Creation and Screening

Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

High-throughput Screening for Protein-based Inheritance in S. cerevisiae