Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

The overall goal of this procedure is to determine the variations in protein degradation rates in a systematic fashion by coupling them to the kinetics of yeast growth in liquid culture. This is accomplished by first growing cells expressing a reporter protein comprised of a degradation signal coupled to a metabolic marker overnight to stationary phase in a suitable selective medium. Next, the optical density of the cells is measured to determine the growth kinetics of the cells, and then the minimal doubling time of the cells during the log phase is calculated from the growth kinetics using a designated software program.

Ultimately, the calculated minimal doubling time can be used to define variations in the protein degradation kinetics. The main advantages of this technique over existing methods like cyclamate or pulse chase degradation assays added our technique is simple to set up. The data acquisition and analysis are straightforward, and the essay design is extremely modular.

This method can help answer key questions in the pot degradation field, such as which elements required for misfolded pot recognition by the ubiquitin pot system. I first had the idea for this method when I wanted to do a screen to identify the proteins involving ubiquitin removal from their subset. So I established a simple way to compare multiple samples To prepare the cells for the analysis.

Begin by transforming the appropriate ox atrophic yeast cells such as TRY 4 67 with a plasmid containing a U three Deron fusion and an additional metabolic marker for plasmid selection and maintenance. For plasmid selection and maintenance, grow the cells on agar plates under a suitable synthetic defined medium, lacking amino acid selection markers, transfer surviving colonies into patches, transfer cells from patches into liquid media and incubate overnight at 30 degrees Celsius until a stationary phase is reached. Then for 15 samples or less, dilute 50 microliters of the cells from the overnight culture with 150 microliters of synthetic defined medium per well.

In a clear bottom 96 well plate, use a microplate reader to determine the OD 600 values of the samples. Then after calculating the exact volume needed for obtaining yeast cells at an amount equivalent to an OD 600 of 0.25, spin down the cells for one minute at 12, 000 times G and room temperature resuspend the cells in one milliliter of the appropriate selective medium to obtain an OD 600 of 0.25 and transfer 200 microliters of the diluted strain into one well of a new 96 well plate when greater than 15 samples are to be tested. Transfer 10 microliters of the overnight grown stationary cells into one well of a new 96 well plate containing 190 microliters of the appropriate selective media to measure the growth kinetics of the transformed yeast cells under selective conditions.

Next, set a multi-mode microplate reader to an incubation temperature of 30 degrees Celsius OD 600 measurement intervals of 15 minutes and orbital and linear shaking cycles of one minute every seven minutes. Then incubate the cells at 30 degrees Celsius in the Microplate reader for 12 to 24 hours. When the cells have reached the stationary phase, export the raw data into a spreadsheet file to determine the yeast growth kinetics using MDT Calc.

Make sure the spreadsheet file containing the growth data is saved and closed. Then open the MDT Calc application program under file path. Click on the rectangle to locate the spreadsheet file under sheet name.

Enter the spreadsheet name where the raw data is located under starting position. Enter the spreadsheet coordinate of time, zero of the first sample under time column. Enter the letter defining the location of the time point column in seconds under blank column, enter the letter defining the location of the blank column.

For example, the A one well of the 96 well plate.Next. Under OD value, choose the minimal transformed OD 600 value required for the initiation of the MDT calculation. Usually 0.15 to 0.25 to ensure that the MDT is calculated only for the samples that reach the defined D 600 value or higher.

Once the last value has been entered, click the calculate button and confirm that the progress bar on the right gradually changes to green. Finally, export the two sets of data that appear automatically on the screen at the completion of the MDT calculation into a new spreadsheet. Confirm that the data includes the MDT value for each well that passes the minimal OD value test and the start time of the interval from which the MDT was calculated in this representative experiment.

The relative differences in the kinetics of protein degradation between the wild type and various mutant strains were compared initially wild type U BBC six, delta UBC seven delta, or DOA 10 delta cells expressing D one VMAU three were grown on synthetic defined complete medium. Then to measure the gills, the cells were washed and incubated in synthetic defined media. Minus uracil similar growth curves and calculated MDT were observed for all strains grown in synthetic defined complete medium, excluding the possibility that the deletion of any of the examined genes affect cell growth.

In contrast, incubation in synthetic defined media minus uracil resulted in poor growth of wild type cells, whereas only a minor growth defect was observed in the mutant strains. UBC seven is absolutely required for DEG one VMAU three degradation enabling an accurate assessment of even the partial effects of various E two mutants. Accordingly, fast growth kinetics are observed in cells expressing UBC seven with the active site mutants, C 89 s and N 81 A.In addition, two mutants predicted to indirectly hinder DEG one VMA ETH three degradation V 20 5G and H 90 4K were also found to enhance cell growth compared to wild type UBC seven, albeit to a lesser degree than the active site mutants.

Thus, the gills method can be used for accurate measurement of the relative contribution of various degradation factors to the stability of a reporter substrate. While attempting this procedure, it is important to remember that the protocol is sensitive to small differences in protein degradation rates. This is an advantage, but it also mean that care should be taken in the design and verification of the Deron to be used Following this procedure.

Other methods like high throughput fluorescence microscopy can be performed to answer additional questions such as what is the aggregation propensity of reporter proteins. For this reason, it is a good idea to also include a GFP tag in the reporter. After watching this video, you should have a good understanding of how to determine the changes in protein degradation rate in a systematic fashion by using a MICROPLATE reader and our our designated software program.