Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Summary

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

Abstract

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.

Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.

This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Introduction

The ubiquitin-proteasome degradation system is a major regulatory machine, which has been implicated in the maintenance of protein homeostasis in all eukaryotes. The UPS initially conjugates multiple ubiquitin molecules to a target protein after which the poly-ubiquitin-tagged protein is degraded by the 26S proteasome. In most cases, the rate limiting step for ubiquitin mediated degradation is substrate ubiquitylation, mediated by E2 conjugating enzymes and E3 ligating enzymes (E3 Ligases)¹. Consequently, intracellular stability of specific proteins reflects their susceptibility to ubiquitin-conjugation and the activity of their cognate ubiquitylation enzymes.

E3 ligases are the principal substrate recognition components of the UPS. As such, these enzymes recognize degrons within their substrates that are either absent or not exposed in their stable counterparts ². For example, many regulators of the cell cycle must be synthesized and degraded in a temporally specific manner in order to keep cell cycle progression in order. The degradation of these proteins is often controlled by phosphorylation, mediated by cell-signaling regulated kinases ^3,4. On the other hand, aberrantly-folded proteins are recognized through cryptic degrons. These are regions that are normally hidden in the native structure and are exposed upon structure perturbation. Such degrons include hydrophobic domains ^5-7 and intrinsically disordered segments⁸. 

Since the seminal discovery of the ubiquitin-system for protein degradation and characterization of its fundamentals in reticulocyte lysates⁹, yeast genetics was instrumental in discovering many of the components of the ubiquitin system¹⁰. The success of yeast as a model organism for systematic analysis of protein degradation by the UPS is mainly due to the fact that the UPS is highly conserved in all eukaryotes⁴, coupled with their amenability as an experimental system. Indeed, yeast-based systems are commonly employed to decipher the mechanisms of action of the ubiquitylation machinery.

Studying protein degradation by biochemical means usually requires preparation of cell extracts. While animal cell proteins can be extracted under relatively mild conditions that preserve protein interactions and function, the presence of a robust cell wall in yeast¹¹ requires considerably harsher disruption conditions which may affect protein recovery. Indeed, different procedures for yeast cell disruption vary considerably in their capacity to recover intact proteins in amounts that correctly represent their relative cellular abundance. Further inaccuracy is inherent in the different methods employed for determining degradation rates of specific proteins: Metabolic labeling-based 'pulse-chase' experiments followed by immunoprecipitation, to isolate specific proteins¹², is often not strictly quantitative. Thus, when protein degradation is compared by this method, extra caution should be exercised in interpreting the results. To circumvent this drawback, an alternative cycloheximide (CHX) chase assay can be employed¹². In this assay, the translation inhibitor is added to cell cultures and temporal changes in protein steady-state levels are subsequently monitored. Nevertheless, the usage of CHX is limited to proteins with relatively short half-lives (< 90 min), as long-term inhibition of protein synthesis is cytotoxic. Notably, both of the above-mentioned assays require the use of protein-specific antibodies, which are not always available.

To overcome these technical limitations, researchers have developed several approaches that do not require cell extraction and direct protein handling. One approach is based on the establishment of auxotrophic yeast strains, obtained by the deletion of genes encoding essential metabolic enzymes. Such genes include HIS3, LEU2, LYS2 and TRP1, encoding for enzymes required for amino acid biosynthesis, as well as URA3 that encodes OMP decarboxylase (Ura3), an essential enzyme of pyrimidine ribonucleotide biosynthesis. Ura3 has been widely used in protein degradation studies. In these assays, constitutive expression of Ura3 rescues growth of ura3 cells in uracil-deficient medium¹³. Consequently, destabilizing Ura3 through the fusion of a degron can diminish cell growth on minimal medium lacking uracil. This method has been used in various protein degradation studies, including the identification of degradation determinants⁵, E3 ligases¹⁴ and auxiliary ubiquitylation factors¹⁵ and the discovery of novel UPS degrons¹⁶. All of these methods employed cell growth on agar plates as assay readout. However, the growth criterion (positive/negative growth), while robust and efficient, is mostly qualitative and does not provide quantitative information that is important for evaluating a degron's potency or the relative contribution of various auxiliary degradation factors.

We have therefore developed and utilized yeast vectors and screening methods enabling systematic and quantitative analysis of protein degradation by the URA3-degron fusion system. The protocol is based on an easy-to-handle assay that measures Growth kinetics in Liquid culture under Selective conditions (GiLS) and on the generation of standard growth curves. Yeast growth kinetics are characterized by three main phases — the lag, the exponential (log) and the stationary phase. Calculation of yeast replication kinetics during the log phase under selective conditions, which is determined by the levels of expression of the Ura3-degron, provides an unbiased quantitative measurement of protein degradation. This method can be applied to measuring and comparing degradation rates of multiple UPS substrates simultaneously in multiple strains and under various conditions.

Protocol

1. Cell Culture

1. Transform the appropriate auxotrophic yeast cells, such as Try467 (Table 2), with a plasmid containing (a) a URA3-degron fusion and (b) an additional metabolic marker for plasmid selection and maintenance.
   NOTE: An example of a suitable plasmid is YDpK-MET25p-Deg1-FLAG-Vma12-Ura3 (LYS2) (Deg1-UV). This is a yeast integrative plasmid containing a fusion protein comprising of Deg1 — a degron derived from the yeast transcription factor Mat2¹⁷, FLAG epitope — for detection by immunoblotting, Vma12 — a stable ER protein, and Ura3^15,18 (Figure 3A). (See Table 3 for plasmids used in this study and for additional examples of suitable plasmids.)
2. For plasmid selection and maintenance, keep cells in agar plates under a suitable Synthetic Defined (SD) medium lacking amino acid selection markers¹⁹ (For example, Deg1-UV is selected and maintained under SD-LYS selective media).
3. Grow cells O/N at 30 °C in a suitable selective SD medium for plasmid maintenance until a stationary phase is reached. Grow the cells in test tubes or in a 96-well plate, depending on the number of samples to be examined (section 2).

2. Cell Preparation for Analysis

1. When a small number of samples (N≤15) are to be assayed, dilute the cells and replace the medium as follows:
   1. Dilute 50 µl of cells from an O/N culture with 150 µl of SD medium per well in a 96-well plate (clear bottom). Note, the first sample is designated as blank contains medium only.
   2. Determine the OD₆₀₀ values of the samples in the 96-well plate using a microplate reader.
   3. Calculate the actual OD₆₀₀ in each well by multiplying the OD₆₀₀ reading by the dilution factor (x4), from which the value of the blank reading is subtracted. Normalize the resulting values by dividing by 0.55 to obtain a calculated path-length of 1 cm according to the Beer-Lambert law. Note, this value is constant when measuring the OD₆₀₀ of 200 µl of cells in a standard 96-well plate.
   4. Calculate the exact volume needed for obtaining yeast cells at amount equivalent to OD₆₀₀ of 0.25 and transfer it to an Eppendorf tube.
   5. Spin down the cells at high speed (12,000 x g) for 1 min.
   6. Remove the supernatant and resuspend the cells in 1 ml of the appropriate selective medium (see section 3.1) to obtain OD₆₀₀ of 0.25.
   7. Transfer 200 µl of the diluted strains to a new well in a 96-well plate.
2. When a large number of samples (N>15) are to be tested, dilute the cells and replace the medium without prior OD₆₀₀ measurement, as follows:
   1. Transfer 10 μl of stationary cells grown O/N in a 96-well plate into a new 96-well plate containing 190 μl (dilution 1:20) of the appropriate selective media (see section 3.1).

3. Growth Kinetics in Liquid Culture Under Selective Conditions (GiLS)

1. Use the following media for growth measurements using the Ura3-degron reporter:
   1. For positive controls of yeast growth under non-restrictive conditions, use plasmid selective SD media (see section 1.2). If no plasmid selection is required, for example, when using an integrative plasmid such as Deg1-UV, SD medium containing all necessary amino acids (SD complete) can be used.
   2. For experimental samples measuring growth under restrictive conditions, use SD-URA medium.
2. Set a multimode microplate reader to: an incubation temperature of 30 °C, OD₆₀₀ measurement intervals of 15 min, and an orbital and linear shaking cycles of 1 min every seven min.
   NOTE: This dual-mode shaking was optimized to obtain homogenous distribution of cells; however, other shaking modes or even no shaking may work as well.
3. Incubate the cells at 30 °C in a microplate reader O/N or until cells have reached the stationary phase (12-24 hr).
4. Export the raw data to a spreadsheet file.
5. Determine yeast growth kinetics using MDTcalc, a customized software program that calculates the minimal time (in hr) for cells to double their population size (Minimal Doubling Time (MDT); for details see section 4).

4. Calculation of Minimal Doubling Time (MDT): Principles of the MDTcalc Algorithm

NOTE: Principles of the MDTcalc algorithm, using a representative sample of Try467 yeast cells (Table 2) grown in SD complete media, are illustrated in Table 1 and Figure 1, showing a data spreadsheet and growth plots, respectively.

1. Subtract OD₆₀₀ blank values, obtained in the respective well, from each of the readings obtained in the sample well. Divide the resulting differences by 0.55 (for 96-well plates) to correct for the light path length. Plot the final values (Table 1, Trans.) against time to produce the actual yeast growth curve (OD₆₀₀/hr) (Figure 1A).
2. Calculate log₂ (OD₆₀₀) for each of the transformed values to convert the logarithmic growth phase into a linear curve (Figure 1B).
3. Calculate the slope of a 10 time-points interval (N=10) starting at time 0 and moving one time-point at a time until N<10 (Table 1, Slope; Figure 1C).
4. Calculate the inverse value of each slope to obtain the time required for the cell population to double (Table 1, 1/Slope; Figure 1C). Calculate the minimal 1/Slope value to determine the MDT (Table 1, black rectangle; Figure 1C). Extract the calculated MDT for cells in the sample (Table 1, 1/Slope, black rectangle) from the selected time interval (Table 1, outlined by red rectangle, Figures 1B, 1D).
   NOTE: The rate of cell growth on SD-URA medium is inversely proportional to the MDT.

5. Using MDTcalc

1. Copy the supplemented program files into a designated folder in the computer.
2. Make sure the spreadsheet file containing the growth data is saved and closed. Open the MDTcalc application program to see the start screen appears.
   1. Insert the required information, as demonstrated in Figure 2:
      1. Under 'File path', click on the rectangle on the right to locate the spreadsheet file.
      2. Under 'Sheet name', write the spreadsheet name where the raw data is located.
      3. Under 'Starting position', insert the spreadsheet coordinate of time 0 of the first sample.
      4. Under 'Time column', insert the letter defining the location of the time point column (time should be provided in seconds).
      5. Under 'Blank column', insert the letter defining the location of the blank column (usually but not necessarily in well A1 of the 96-well plate).
      6. Under 'OD value', choose the minimal transformed OD₆₀₀ value required for the initiation of MDT calculation (usually 0.15-0.25). Setting this value prevents calculations of MDT for cultures in lag phase due to excessive dilution so that MDT is calculated only for samples that reach the defined OD₆₀₀ value or higher.
   2. Once the last value has been entered, press the 'calculate' button at the bottom of the screen. Ensure that the progress bar on the right gradually changes to green. Do not open the spreadsheet file during the calculation process.
   3. Export the two sets of data that appear automatically on the screen at the completion of the MDT calculation into a spreadsheet. Ensure that the data includes: (a) the MDT value for each well that passes the minimal OD value test (as set in section 5.2.1.6) and (b) the initial time point of the interval from which the MDT was calculated (Start time).

Results

Investigating the role of enzymes of the Doa10 pathway in the degradation of a reporter substrate

To test the validity of the GiLS method it was compared to a traditional degradation assay. This experiment assesses the relative contribution of components of the ER-membrane localized Doa10 E3-ligase complex^20,21 to the degradation of the protein quality control reporter substrate Deg1-VU (Figure 3A). The Deg1-VU plasmid was integra...

Discussion

Here we describe an assay based on cell growth for determining relative protein degradation rates, termed 'Growth kinetics in Liquid culture under Selective conditions' (GiLS). The GiLS assay has several advantages: It is simple to set up, data acquisition and analysis is straightforward and it is extremely modular. Consequently, GiLS can be applied simultaneously to multiple samples in a user friendly multi-well plate format that can be adapted to automation for high throughput applications. Most importantly, Gi...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Difco yeast nitrogen base w/o amino acids and ammonium sulfate	BD Biosciences	233520	For yeast growth on SD minimal media.  Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan
Ammonium sulfate	Sigma - Aldrich	A4418
Glucose	Sigma - Aldrich	16325
Adenine	Sigma - Aldrich	A8626
Uracil	Sigma - Aldrich	U0750
Amino acids	Highest purity available
96-well plates	Nunc	167008	Any other compatible brand can be used
Cyclohexamide	Sigma - Aldrich	C7698	Working conc. 0.5 mg/ml
Infinite 200 PRO series	Tecan		For yeast incubation and OD600 measurements. Any other compatible temp-controled reader can be used.
MDTcalc			Experimental software