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

Summary

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.

Abstract

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.

Introduction

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 fibrosis^1-5. The ubiquitin-proteasome system (UPS), which catalyzes selective protein degradation, is an emerging therapeutic target for these conditions^6-10. 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 MATα2^13,14, 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.

Protocol

1. Yeast Growth Assay to Identify Candidate Mutants Defective in Protein Degradation

1. Transform wild-type and mutant yeast cells with a plasmid encoding an unstable protein fused, in frame, to a reporter metabolic enzyme.
2. Inoculate transformants in 5 ml of synthetic defined (SD) minimal medium that is selective for cells harboring plasmid molecules. Incubate overnight at 30 °C, rotating.
3. 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.
4. 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.
   1. 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 µl. 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 µl.
   2. For each row of yeast, add 125 µl 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.
   3. Mix the contents of the first column (yeast diluted to an OD₆₀₀ of 0.2) by pipetting up and down with a multichannel pipettor.
   4. Transfer 25 µl of yeast from Column 1 to Column 2, using a multichannel pipettor. Mix by pipetting up and down. Transfer 25 µl from Column 2 to Column 3, and 25 µl from Column 3 to Column 4 (mixing well at each step).
5. Mix each sample with a multichannel pipettor. Proceeding from most dilute to least dilute columns of yeast, pipette 4 µl 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 – 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.
6. Allow plates to dry on the bench top.
7. Incubate plates at 30 °C for 2 – 6 days.
8. Photograph each plate after incubation.

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. Please click here to view a printable version of this figure with 100-mm templates.

2. Biochemical Confirmation of Yeast Growth Assay

1. Growth of Yeast Cells and Post-Alkaline Protein Extraction (modified from ³¹)
   1. Transform wild-type and mutant yeast cells with a plasmid encoding the unstable protein.
   2. Inoculate transformants in 5 ml of SD medium that is selective for cells harboring plasmid molecules. Incubate overnight at 30 °C, rotating.
   3. 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.
   4. Dilute yeast cells to an OD₆₀₀ of 0.2 in 10 ml fresh selective medium.
   5. Continue to incubate cells at 30 °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.
   6. 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
   7. Resuspend cells in 1 ml distilled water. Transfer suspended cells to a microcentrifuge tube.
   8. Pellet cells by centrifugation at 6,500 x g for 30 sec at room temperature. Remove supernatant by pipetting or aspiration.
   9. Resuspend cells in 100 µl distilled water by pipetting up and down or vortexing, and add 100 µl 0.2 M NaOH. Mix by pipetting up and down. Incubate samples for 5 min at room temperature.
   10. 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.
   11. Resuspend pellet in 50 – 100 µl 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.
   12. To fully denature proteins, incubate lysates at 95 °C for 5 min.
       NOTE: Aggregation-prone proteins (e.g. proteins with several transmembrane segments) may become insoluble when incubated at 95 °C. Therefore, lysates should be incubated at lower temperatures (e.g. 37 °C – 70 °C) for 10 – 30 min, as empirically determined, for the analysis of such proteins.
   13. Cool lysates by placing on ice for 5 min.
   14. 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 °C.
2. Representative Western Blotting Protocol
   1. Load empirically determined volume of lysates in an SDS-PAGE gel.
   2. Run gel at 200 V until dye front has reached the bottom of the gel.
   3. Transfer proteins from gel to polyvinylidene fluoride (PVDF) membrane by wet transfer at 20 V for 60 – 90 min at 4 °C.
   4. Block membrane by incubating in 5% skim milk in Tris-Buffered Saline (TBS), rocking, for 1 hr at room temperature or overnight at 4 °C.
   5. Decant blocking solution.
   6. 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.
   7. Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.
   8. 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.
   9. Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.
   10. Acquire image of membrane using Li-Cor Odyssey CLx and Image Studio software (or comparable imaging equipment and software), according to manufacturer recommendations.
   11. 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.
   12. Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.
   13. Incubate membrane with appropriate fluorophore-conjugated secondary antibody in 1% skim milk in TBS/T for 1 hr at room temperature, rocking.
   14. Decant antibody solution, and wash membrane 3 x 5 min with TBS/T at room temperature, rocking.
   15. Acquire image of membrane using Li-Cor Odyssey CLx and Image Studio software (or comparable imaging equipment and software), according to manufacturer recommendations.

Results

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

Discussion

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...

Materials

List of materials used in this article

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