This work was supported by a grant from the National Institutes of Health (NIGMS-074696) to K.A.M. and an American Society of Microbiology Robert D. Watkins Graduate Student Research Fellowship to J.L.A. We also thank John Glover at the University of Toronto for providing the FFL-GFP source plasmid.

1. Construction of Strains Containing FFL-GFP Plasmid
For this study, Saccharomyces cerevisiae strain BY4741 (MATa, his3&Delta;1, leu2&Delta;0, met15&Delta;0, ura3&Delta;0) was used along with an HSP104 deletion strain from the yeast knockout collection (Open Biosystems/Thermo Scientific). The deletion was confirmed using an Hsp104-specific antibody for Western blot analysis.
FFL-GFP was expressed from p426MET25-FFL-GFP, constructed from a LEU2-based source plasmid obtained from the Glover laboratory at the University of Toronto 17 and obtainable upon request to the authors. Expression of the FFL-GFP plasmid fusion protein is controlled by the MET25 methionine-repressible promoter. The plasmid was transformed into each strain using a protocol was adapted from Gietz et. al, 1995:
<ol>
 <li>Centrifuge 25 ml of log phase cells in a 50 ml conical tube at 4,400 rpm for 2 min, wash with 500 &mu;l of double distilled H20 (ddH2O), and discard supernate.</li>
 <li>Resuspend cells in 250 &mu;l of TE/LiAc (Tris-HCl 10 mM, pH=7.5, 1 mM EDTA, 0.1 M lithium acetate). Incubate at 30 &deg;C without mixing for 20 min.</li>
 <li>Transfer 50 &mu;l of competent cells to a new tube along with 5 ml (50 &mu;g) of carrier DNA and 5 &mu;l (0.1-5 &mu;g) of plasmid DNA. Add 300 &mu;l of PEG/TE/LiAc (same as TE/LiAc plus 40% polyethylene glycol) to this mixture, and incubate cells at 30 &deg;C for another 30 min.</li>
 <li>Add 1/10 volume (v/v) DMSO (36 &mu;l) to this mixture and heat shock at 42 &deg;C for 6 min.</li>
 <li>Centrifuge mixture at 6,000 rpm for 30 sec and remove supernate. Resuspend cells in 100 &mu;l of sterile ddH20 and plate cells onto synthetic complete media lacking uracil (SC-URA).</li>
</ol>
2. Induction of FFL-GFP
FFL-GFP is induced once cells are in log phase so the majority of the protein prior to heat-shock is newly made to avoid proteins that have terminally aggregated over time due to aging-associated cellular inadequacies.
<ol>
 <li>Day 1: Incubate cells in 5 ml of SC-URA at 30 &deg;C rotating O/N.</li>
 <li>Day 2: Measure OD600 and inoculate fresh cultures in 5 ml of SC-URA OD600~0.2.</li>
 <li>Incubate cultures with rotation at 30 &deg;C until they reach log phase OD600~0.8-1.0.</li>
 <li>Centrifuge cells in 15 ml conical tubes at 4,400 rpm for 3 min, decant supernate and resuspend cell pellet in 500 &mu;l of ddH20; transfer solution to a microcentrifuge tube.</li>
 <li>Centrifuge at 6,000 rpm and discard supernate.</li>
 <li>Resuspend cells in 5 ml of SC-URA-MET. Cells should be incubated rotating in this media for 1 hr at 30 &deg;C.</li>
 <li>Centrifuge cells and repeat wash in ddH20 and discard supernate.</li>
 <li>Resuspend cells in 5 ml SC-URA media containing 100 &mu;g/ml of cycloheximide to inhibit protein expression, and proceed immediately to Step 3.</li>
</ol>
3. Enzymatic FFL-GFP Recovery Assay
This assay is a quantitative approach to determine the levels of active enzyme in a population.
<ol>
 <li>Preparation for this assay:
 <ol class="lroman">
 <li>Measure OD600 for each sample.</li>
 <li>Prepare necessary amount of D-luciferin needed based on the number of samples and desired number of replicates plus approximately 1,200 &mu;l for the tubing. Final concentration of D-luciferin is ~23 &mu;g per 100 &mu;l of cells. Initially it is dissolved in water at a stock solution concentration of 7.5 mg/ml and diluted to 455 &mu;g/ml in SC prior to experiment. In Figure 2, three replicates for each sample were analyzed resulting in a total of 2.4 ml of D-luciferin being used.</li>
 <li>Program plate reader for flash luminescence assay (adapted from BioTek Gen5 "Luminescence Flash Assay with Injection"; for other instruments follow manufacturer's instructions)
 <ol class="lalpha">
 <li>Set temperature to 30 &deg;C</li>
 <li>Set plate reader to add D-luciferin, shake, and read each well individually.</li>
 <li>Read luminescence (endpoint reading, 5 sec integration time, 180 sensitivity level)</li>
 <li>For recovery assay between each reading shake for 5 min, delay 20 min, and shake an additional 5 min.</li>
 <li>Dispense 50 &mu;l of D-luciferin from an injector.</li>
 </ol>
 </li>
 </ol>
 </li>
 <li>Preheat-shock luminescence is measured immediately after cells are added to media containing cycloheximide to halt protein synthesis.
 <ol class="lroman">
 <li>Transfer 100 &mu;l of cells for each sample with desired number of replicates into a solid white 96 well plate.</li>
 <li>Controls should include a water blank and cells containing an empty vector.</li>
 <li>Start the read with the specifications listed above.</li>
 </ol>
 </li>
 <li>To denature the FFL moiety of FFL-GFP, incubate the 5 ml culture in a glass culture tube at 42 &deg;C shaking for 25 min.</li>
 <li>Recovery assay luminescence readings start immediately after cells are removed from the incubator, which is time point zero. Measure luminescence same as described above for the first time point and every 30 min for 90 min.</li>
 <li>Normalize samples to preheat-shock luminescence values that have been adjusted based on the OD600 (divide the preheat-shock values by OD600).</li>
</ol>
4. Fluorescence Microscopy
This assay is a semi-quantitative method to determine solubilization of aggregates over time in a population of cells.
<ol>
 <li>Induction is the same as described in Section 2, Steps 1-8 of protocols.</li>
 <li>The same time points are taken as described above (Section 3, Step 4), but for microscopy collect 1 ml of cells at preheat-shock and each recovery time point, and incubate samples rotating at 30 &deg;C in culture tube.</li>
 <li>Visualization:
 <ol class="lroman">
 <li>Centrifuge 1 ml of cells at 6,000 rpm for 30 sec and remove supernate.</li>
 <li>Resuspend cell pellet in 2 &mu;l of ddH2O.</li>
 <li>Mix 2 &mu;l of cells plus 2 &mu;l of 2% low melt agarose on the slide and add a cover slip. In order to obtain a single plane of cells lightly press finger down in the center of the cover slip and rotate until the cover slip is difficult to move.</li>
 <li>Cells are visualized using 100X oil objective on fluorescent microscope; use DIC to visualize cells and the FITC filter to visualize GFP fluorescence.</li>
 <li>Take multiple pictures for each strain at each time point for quantitation. Fields for pictures should contain ~15-30 cells for quantitative post-experimental analysis.</li>
 <li>To calculate percentage of cells containing aggregates, count 50-100 cells total and divide the number of cells containing aggregates.</li>
 </ol>
 </li>
</ol>
5. Single Cell Microscopy
This method is used to follow the solubilization of individual aggregates in a single cell over time.
<ol>
 <li>Induce FFL-GFP expression as described in Section 2, Steps 1-8.</li>
 <li>After heat-shock, centrifuge cells in 15 ml conical tube at 4,400 rpm for 2 min.</li>
 <li>Wash cells with 500 &mu;l of water and resuspend in 20 &mu;l of ddH2O.</li>
 <li>For each strain, cut out a 5x5 mm section of an SC-URA agar plate and using the surface that was in contact with Petri dish, pipette 4 &mu;l of cells and spread around surface of agar with pipette tip. Let stand for ~1 min.</li>
 <li>Use tweezers to place agar cube section face-down on a ~55 mm glass bottom dish No. 0 and press down lightly.</li>
 <li>Visualization:
 <ol class="lroman">
 <li>See i-iii in Section 4.</li>
 <li>Set coordinates for 2-4 focal points for each strain depending on density of culture.</li>
 <li>Set camera to take pictures with a Z-stack with appropriate range (-/+ 15 &mu;m with a 0.5-1.0 &mu;m slice thickness) every 5 min for a 90 min period.</li>
 </ol>
 </li>
</ol>

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No conflicts of interest declared.

In this article the model protein FFL-GFP was used to show that the yeast disaggregase, Hsp104 contributes to protein re-solubilization and repair. The enzymatic assays and microscopy differentially interrogated the status of the same substrate protein to determine refolding efficiency and yield. Results of the enzymatic recovery assays suggest that not only is the maximal recovery in the hsp104D mutant strain inefficient, but the initial magnitude of the unfolding stress was greater in the mutant strain (<stron...

In humans neurodegenerative disorders including Alzheimer's, Parkinson's, and Huntington's diseases have been linked to protein misfolding and aggregation 10. Cells employ molecular chaperones to prevent kinetic trapping of cellular proteins into misfolded inactive structures 6. Chaperones participate in intricate interaction networks within the cell, but it is not completely understood how the sum of these interactions contributes to organismal proteostasis. One of the main chaperones responsible for the majority of cytosolic protein folding is the 70 kD heat shock protein (Hsp70) family 19. It has been shown that in yeast loss of Hsp70 decreases the ability to fold nascent heterologously expressed FFL and to refold the endogenous protein, ornithine transcarbamoylase, in vivo 18, 7. The ability to analyze folding with near-real time resolution will facilitate understanding how additional cellular factors contribute to this Hsp70-dependent process. In addition, folding/refolding reactions may not be completely dependent on these contributing proteins, so assays must be sensitive enough to detect both large and small changes in kinetics and efficiency.
The yeast cell disaggregase, Hsp104, plays a vital role in repairing aggregated misfolded proteins. Although Hsp104 homologs have been identified in fungi and plants, this family appears to be absent in metazoans. It has been proposed that other chaperones such as those of the Hsp110 family perform some of the known Hsp104 activities in mammals 16. Hsp104 is an AAA+, hexameric protein complex that functions in yeast to remodel protein aggregates, contributing to refolding and repair 13. Hsp104, along with the yeast Hsp70, Ssa1, and the yeast Hsp40, Ydj1, is required for recovery of denatured FFL in yeast cells 5, 8. The small heat shock protein, Hsp26, has also been shown to be required for Hsp104-mediated disaggregation of FFL 2.
FFL is a two-domain protein that binds the substrate luciferin in the active site and following a conformational change that requires ATP and oxygen, decarboxylates the substrate releasing oxyluciferin, carbon dioxide (CO2), adenosine monophosphate (AMP), and light 11, 9, 3. The commercially available FFL substrate, D-luciferin, results in light emission between 550-570 nm that can be detected using a luminometer 15. FFL is exquisitely sensitive to denaturation from chemical or heat treatments and aggregates rapidly upon unfolding. When exposed to temperatures between 39-45 &deg;C FFL is reversibly unfolded and inactivated 12. In contrast, GFP and its derivatives are highly resistant to protein unfolding stresses 14. Therefore fusion of these two proteins allows FFL to function as an experimentally labile moiety capable of targeting functional GFP to intracellular deposits that can be visualized using fluorescence microscopy at both the population and single cell levels. Application of the enzymatic assay in a semi-automated multimode plate reader coupled with automated microscopy allows unprecedented simultaneous assessment of kinetics and yield of refolding reactions. In addition, the facile molecular genetics of the model eukaryote Saccharomyces cerevisiae allows both precise manipulation of the protein quality control network and the opportunity for discovery-based approaches to identify novel players contributing to cellular stress response and proteostasis.
In this study, wild-type (WT) and HSP104 deletion strains expressing FFL-GFP are subject to protein denaturing heat shock. FFL-GFP refolding is monitored through both an enzymatic assay and microscopy as a proxy readout for repair of the expressed proteome over a recovery time course. When compared to WT cells, we show the Hsp104 deletion strain is ~60% less efficient at refolding FFL-GFP, supporting previous findings establishing a role for Hsp104 in reactivation of denatured FFL 2.

<table border="1">
 <tbody>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Reagents</td>
 </tr>
 <tr>
 <td>Synergy MX Microplate Reader</td>
 <td>BioTek</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Olympus IX81-ZDC Confocal Inverted Microscope</td>
 <td>Olympus, Tokyo Japan</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>Lumitrac 200 white 96-well plates</td>
 <td>USA Scientific</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td>SlideBook 5.0 digital microscopy software</td>
 <td>Intelligent Imaging Innovations, Inc. Denver, CO, USA</td>
 <td></td>
 <td></td>
 </tr>
 <tr>
 <td></td>
 <td></td>
 <td></td>
 <td>Equipment</td>
 </tr>
 <tr>
 <td>Tris Base</td>
 <td>Fisher</td>
 <td>BP152-1</td>
 <td></td>
 </tr>
 <tr>
 <td>(LiAc) Lithium Acetate</td>
 <td>Sigma</td>
 <td>L6883</td>
 <td></td>
 </tr>
 <tr>
 <td>PEG (polyethelene glycol)</td>
 <td>Fisher</td>
 <td>BP233-1</td>
 <td></td>
 </tr>
 <tr>
 <td>EDTA</td>
 <td>Fisher</td>
 <td>BP120-1</td>
 <td></td>
 </tr>
 <tr>
 <td>DMSO</td>
 <td>Malinckrodt</td>
 <td>4948</td>
 <td></td>
 </tr>
 <tr>
 <td>SC</td>
 <td>Sunrise</td>
 <td>1300-030</td>
 <td></td>
 </tr>
 <tr>
 <td>SC-URA</td>
 <td>Sunrise</td>
 <td>1306-030</td>
 <td></td>
 </tr>
 <tr>
 <td>cycloheximide</td>
 <td>Acros Organics</td>
 <td>357420050</td>
 <td></td>
 </tr>
 <tr>
 <td>D-luciferin</td>
 <td>Sigma</td>
 <td>L9504</td>
 <td></td>
 </tr>
 <tr>
 <td>low melt agarose</td>
 <td>NuSieve</td>
 <td>50082</td>
 <td></td>
 </tr>
 <tr>
 <td>immersion oil</td>
 <td>Cargille</td>
 <td>16484</td>
 <td></td>
 </tr>
 </tbody>
</table>

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.

Yeast is dependent on the disaggregase, Hsp104 to efficiently refold heat-denatured proteins. The activity of FFL-GFP was monitored after a 25 min heat shock, using a luminescence flash assay Figure 1. The results of this automated assay shown in Figure 2 revealed a stepwise increase in activity over 90 min that ultimately led to a &gt;80% recovery in WT cells. The hsp104&Delta; strain recovered 19% of the original activity over the same time frame. Moreover, the extent of initi...

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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.

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 ...

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10.2K 조회수.  University of Texas Medical School. 이 문서에서는 조사 반딧불 루시 페라 제 - GFP 융합 단백질의 사용을 설명합니다 생체 내의 단백질 폴딩 사카 cerevisiae의. 모델 열 변성 된 단백질의 접힘이 시약을 사용하면 형광 현미경 및 단백질 품질 관리 proteostasis 네트워크 구성 요소의 역할을 조사하는 효소 분석에 의해 동시에 모니터링 할 수 있습니다.

비디오: Coupled Assays for Monitoring Protein Refolding 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 ...

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 ...

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the correct amount of protein is synthesized for the cell&#8217;s specific growth conditions. One approach for measuring mRNA decay rates is inhibiting transcription and subsequently monitoring the disappearance of the already present mRNA. The rate of mRNA decay can then be quantified, and an accurate half-life can be determined utilizing several techniques. In S. cerevisiae, protocols that measure mRNA half-lives have been developed and include inhibiting transcription of mRNA using strains that harbor a temperature sensitive allele of RNA polymerase II, rpb1-1. Other techniques for measuring mRNA half-lives include inhibiting transcription with transcriptional inhibitors such as thiolutin or 1,10-phenanthroline, or alternatively, by utilizing mRNAs that are under the control of a regulatable promoter such as the galactose inducible promoter and the TET-off system. Here, we describe measurement of S. cerevisiae mRNA decay rates using the temperature sensitive allele of RNA polymerase II. This technique can be used to measure mRNA decay rates of individual mRNAs or genome-wide.

Measurement of mRNA Decay Rates in Saccharomyces cerevisiae Using rpb1-1 Strains

The steady state level of specific mRNAs is determined by the rate of synthesis and decay of the mRNA. Genome-wide mRNA degradation rates or the decay rates of specific mRNAs can be measured by determining mRNA half-lives. This protocol focuses on measurement of mRNA decay rates in Saccharomyces cerevisiae.

mRNA steady state levels vary depending on environmental conditions. Regulation of the steady state accumulation levels of an mRNA ensures that the ...

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. ...

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.

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.