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 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.
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.
1. Growth and Harvest of Yeast Cells
2. Cycloheximide Chase
3. Post-alkaline Protein Extraction (Modified from 16,40)
4. Representative SDS-PAGE and Western Blotting Procedure (Adapted from 16)
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, cytosolic protease 45. The Deg1-Sec62 protein is targeted for degradation after it persistently and aberrantly engages the translocon, a channel that facilitates protein translocation into the ER lumen or membrane 43. Similarly, mammalian apolipoprotein B, a protein component of low-density lipoproteins, persistently engages the ER translocon and subsequently undergoes degradation by a related mechanism when its lipid binding partners are absent 46-48. Thus, analyses of Deg1-Sec62 degradation in yeast cells may yield insight into the degradation of proteins that persistently or aberrantly engage the translocon, provisionally termed ERAD-T (for translocon-associated) substrates 43.
Previous studies have indicated that the ER-resident ubiquitin ligase Hrd1 functions with the ubiquitin-conjugating enzyme Ubc7 to catalyze degradation of Deg1-Sec62 16,42-44. The transmembrane protein Cue1 anchors Ubc7 to the ER membrane and activates the enzyme 49-51. However, a requirement for Cue1 in the degradation of Deg1-Sec62 has not been directly investigated. To test the hypothesis that Cue1, like Hrd1 and Ubc7, is required for efficient degradation of Deg1-Sec62, wild-type and mutant yeast cells expressing Deg1-Sec62 were subjected to cycloheximide chase and western blot analysis (Figure 2A). The Deg1-Sec62 protein migrates on SDS-PAGE as multiple species. This is consistent with earlier studies indicating extensive post-translational modification of the protein 43,44,52. In wild-type yeast cells, Deg1-Sec62 was readily degraded. As previously observed, loss of either Hrd1 or Ubc7 substantially stabilized the Deg1-Sec62 protein 42-44. As predicted, Deg1-Sec62 was stabilized in the absence of Cue1 (to a similar extent as in the absence of Ubc7), confirming a role for the Ubc7-interacting protein in ERAD-T.
The proportion of Deg1-Sec62 protein remaining at each time point was quantified and is plotted in Figure 2B. We note that the apparent rate of disappearance of Deg1-Sec62 in wild-type cells may be an underestimation of the rate of degradation of nascent Deg1-Sec62 (i.e., the protein's half-life, which can be most directly determined by pulse chase analysis 43). Because wild-type yeast actively degrade the Deg1-Sec62 protein prior to cycloheximide addition, steady state abundance of Deg1-Sec62 is substantially reduced in these cells (compared to cells in which degradation is impaired). This can be appreciated by comparing the abundance of Deg1-Sec62 at the initial time points for each strain in Figure 2A. Further, any degradation-resistant subpopulation of the substrate protein (e.g., if the protein accumulates in intracellular pools from which degradation is prevented) might appear relatively stable in wild-type cells over the time course of the experiment.
Figure 1. Model for Degradation of Deg1-Sec62 following Aberrant Engagement of Translocon. (A) Schematic depiction of Deg1-Sec62. Deg1-Sec62 consists of the following elements, in sequence: Deg1 (the amino-terminal 67 amino acids from the yeast transcriptional repressor MATα2), a Flag (F) epitope, the 2-transmembrane protein Sec62, and two copies of the Staphylococcus aureus Protein A (PrA). (B) Following normal co-translational insertion of its two transmembrane segments into the endoplasmic reticulum (ER) membrane, persistent association of Deg1-Sec62 with the translocon triggers abnormal, Deg1-dependent, post-translational translocon engagement. A portion of the cytosolic amino terminus aberrantly enters-and likely remains within-the translocon. (C) Following translocon engagement, the Hrd1 ubiquitin ligase ubiquitylates Deg1-Sec62. Hrd1 is assisted by the ubiquitin-conjugating enzyme Ubc7, which is anchored in the ER membrane by Cue1. Ub, ubiquitin protein monomers. (D) Ubiquitylated Deg1-Sec62 is extracted from the ER membrane and degraded by the proteasome, relieving translocon obstruction. Please click here to view a larger version of this figure.
Figure 2. Cycloheximide Chase followed by Western Blotting Indicates that Cue1 is Required for Efficient Deg1-Sec62 Degradation. (A) Yeast cells of the indicated genotypes were transformed with a yeast centromeric plasmid encoding Deg1-Sec62 (pRS416-PMET25–Deg1-Flag-Sec62-2xProtA; AmpR/URA343) and subjected to cycloheximide chase analysis. Proteins from each time point (equivalent to 0.125 OD600 units) were separated on an 8% SDS-PAGE gel and detected by western blotting with rabbit anti-mouse secondary antibodies, which directly bind the C-terminal Protein A epitopes of the Deg1-Sec62 fusion protein. As a loading control, the membrane was subsequently incubated with antibodies specific to Pgk1. This experiment has been replicated three times; representative results are pictured. (B) Quantification of data in (A) was performed using imaging software (see Table of Materials). The total fluorescence intensity of an area encompassing the Deg1-Sec62 protein was determined for each sample. Background fluorescence intensity for each sample was extrapolated from the average fluorescence intensity of pixels near the Deg1-Sec62 protein. This extrapolated background fluorescence intensity was subtracted from the total fluorescence intensity to yield adjusted fluorescence intensity for each sample. Similar quantifications for total, background, and adjusted fluorescence intensities were performed for Pgk1, a loading control protein whose abundance does not vary in the conditions assayed. To compare the abundance of Deg1-Sec62 protein among samples, a ratio of the adjusted signal intensities of Deg1-Sec62 to Pgk1 was determined for each sample. The Deg1-Sec62/Pgk1 ratio was defined as 100% for the first time point (i.e., immediately after cycloheximide addition) for each strain under analysis. The amount of Deg1-Sec62 remaining at subsequent time points (i.e., the Deg1-Sec62/Pgk1 ratios) was calculated as a percentage of the Deg1-Sec62/Pgk1 ratio at the first time point. Please click here to view a larger version of this figure.
Synthetic Defined (SD) Minimal Yeast Medium | 2% dextrose, 0.67% yeast nitrogen base without amino acids, 0.002% adenine, 0.004% uracil, 0.002% arginine, 0.001% histidine, 0.006% isoleucine, 0.006% leucine, 0.004% lysine, 0.001% methionine, 0.006% phenylalanine, 0.005% threonine, 0.004% tryptophan. For solid (plate) medium, include 2% agar. | 1. Selective medium is prepared by omitting appropriate amino acid(s) or nitrogenous bases. |
2. For convenience, these ingredients may be maintained as concentrated stock solutions as follows. Amino acids may be maintained as 100x stock solution containing all desired amino acids. Yeast nitrogen base may be maintained in a 20x stock solution (13.4%). Dextrose may be maintained in a 40% stock solution. Adenine and uracil may be maintained as 1% stock solutions in 0.1 M NaOH. | ||
3. Sterilize medium by autoclaving. | ||
Yeast Extract-Peptone Dextrose (YPD) Rich Yeast Medium | 1% yeast extract, 2% peptone, 2% dextrose (Optional: 0.002% adenine). For solid (plate) medium, include 2% agar. | 1. To prevent the slow growth and red coloration characteristic of particular laboratory yeast strains in the absence (or low abundance) of adenine, adenine may be added to YPD growth medium. |
2. For convenience, some ingredients may be maintained as concentrated stock solutions as follows. Dextrose may be maintained in a 40% stock solution. Adenine may be maintained as a 1% stock solution in 0.1 M NaOH. | ||
3. Sterilize medium by autoclaving. | ||
20x Stop Mix | 200 mM sodium azide, 5 mg/ml bovine serum albumin | Sodium azide 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. |
20 mg/ml Cycloheximide | 1. Prepare in ethanol. Store at -20 °C. | |
2. 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. | ||
0.2 M Sodium Hydroxide | Prepare in water. Sodium hydroxide reacts with glass. Therefore, for long-term storage, 0.2 M sodium hydroxide should be maintained in plastic containers. | |
Laemmli Sample Buffer | 2% SDS, 10% glycerol, 5% β-mercaptoethanol, 60 mM Tris HCl pH 6.8, 0.008% bromophenol blue | 1. Laemmli Sample Buffer is often prepared by diluting a more concentrated (e.g., 5x) stock. |
2. The dye bromophenol blue may be added to desired intensity. A "pinch" (very small amount tapped from the edge of a spatula) is typically sufficient. | ||
Laemmli Running Buffer (5x) | 125 mM Tris, 960 mM glycine, 0.5% SDS | To prepare 1 L of 1x Laemmli Running Buffer, dilute 1:5 in dH2O. |
Tris Acetate-SDS Transfer Buffer (5x) | 125 mM Tris acetate (pH 8.8), 960 mM glycine, 0.05% SDS | To prepare 20 L of 1x Tris Acetate-SDS Transfer Buffer, combine 4 L of 5x stock, 4 L of methanol, and 12 L of dH2O. |
10x Tris-Buffered Saline (TBS) | 500 mM Tris, 1.5 M NaCl; pH adjusted to 7.5 | To prepare 1 L of 1x TBS, dilute 1:10 in dH2O. 1x TBS may be supplemented with the detergent Tween-20 and powdered skim milk, as appropriate. |
Table 1. Solutions Used in this Study.
Strain Name | Alias | Relevant Genotype | Referenzen |
VJY42 | MHY501 | MATα | Chen et al., 1993 |
his3-Δ200 | |||
leu2-3,112 | |||
ura3-52 | |||
lys2-801 | |||
trp1-1 | |||
gal2 | |||
VJY47 | YWO55; MHY507 | MATα | Jungmann et al., 1993; Rubenstein et al., 2012 |
his3-Δ200 | |||
leu2-3,112 | |||
ura3-52 | |||
lys2-801 | |||
trp1-1 | |||
gal2 | |||
ubc7Δ::LEU2 | |||
VJY56 | YTX105; MHY1364 | MATα | Biederer et al., 1997; Ravid et al., 2006 |
his3-Δ200 | |||
leu2-3,112 | |||
ura3-52 | |||
lys2-801 | |||
trp1-1 | |||
gal2 | |||
cue1Δ::HIS3 | |||
VJY172 | MHY6199 | MATα | This study |
his3-Δ200 | |||
leu2-3,112 | |||
ura3-52 | |||
lys2-801 | |||
trp1-1 | |||
gal2 | |||
hrd1Δ::kanMX4 |
Table 2. Yeast Strains Used in this Study. All strains are congenic with MHY500 53. VJY42, VJY47, and VJY56 were described previously 49,53,54. A detailed strain generation and confirmation strategy for VJY172 (prepared for this study) is available upon request.
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. In such experiments, nascent proteins are briefly pulse-labeled (e.g., with radioactive amino acids). Changes in the abundance of the labeled nascent proteins (which may or may not mirror changes in steady state protein abundance) may be tracked over time.
Cycloheximide chase is suitable for analyzing protein stability over a relatively short time course (i.e., up to two hours). Over longer time courses (i.e., two hours to days), cycloheximide, a global inhibitor of translation, is toxic to cells, likely due to depletion of ubiquitin 56. Further, analyses of protein stability over longer time courses are more likely to be compromised by indirect effects of globally impaired protein synthesis on the degradation of the protein of interest (e.g., degradation of a short-lived protein involved in the degradation of the protein of interest). Other techniques, such as pulse chase metabolic labeling experiments, are therefore better suited for studying the degradation of long-lived proteins and may be performed to corroborate results obtained in cycloheximide chase experiments.
The timing of addition of cycloheximide and collection of cells is an important consideration in this procedure. Precision is especially important in the analysis of very short-lived proteins, as small deviations in the time elapsed from cycloheximide addition to cell harvest can have a substantial impact on apparent degradation kinetics. Further, without a clear plan for executing these time-sensitive steps, an experiment may rapidly devolve into chaos and frustration. For this reason, it is recommended to add cycloheximide to cultures at planned, regular intervals. 30-sec intervals are suggested in this protocol, but the timing may be adjusted to match the comfort level and dexterity of the investigator. Novice investigators may find it useful to write a schedule of sample collection times prior to initiating the experiment and to cross off each scheduled collection as it occurs.
In Steps 1.7 and 1.8 of the protocol described here, yeast cell cultures are concentrated in reduced volumes of growth medium for ease of manipulation during the subsequent cycloheximide chase procedure. However, centrifugation of yeast cells rapidly induces certain cellular stress responses (e.g., signaling pathways that are activated by glucose deprivation) 57,58. Investigators are therefore cautioned against extended periods of centrifugation. When analyzing the stability of proteins that may be sensitive to centrifugation, investigators may wish to add cycloheximide directly to mid-log phase cultures without prior centrifugation. In such cases, cycloheximide should be added to the same final concentration (250 μg/ml), and yeast cell samples may be harvested by methodologies that do not require an initial centrifugation step (e.g., rapid filtration) 57. Further, in Steps 2.3 – 2.5, samples collected at each time point are transferred to a solution containing sodium azide and placed on ice. These conditions inhibit continued protein degradation for the duration of the chase. It should be noted that azide reduces the cellular concentration of ATP 59, thereby specifically inhibiting the activity of ATP-dependent proteases. While reduced temperature should also reduce the rate at which non-ATP-dependent proteolytic processes function, investigators studying proteins degraded by such mechanisms may alternatively choose to snap-freeze cell pellets in liquid nitrogen as each sample is harvested.
A sample protocol for cell lysis and a representative protocol for western blotting included in this manuscript have been adapted from previous reports 16,40. In the post-alkaline lysis method described here, NaOH pretreatment enhances extraction of yeast proteins upon subsequent addition of Laemmli sample buffer. The mechanism by which this enhancement occurs is poorly characterized 40. However, polysaccharides such as those found in yeast cell walls are solubilized in alkali conditions 60. It is therefore tempting to speculate that incubation in the presence of NaOH partially or completely spheroplasts yeast cells (i.e., removes cell wall components), rendering them more sensitive to the SDS detergent present in the Laemmli sample buffer. Because proteins are released under highly denaturing conditions, protease inhibitors need not be included in the Laemmli sample buffer. The specific method of cell lysis may be modified as appropriate for a given experiment. For instance, the post-alkaline lysis method may not be suitable for low-abundance proteins or proteins that are poorly detected by western blot. In such cases, methods that include a trichloroacetic acid precipitation step to concentrate protein samples may be most appropriate 61. Moreover, several important considerations regarding antibody selection, loading control choice, and alternative means of detection (e.g., chemiluminescent western blotting using film or digital imaging systems) have been recently discussed 16. Quantitation of protein abundance following cycloheximide treatment may be performed. Many imaging systems are sold with software packages that facilitate this analysis.
Cycloheximide chase may be implemented to analyze the degradation of a variety of yeast proteins and may be adapted to study protein stability in other eukaryotic cells. As described in this protocol, cycloheximide treatment is followed by cell lysis and detection of protein abundance by western blot analysis. Depending on the application, however, protein abundance following cycloheximide treatment may be assessed by a range of techniques, as appropriate for research objectives. For example, if protein size is not a relevant factor for analysis, cell lysates may be subjected to dot blot or enzyme-linked immunosorbent assay (ELISA) analysis, which report on protein abundance, but not apparent molecular weight 62. For proteins on the cell surface (or internal fluorescently tagged internal proteins), flow cytometry may be used to quantify protein abundance of samples at different times after cycloheximide addition 51. Fluorescence microscopy following cycloheximide treatment would provide information on both protein abundance and localization 18. The range of substrates, organisms, and downstream applications amenable to cycloheximide chase makes the technique a highly versatile and informative means of studying protein degradation.
The authors have nothing to disclose.
The authors thank current and former members of the Rubenstein lab for providing a supportive and enthusiastic research environment. The authors thank Mark Hochstrasser (Yale University) for sharing reagents and expertise. E.M.R. thanks Stefan Kreft (University of Konstanz) and Jennifer Bruns (University of Pittsburgh) for sharing invaluable expertise in kinetic analysis of proteins. This work was supported by a National Institutes of Health grant (R15 GM111713) to E.M.R., a Ball State University ASPiRE research award to E.M.R, a research award from the Ball State University chapter of Sigma Xi to S.M.E., and funds from the Ball State University Provost’s Office and Department of Biology.
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 |