Cycloheximide Chase Analysis of Protein Degradation in Saccharomyces cerevisiae

Podsumowanie

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).

Streszczenie

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.

Wprowadzenie

Proteins perform crucial functions in virtually every cellular process. Many physiological processes require the presence of a specific protein (or proteins) for a defined period of time or under particular circumstances. Organisms therefore monitor and regulate protein abundance to meet cellular needs ¹. For example, cyclins (proteins that control cell division) are present at specific phases of the cell cycle, and the loss of regulated cyclin levels has been associated with malignant tumor formation ². In addition to regulating protein levels to meet cellular needs, cells employ degradative quality control mechanisms to eliminate misfolded, unassembled, or otherwise aberrant protein molecules ³. Control of protein abundance involves regulation of both macromolecular synthesis (transcription and translation) and degradation (RNA decay and proteolysis). Impaired or excessive protein degradation contributes to multiple pathologies, including cancer, cystic fibrosis, neurodegenerative conditions, and cardiovascular disorders ^4-8. Proteolytic mechanisms therefore represent promising therapeutic targets for a range of illnesses ^9-12.

Analysis of proteins at a single time point (e.g., by western blot ¹³, flow cytometry ¹⁴, or fluorescence microscopy ¹⁵) provides a snapshot of steady state protein abundance without revealing the relative contributions of synthesis or degradation. Similarly, growth-based reporter assays reflect steady state protein levels over an extended time period without discriminating between the influences of synthesis and degradation ^15-20. It is possible to infer the contribution of degradative processes to steady state protein levels by comparing abundance before and after inhibiting specific components of the degradative mechanism (e.g., by pharmacologically inactivating the proteasome ²¹ or knocking out a gene hypothesized to be required for degradation ¹³). A change in steady state protein levels after inhibiting degradative pathways provides strong evidence for the contribution of proteolysis to the control of protein abundance ¹³. However, such an analysis still does not provide information regarding the kinetics of protein turnover. Cycloheximide chase followed by western blotting overcomes these weaknesses by allowing researchers to visualize protein degradation over time ^22-24. Further, because protein detection following cycloheximide chase is typically performed by western blotting, radioactive isotopes and lengthy immunoprecipitation steps are not required for cycloheximide chase, unlike many commonly used pulse chase techniques, which are also performed to visualize protein degradation²⁵.

Cycloheximide was first identified as a compound with anti-fungal properties produced by the gram-positive bacterium Streptomyces griseus ^26,27. It is a cell-permeable molecule that specifically inhibits eukaryotic cytosolic (but not organellar) translation by impairing ribosomal translocation ^28-31. In a cycloheximide chase experiment, cycloheximide is added to cells, and aliquots of cells are collected immediately and at specific time points following addition of the compound ²². Cells are lysed, and protein abundance at each time point is analyzed, typically by western blot. Decreases in protein abundance following the addition of cycloheximide can be confidently attributed to protein degradation. An unstable protein will decrease in abundance over time, while a relatively stable protein will exhibit little change in abundance.

Mechanisms of selective protein degradation have been highly conserved throughout Eukarya. Much of what is known about protein degradation was first learned in the model unicellular eukaryote, Saccharomyces cerevisiae (budding yeast) ^25,32-36. Studies with yeast are likely to continue providing novel and important insights into protein degradation. A method for cycloheximide chase in yeast cells followed by western blot analysis of protein abundance is presented here.

Protokół

1. Growth and Harvest of Yeast Cells

1. If not analyzing degradation kinetics of an endogenous yeast protein, transform desired yeast strain(s) with a plasmid encoding the protein of interest. Reliable methods for yeast transformation have been previously described ³⁷.
2. Inoculate yeast in 5 ml of appropriate medium (e.g., selective synthetic defined (SD) medium for plasmid maintenance of transformed cells or non-selective yeast extract-peptone-dextrose (YPD) medium for non-transformed cells). Incubate overnight at 30 °C, rotating.
   NOTE: 30 °C is the optimal growth temperature for typical wild-type laboratory yeast strains ³⁸. However, because some mutant yeast strains do not grow optimally at 30 °C and some proteins undergo temperature-dependent degradation, the temperatures used for yeast cell growth and cycloheximide chase should be empirically determined ³⁹.
3. Measure the optical density at 600 nm (OD₆₀₀) of each overnight culture.
   NOTE: Cells may be in logarithmic or stationary growth phase but should have minimally reached a density that will allow dilution to an OD₆₀₀ = 0.2 in 15 ml of fresh medium.
4. Dilute the overnight cultures to an OD₆₀₀ value of 0.2 in 15 ml of fresh medium.
5. Incubate yeast at 30 °C, shaking until the cells reach mid-logarithmic growth phase (i.e., an OD₆₀₀ between 0.8 and 1.2).
6. During yeast cell growth, perform the following in preparation for the cycloheximide chase procedure:
   1. Set a heat block that can accommodate 15 ml conical tubes to 30 °C for incubation of cells in the presence of cycloheximide. Add water to the wells of the heat block to allow efficient heat distribution to cultures. Add water to each well such that a 15 ml conical tube will cause the water level to rise to, but not overflow, the lip of the well.
   2. Set a second heat block that can accommodate 1.5 ml microcentrifuge tubes to 95 °C for protein denaturation following cell lysis.
   3. Pre-warm fresh growth medium (1.1 ml per time point per culture to be assayed) to 30 °C.
   4. Add 50 µl 20x Stop Mix to pre-labeled microcentrifuge tubes (one tube per time point per culture to be assayed). Place tubes on ice.
      CAUTION: Sodium azide, an ingredient in 20x Stop Mix, is toxic via oral ingestion or dermal contact. Follow manufacturer recommendations when preparing, storing, and handling sodium azide. In the event of accidental exposure, consult material safety data sheet provided by manufacturer.
7. When cells have reached mid-logarithmic growth, collect 2.5 OD₆₀₀ units of each culture per time point to be assayed (e.g., 7.5 OD₆₀₀ units to analyze protein abundance at three time points). Centrifuge collected cells in 15 ml conical tubes at 3,000 x g at room temperature for 2 min.
   NOTE: One OD₆₀₀ unit is equal to the amount of yeast present in 1 ml culture at an OD₆₀₀ of 1.0. The volume of culture (in ml) required to harvest X OD₆₀₀ units (V) may be determined by using the following equation: V = X OD₆₀₀ units/Measured OD₆₀₀. For example, to harvest 7.5 OD₆₀₀ units of yeast cell culture at an OD₆₀₀ of 1.0, collect 7.5 OD₆₀₀ units/1.0 = 7.5 ml yeast culture.
8. Resuspend each cell pellet in 1 ml of 30 °C (pre-warmed) fresh growth medium per 2.5 OD₆₀₀ units of cells (e.g., 3 ml for 7.5 OD₆₀₀ units).

2. Cycloheximide Chase

1. Equilibrate yeast cell suspensions by incubation for 5 min in the 30 °C heat block.
2. Prepare a timer to count up from 0:00.
3. To begin the cycloheximide chase, press "Start" on the timer. Swiftly, but carefully, perform the following steps:
   1. Add cycloheximide to a final concentration of 250 µg/ml to the first yeast cell suspension (e.g., add 37.5 µl of 20 mg/ml cycloheximide stock to 3 ml of cell suspension), and vortex briefly to mix.
      CAUTION: Cycloheximide is a dermal irritant and is toxic via oral ingestion. Follow manufacturer recommendations when preparing, storing, and handling cycloheximide. In the event of accidental exposure, consult material safety data sheet provided by manufacturer.
   2. Immediately after adding cycloheximide and vortexing, transfer 950 µl (~2.4 OD₆₀₀ units) of the yeast cell suspension with added cycloheximide to a pre-labeled microcentrifuge tube containing 50 µl ice-cold 20x stop mix. Vortex the microcentrifuge tube, and place on ice until all samples have been collected.
   3. Return the yeast cell suspension to 30 °C.
4. Repeat steps 2.3.1 through 2.3.3 for each of the remaining yeast cell suspensions at regular time intervals (e.g., every 30 sec, such that cycloheximide is added to Sample #1 at 0:00, Sample #2 at 0:30, Sample #3 at 1:00, etc.).
5. At each subsequent time point, vortex yeast cell suspensions and transfer 950 μl to labeled microcentrifuge tubes containing 50 μl pre-chilled 20x Stop Mix. Vortex and place collected cells on ice. Return 15 ml conical tubes to 30 °C heat block.
   1. For example, for collection of cells 30 min after cycloheximide addition (assuming 30-sec intervals between cycloheximide addition to yeast cell suspensions at the beginning of the time course), vortex and remove 950 μl of cell suspension from Sample #1 at 30:00. Repeat for Sample #2 at 30:30, and so on.
      NOTE: To prevent settling of yeast, vortex cell suspensions in 15 ml conical tubes approximately every 5 min throughout the course of the chase. Alternatively, yeast cell suspensions may be maintained in a continuously agitating water bath for the duration of the cycloheximide chase experiment.
6. When all samples have been collected, pellet collected cells by centrifugation at 6,500 x g at room temperature for 30 sec. Remove the supernatant by pipetting or aspiration. Cells are now ready for alkaline lysis. Alternatively, snap-freeze pelleted cells in liquid nitrogen and store at -80 °C.

3. Post-alkaline Protein Extraction (Modified from ^16,40)

1. Add 100 µl of distilled water to each cell pellet. Resuspend by pipetting up and down or vortexing.
2. Add 100 µl of 0.2 M NaOH to each sample. Mix by pipetting up and down or vortexing.
3. Incubate suspended cells at room temperature for 5 min. At this stage, yeast cells have not been lysed, and proteins have not been released ⁴⁰.
4. Pellet cells by centrifugation at 18,000 x g at room temperature for 30 sec. Remove supernatant by pipetting or aspiration.
5. To lyse cells, add 100 µl of Laemmli sample buffer to each cell pellet. Resuspend by pipetting up and down or vortexing.
   NOTE: Sequential incubation of cells with NaOH and Laemmli sample buffer releases proteins in a form compatible with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a Tris-glycine running buffer system.
6. Incubate at 95 °C for 5 min to fully denature proteins.
   NOTE: Incubation at 95 °C may not be suitable for analysis of proteins that are prone to aggregation (e.g., proteins with several transmembrane segments). These proteins may become insoluble when incubated at elevated temperatures ⁴¹. Thus, for the analysis of such proteins, lysates should be incubated at lower temperatures (e.g., 37 °C - 70 °C) for 10 - 30 min, as empirically determined.
7. Centrifuge lysates at 18,000 x g at room temperature for 1 min to pellet insoluble material. The supernatant (solubilized extracted protein) is ready for separation by SDS-PAGE and subsequent western blot analysis. Alternatively, store lysates at -20 °C.

4. Representative SDS-PAGE and Western Blotting Procedure (Adapted from ¹⁶)

1. Load protein molecular weight standards and empirically determined volume of lysates onto an SDS-PAGE gel.
   NOTE: Choose the percentage of acrylamide and bis-acrylamide in the SDS-PAGE gel based on the molecular weight of the protein of interest. In general, lower percentage gels are better suited for resolving higher molecular weight proteins.
2. Run gel at 200 V until the dye front has reached the bottom edge of the gel.
3. Transfer proteins from the 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 Tris-Buffered Saline (TBS) containing 5% skim milk, rocking, at room temperature for 1 hr or at 4 °C overnight.
   NOTE: To prevent microbial growth in the presence of a membrane incubated overnight, it is recommended to include sodium azide in the blocking solution at a final concentration of 0.02%.
5. Incubate membrane with primary antibody specific for protein of interest in TBS with 0.1% Tween-20 (TBS/T) and 1% skim milk, rocking, at room temperature for 1 hr.
6. Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.
7. Incubate membrane with appropriate fluorophore-conjugated secondary antibody in TBS/T with 1% skim milk at room temperature for 1 hr, rocking.
   NOTE: Fluorophores are light-sensitive. Therefore, dilutions of antibodies conjugated to fluorophores should be prepared in the dark, and incubation of membranes in the presence of antibodies conjugated to fluorophores and subsequent wash steps should occur in lightproof (e.g., foil-wrapped) containers.
8. Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.
9. Image membrane using LI-COR Odyssey CLx and Image Studio software (or comparable imaging equipment and software), following manufacturer recommendations.
10. After acquiring membrane image, incubate the membrane with a primary antibody specific for a loading control protein in TBS/T with 1% skim milk at room temperature for 1 hr, rocking.
11. Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.
12. Incubate membrane with appropriate fluorophore-conjugated secondary antibody in TBS/T with 1% skim milk at room temperature for 1 hr, rocking.
13. Wash membrane at room temperature 3 x 5 min with TBS/T, rocking.
14. Image membrane as in step 4.9.

Wyniki

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

Dyskusje

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

Materiały

Name	Company	Catalog Number	Comments
Desired yeast strains, plasmids, standard medium and buffer components
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	A tube roller is recommended to maintain overnight  starter cultures of yeast cells in suspension. Alternatively, if a tube roller is unavailable, a platform shaker in a temperature-controlled incubator may be used for overnight starter cultures. A platform shaker or tube roller may be used to maintain larger cultures in suspension.
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
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- and 50-ml conical tubes
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 Heaters	Fisher Scientific	1172011AQ	It is recomended that two heaters are available (one for incubating cells during cycloheximide treatment and one for boiling lysates to denature proteins). Alternatively, 30 °C water bath may be used for incubation of cells in the presence of cycloheximide. Boiling water bath with hot plate may altertnatively be used to denature proteins.
Heating block for 12 x 15-ml conical tubes	Fisher Scientific	11-473-70	For use with Dri-Bath Heater during incubation of cells in the presence of cycloheximide.
Heating block for 20 x 1.5-ml conical tubes	Fisher Scientific	11-718-9Q	For use with Dri-Bath Heater to boil lysates for protein denaturation.
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