The overall goal of this procedure is to visualize the degradation of the steady state population of a specific protein in Saccharomyces cerevisiae. This method can be used to determine the genetic requirements for, and environmental effects on, the degradation of a protein of interest. The main advantage of this technique is that radioactive isotopes and lengthy immunoprecipitation steps are not required, unlike pulse-chase techniques, which are also used to visualize protein degradation.
This procedure can be used to analyze either an endogenous yeast protein or a protein expressed from a plasmid. For the latter, the yeast strain is transformed with a plasmid encoding the protein of interest following a standard yeast transformation protocol. Inoculate the yeast in five milliliters of appropriate medium.
Incubate overnight at 30 degrees Celsius with rotation. On the following morning, measure the optical density at 600 nanometers, or OD600, of each overnight culture. Dilute the cultures to an OD600 of 0.2 in 15 milliliters of fresh medium.
Incubate at 30 degrees Celsius with shaking until the cells reach mid-logarithmic growth phase. While the yeast cells are growing, prepare for the cycloheximide chase procedure. Set a heat block that can accommodate 15 milliliter conical tubes to 30 degrees Celsius for the incubation of cells in the presence of cycloheximide.
To ensure efficient heat distribution to the cultures, add water to each well of the heat block such that a 15 milliliter conical tube will cause the water level to rise to, but not overflow, the lip of the well. Set a second heat block that can accommodate 1.5 milliliter microcentrifuge tubes to 95 degrees Celsius for protein denaturation following cell lysis. Pre-warm fresh growth medium to 30 degrees Celsius.
1.1 milliliters of medium is needed per time point per culture to be assayed. Add 50 microliters of 20x Stop Mix to pre-labeled microcentrifuge tubes. Prepare one tube per time point per culture to be assayed.
Place the tubes on ice. When the yeast cells have reached mid-logarithmic growth, collect 2.5 OD600 units of each culture per time point to be assayed. One OD600 unit is equal to the amount of yeast present in one milliliter of culture at an OD600 of 1.0.
Centrifuge the collected cells in 15 milliliter conical tubes at 3, 000 times g at room temperature for two minutes. Remove the supernatant. Re-suspend each cell pellet in one milliliter of 30 degree Celsius fresh growth medium per 2.5 OD600 units of cells.
Prior to starting the cycloheximide chase, equilibrate the yeast cell suspensions by incubating in the 30 degree Celsius heat block for five minutes. The most difficult aspect of this procedure is the timing of cycloheximide addition and collection of cells for each sample. We ensure success by establishing and adhering to a time table for addition of cycloheximide and collection of cells.
To begin the cycloheximide chase, press Start"on the timer. Swiftly but carefully add cycloheximide to a final concentration of 250 micrograms per milliliter to the first yeast cell suspension and vortex briefly to mix. Immediately transfer 950 microliters, or approximately 2.4 OD600 units, of the yeast cell suspension with added cycloheximide to a pre-labeled microcentrifuge tube containing 50 microliters of ice-cold 20x Stop Mix.
Vortex the microcentrifuge tube and place on ice until all samples have been collected. Begin the cycloheximide chase as demonstrated for each of the remaining yeast cell suspensions at regular time intervals. At each subsequent time point, vortex the yeast cell suspensions and transfer 950 microliters to labeled microcentrifuge tubes containing 50 microliters of pre-chilled 20x Stop Mix.
Vortex and place the collected cells on ice. To prevent settling of the yeast cells, vortex the cell suspensions in the 15 milliliter conical tubes approximately every five minutes throughout the course of the chase. When all samples have been collected, pellet the collected cells by centrifugation at 6, 500 times g and room temperature for 30 seconds.
Remove the supernatant by pipetting or aspiration. The cells are now ready for alkaline lysis. Prepare the cells for alkaline lysis by adding 100 microliters of distilled water to each cell pellet, and re-suspend by pipetting up and down.
Add 100 microliters of 0.2 molar sodium hydroxide to each sample. Mix by vortexing. Incubate the cells at room temperature for five minutes.
At this stage, the yeast cells have not been lysed, and proteins have not been released. Next, pellet the cells by centrifugation at 18, 000 times g at room temperature for 30 seconds. Remove the supernatant by pipetting or aspiration.
To lyse the cells, add 100 microliters of Laemmli Sample Buffer to each cell pellet. Re-suspend by pipetting up and down. Incubate at 95 degrees Celsius for five minutes to fully denature the proteins.
Centrifuge the lysates at 18, 000 times g and room temperature for one minute to pellet insoluble material. The supernatant, which is solubilized extracted protein, is ready for separation by SDS PAGE and subsequent Western blot analysis. Alternatively, the lysates can be stored at negative 20 degrees Celsius.
The stability of Deg1-Sec62, a model yeast endoplasmic reticulum-associated degradation substrate, was analyzed by the cycloheximide chase methodology. The Deg1-Sec62 protein migrates on SDS PAGE as multiple species and is readily degraded in wild-type cells. Pgk1 is a loading control whose abundance does not vary in the conditions assayed.
Loss of either the ER resident ubiquitin ligase hrd1 or the ubiquitin-conjugating enzyme ubc7 substantially stabilized the Deg1-Sec62 protein, which confirms the previously observed role of these proteins in Deg1-Sec62 degradation. Cue1 is a transmembrane protein that anchors ubc7 to the ER membrane and activates the enzyme, but a requirement for cue1 in Deg1-Sec62 degradation has not been directly investigated. The observation from this study that Deg1-Sec62 was stabilized in the absence of cue1, confirmed a role for cue1 in the ER associated degradation of Deg1-Sec62.
The result from the Western blot was quantified using imaging software. To compare the abundance of Deg1-Sec62 protein amongst samples, a ratio of the adjusted signal intensities of Deg1-Sec62 to Pgk1 was determined for each sample. It's important to remember that this procedure most directly reports on the degradation kinetics of a steady state population of a protein of interest.
To visualize the degradation of nascent population of a protein of interest, other techniques, such as pulse-chase experiments, can be used. Don't forget that cycloheximide and sodium azide are extremely toxic and precaution should be taken to avoid direct contact with these chemicals while performing this procedure.
