Name: reporter-based growth assay for systematic analysis of protein degradation
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We thank Dr. Yuval Reiss and Dr. William Breuer for critically reviewing the manuscript and Omri Alfassy for helping in the development of the Ura3-GFP screen assay. We also thank Dr. M. Hochstrasser and Dr. R. Kulka for plasmids and strains. This work was funded by the Israeli Academy of Sciences (grant 786/08) and by the United States-Israel Binational Scientific foundation (grant 2011253).

1. Cell Culture
<ol>
	<li>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 &#8212; a degron derived from the yeast transcription factor Mat217, FLAG epitope &#8212; for detection by immunoblotting, Vma12 &#8212; a stable ER protein, and Ura315,18 (Figure 3A). (See Table 3 for plasmids used in this study and for additional examples of suitable plasmids.)</li>
	<li>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).</li>
	<li>Grow cells O/N at 30 &#176;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).</li>
</ol>
2. Cell Preparation for Analysis
<ol>
	<li>When a small number of samples (N&#8804;15) are to be assayed, dilute the cells and replace the medium as follows:
	<ol>
		<li>Dilute 50 &#181;l of cells from an O/N culture with 150 &#181;l of SD medium per well in a 96-well plate (clear bottom). Note, the first sample is designated as blank contains medium only.</li>
		<li>Determine the OD600 values of the samples in the 96-well plate using a microplate reader.</li>
		<li>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 &#181;l of cells in a standard 96-well plate.</li>
		<li>Calculate the exact volume needed for obtaining yeast cells at amount equivalent to OD600 of 0.25 and transfer it to an Eppendorf tube.</li>
		<li>Spin down the cells at high speed (12,000 x g) for 1 min.</li>
		<li>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.</li>
		<li>Transfer 200 &#181;l of the diluted strains to a new well in a 96-well plate.</li>
	</ol>
	</li>
	<li>When a large number of samples (N&#62;15) are to be tested, dilute the cells and replace the medium without prior OD600 measurement, as follows:
	<ol>
		<li>Transfer 10 &#956;l of stationary cells grown O/N in a 96-well plate into a new 96-well plate containing 190 &#956;l (dilution 1:20) of the appropriate selective media (see section 3.1).</li>
	</ol>
	</li>
</ol>
3. Growth Kinetics in Liquid Culture Under Selective Conditions (GiLS)
<ol>
	<li>Use the following media for growth measurements using the Ura3-degron reporter:
	<ol>
		<li>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.</li>
		<li>For experimental samples measuring growth under restrictive conditions, use SD-URA medium.</li>
	</ol>
	</li>
	<li>Set a multimode microplate reader to: an incubation temperature of 30 &#176;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.</li>
	<li>Incubate the cells at 30 &#176;C in a microplate reader O/N or until cells have reached the stationary phase (12-24 hr).</li>
	<li>Export the raw data to a spreadsheet file.</li>
	<li>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).</li>
</ol>
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.

<ol>
	<li>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).</li>
	<li>Calculate log2 (OD600) for each of the transformed values to convert the logarithmic growth phase into a linear curve (Figure 1B).</li>
	<li>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&#60;10 (Table 1, Slope; Figure 1C).</li>
	<li>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.</li>
</ol>
5. Using MDTcalc
<ol>
	<li>Copy the supplemented program files into a designated folder in the computer.</li>
	<li>Make sure the spreadsheet file containing the growth data is saved and closed. Open the MDTcalc application program to see the start screen appears.
	<ol>
		<li>Insert the required information, as demonstrated in Figure 2:
		<ol>
			<li>Under &#39;File path&#39;, click on the rectangle on the right to locate the spreadsheet file.</li>
			<li>Under &#39;Sheet name&#39;, write the spreadsheet name where the raw data is located.</li>
			<li>Under &#39;Starting position&#39;, insert the spreadsheet coordinate of time 0 of the first sample.</li>
			<li>Under &#39;Time column&#39;, insert the letter defining the location of the time point column (time should be provided in seconds).</li>
			<li>Under &#39;Blank column&#39;, insert the letter defining the location of the blank column (usually but not necessarily in well A1 of the 96-well plate).</li>
			<li>Under &#39;OD value&#39;, 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.</li>
		</ol>
		</li>
		<li>Once the last value has been entered, press the &#39;calculate&#39; 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.</li>
		<li>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).</li>
	</ol>
	</li>
</ol>

<ol>
	<li>Hershko, A., Ciechanover, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin+system.">The ubiquitin system.</a> Annual review of biochemistry. 67, 425-479 (1998).</li><li>Ravid, T., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diversity+of+degradation+signals+in+the+ubiquitin-proteasome+system.">Diversity of degradation signals in the ubiquitin-proteasome system.</a> Nature reviews. Molecular cell biology. 9, 679-689 (2008).</li><li>Willems, A. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SCF+ubiquitin+protein+ligases+and+phosphorylation-dependent+proteolysis.">SCF ubiquitin protein ligases and phosphorylation-dependent proteolysis.</a> Philos Trans R Soc Lond B Biol Sci. 354, 1533-1550 (1999).</li><li>Tyers, M., Jorgensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteolysis+and+the+cell+cycle:+with+this+RING+I+do+thee+destroy.">Proteolysis and the cell cycle: with this RING I do thee destroy.</a> Curr Opin Genet Dev. 10, 54-64 (2000).</li><li>Johnson, P. R., Swanson, R., Rakhilina, L., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signal+masking+by+heterodimerization+of+MAT&#945;2+and+MAT+a1+blocks+their+mutual+destruction+by+the+ubiquitin-proteasome+pathway.">Degradation signal masking by heterodimerization of MAT&#945;2 and MAT a1 blocks their mutual destruction by the ubiquitin-proteasome pathway.</a> Cell. 94, 217-227 (1998).</li><li>Furth, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exposure+of+bipartite+hydrophobic+signal+triggers+nuclear+quality+control+of+Ndc10+at+the+endoplasmic+reticulum/nuclear+envelope.">Exposure of bipartite hydrophobic signal triggers nuclear quality control of Ndc10 at the endoplasmic reticulum/nuclear envelope.</a> Molecular Biology of the Cell. 22, 4726-4739 (2011).</li><li>Fredrickson, E. K., Gallagher, P. S., Clowes Candadai, S. V., Gardner, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrate+recognition+in+nuclear+protein+quality+control+degradation+is+governed+by+exposed+hydrophobicity+that+correlates+with+aggregation+and+insolubility.">Substrate recognition in nuclear protein quality control degradation is governed by exposed hydrophobicity that correlates with aggregation and insolubility.</a> J Biol Chem. 10, (2013).</li><li>Rosenbaum, J. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disorder+Targets+Misorder+in+Nuclear+Quality+Control+A+Disordered+Ubiquitin+Ligase+Directly+Recognizes+Its+Misfolded+Substrates.">Disorder Targets Misorder in Nuclear Quality Control A Disordered Ubiquitin Ligase Directly Recognizes Its Misfolded Substrates.</a> Molecular cell. 41, 93-106 (2011).</li><li>Ciechanover, A., Hod, Y., Hershko, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+heat-stable+polypeptide+component+of+an+ATP-dependent+proteolytic+system+from+reticulocytes.">A heat-stable polypeptide component of an ATP-dependent proteolytic system from reticulocytes.</a> Biochemica., &amp; Biophysical Research Communications. 81, 1100-1105 (1978).</li><li>Finley, D., Ulrich, H. D., Sommer, T., Kaiser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ubiquitin-proteasome+system+of+Saccharomyces+cerevisiae.">The ubiquitin-proteasome system of Saccharomyces cerevisiae.</a> Genetics. 192, 319-360 (2012).</li><li>Klis, F. M., Boorsma, A., De Groot, P. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+construction+in+Saccharomyces+cerevisiae.">Cell wall construction in Saccharomyces cerevisiae.</a> Yeast. 23, 185-202 (2006).</li><li>Zhou, W., Ryan, J. J., Zhou, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+analyses+of+sumoylated+proteins+in+Saccharomyces+cerevisiae.+Induction+of+protein+sumoylation+by+cellular+stresses.">Global analyses of sumoylated proteins in Saccharomyces cerevisiae. Induction of protein sumoylation by cellular stresses.</a> J Biol Chem. 279, 32262-32268 (2004).</li><li>Alani, E., Kleckner, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+type+of+fusion+analysis+applicable+to+many+organisms:+protein+fusions+to+the+URA3+gene+of+yeast.">A new type of fusion analysis applicable to many organisms: protein fusions to the URA3 gene of yeast.</a> Genetics. 117, 5-12 (1987).</li><li>Swanson, R., Locher, M., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+ubiquitin+ligase+of+the+nuclear+envelope/endoplasmic+reticulum+that+functions+in+both+ER-associated+and+Matalpha2+repressor+degradation.">A conserved ubiquitin ligase of the nuclear envelope/endoplasmic reticulum that functions in both ER-associated and Matalpha2 repressor degradation.</a> Genes Dev. 15, 2660-2674 (2001).</li><li>Ravid, T., Kreft, S. G., Hochstrasser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Membrane+and+soluble+substrates+of+the+Doa10+ubiquitin+ligase+are+degraded+by+distinct+pathways.">Membrane and soluble substrates of the Doa10 ubiquitin ligase are degraded by distinct pathways.</a> EMBO J. 25, 533-543 (2006).</li><li>Gilon, T., Chomsky, O., Kulka, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+signals+for+ubiquitin+system+proteolysis+in+Saccharomyces+cerevisiae.">Degradation signals for ubiquitin system proteolysis in Saccharomyces cerevisiae.</a> Embo J. 17, 2759-2766 (1998).</li><li>Hochstrasser, M., Varshavsky, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+degradation+of+a+transcriptional+regulator:+the+yeast+2+repressor.">In vivo degradation of a transcriptional regulator: the yeast 2 repressor.</a> Cell. 61, 697-708 (1990).</li><li>Metzger, M. B., Maurer, M. J., Dancy, B. M., Michaelis, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Degradation+of+a+cytosolic+protein+requires+endoplasmic+reticulum-associated+degradation+machinery.">Degradation of a cytosolic protein requires endoplasmic reticulum-associated degradation machinery.</a> J Biol Chem. 283, 32302-32316 (2008).</li><li>Guthrie, C., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Guide to Yeast Genetics and Molecular Biology. 194, (1991).</li><li>Carvalho, P., Goder, V., Rapoport, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+ubiquitin-ligase+complexes+define+convergent+pathways+for+the+degradation+of+ER+proteins.">Distinct ubiquitin-ligase complexes define convergent pathways for the degradation of ER proteins.</a> Cell. 126, 361-373 (2006).</li><li>Denic, V., Quan, E. M., Weissman, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+luminal+surveillance+complex+that+selects+misfolded+glycoproteins+for+ER-associated+degradation.">A luminal surveillance complex that selects misfolded glycoproteins for ER-associated degradation.</a> Cell. 126, 349-359 (2006).</li><li>Wu, P. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+conserved+catalytic+residue+in+the+ubiquitin-conjugating+enzyme+family.">A conserved catalytic residue in the ubiquitin-conjugating enzyme family.</a> Embo J. 22, 5241-5250 (2003).</li><li>Boeke, J. D., Trueheart, J., Natsoulis, G., Fink, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Fluoroorotic+acid+as+a+selective+agent+in+yeast+molecular+genetics.">5-Fluoroorotic acid as a selective agent in yeast molecular genetics.</a> Methods Enzymol. 154, 164-175 (1987).</li><li>Alfassy, O. S., Cohen, I., Reiss, Y., Tirosh, B., Ravid, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Placing+a+disrupted+degradation+motif+at+the+C+terminus+of+proteasome+substrates+attenuates+degradation+without+impairing+ubiquitylation.">Placing a disrupted degradation motif at the C terminus of proteasome substrates attenuates degradation without impairing ubiquitylation.</a> J Biol Chem. 288, 12645-12653 (2013).</li><li>Cronin, S. R., Hampton, R. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+protein+degradation+with+green+fluorescent+protein.">Measuring protein degradation with green fluorescent protein.</a> Methods in Enzymology. 302, 58-73 (1999).</li></ol>

The authors have nothing to disclose.

Here we describe an assay based on cell growth for determining relative protein degradation rates, termed &#39;Growth kinetics in Liquid culture under Selective conditions&#39; (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, GiLS provides highly reproducible, quantitative and robust data and is more flexible than agar-plate based-growth assays that select for positive/negative growth. In addition, GiLS is highly sensitive in that it can detect small variations in protein degradation rates or steady-state levels, reflecting the activity of relevant UPS components or degrons. Finally, GiLS assays require significantly shorter growth periods (&#60;12 hr) compared to growth assays on agar plates (typically 2-4 days).
While the GiLS assay is very useful for studying protein degradation, several important considerations should be taken into account. For example, it is essential to use an isogenic yeast strain background due to strain-specific growth characteristics. Furthermore, to ascertain assay validity, each assay should be repeated at least three times under identical conditions, most importantly, the initial cell density (OD600), growth temperature, and cell culture shaking intensity should not be altered. It is therefore recommended that the specific assay protocol is stored in the microplate reader&#39;s software for repeated uses. An additional variable that should be considered is where the degron is positioned within Ura3. As the degron position may determine its function, positioning at either the C&#39; or N&#39; regions must be empirically tested.
Calibration of reporter protein expression level is also critical for successful application of GiLS. An optimal reporter is one that is efficiently degraded by the system under investigation and thus confers uracil auxotrophy. However, variations in auxotrophy should also faithfully mirror the reporter stability. This can be determined empirically by prior testing of the correlation between reporter protein degradation rates, determined by additional biochemical methods, and MDT values, determined by GiLS (Figure 3B). One common problem arises when the basal steady-state expression of a reporter does not reach a threshold level and is thus too low to enable growth on SD-URA even when it is completely stabilized. Such a problem can be overcome by placing the reporter expression under the control of an inducible promoter that increases basal protein expression without affecting degradation. Promoter activity in such systems should be finely calibrated to avoid permanent growth on SD-URA due to high protein levels and reporter enzyme activity even when it is partially degraded. The promoters and expression conditions described in this study were chosen because they allow sensitive regulation of protein expression: MET25p and CUP1p are metabolite-induced promoters the activity of which can be finely tuned through control of methionine and copper, respectively (Table 3).
Quality control reporter substrates (as those described in this study) can form intracellular aggregates that might sequester Ura3 and prevent its function. In such a case uracil auxotrophy can be mistakenly interpreted as the result of protein degradation. To test the tendency of different degrons to aggregate, an additional reporter should be employed. For example, the Ura3-GFP reporter designed for the degron screen (Figure 5A), enables visualization of intracellular aggregates by fluorescent microscopy of cells growing in the presence of 5-FOA. These aggregates appear as dense GFP foci that are clearly distinguishable from the diffuse appearance of the soluble protein (data not shown). The Ura3-GFP-degron reporter can be similarly used to determine the effect of the degron on protein co-localization and subcellular distribution. It can also be used for following the degradation of selected substrates by flow cytometry, as a secondary screen25.
A most significant advantage of the GiLS method is that it is sufficiently flexible and thus can easily be adapted to a variety of applications in addition to those described in herein. For example, the method can also be used for examining the fitness of the proteasome under various conditions. In this case, a non-ubiquitylated proteasome substrate such as ornithine decarboxylase (ODC) is fused to Ura3. The method can also test protein degradation in distinct intracellular compartments when a specific localization signal is employed. In this study Vma12 anchored the reporter to the ER-membrane (Figures 3, 4). Similarly, a nuclear localization signal (NLS), ER retention signal (KDEL) or cytosol (absence of a specific signal) can be employed. A system enabling simultaneous analysis of protein degradation in various intracellular compartments could have profound impact on our understanding of how protein degradation networks operate. Finally, the principles of the selection method can be applied to research of protein degradation in other model cell-based systems, from bacteria to human cells, all of which require the URA3 gene product for survival.

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&#160;intrinsically disordered segments8.&#160;
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 &#39;pulse-chase&#39; 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 (&#60; 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&#39;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 &#8212; 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.

<table><tbody><tr><td>Difco yeast nitrogen base w/o amino acids and ammonium sulfate</td><td>BD Biosciences</td><td>233520</td><td>For yeast growth on SD minimal media.&amp;nbsp; Amino acids to be supplied are: Arginine, Histidine, Isoleucine, Leucine, Lysine, Methionine, Phenylalanine, Threonine, Tryptophan</td></tr><tr><td>Ammonium sulfate</td><td>Sigma - Aldrich</td><td>A4418</td><td></td></tr><tr><td>Glucose</td><td>Sigma - Aldrich</td><td>16325</td><td></td></tr><tr><td>Adenine</td><td>Sigma - Aldrich</td><td>A8626</td><td></td></tr><tr><td>Uracil</td><td>Sigma - Aldrich</td><td>U0750</td><td></td></tr><tr><td>Amino acids</td><td>Highest purity available&amp;nbsp;</td><td></td><td></td></tr><tr><td>96-well plates</td><td>Nunc</td><td>167008</td><td>Any other compatible brand can be used</td></tr><tr><td>Cyclohexamide&amp;nbsp;</td><td>Sigma - Aldrich</td><td>C7698</td><td>Working conc. 0.5 mg/ml</td></tr><tr><td>Infinite 200 PRO series</td><td>Tecan</td><td></td><td>For yeast incubation and OD&lt;sub&gt;600&lt;/sub&gt; measurements. Any other compatible temp-controled reader can be used.</td></tr><tr><td>MDTcalc</td><td></td><td></td><td>Experimental software</td></tr></tbody></table>

reporter-based growth assay for systematic analysis of protein degradation

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.

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 complex20,21 to the degradation of the protein quality control reporter substrate Deg1-VU (Figure 3A). The Deg1-VU plasmid was integrated into wild type cells or into cells lacking the gene for the E2 conjugating enzyme, UBC7 (ubc7) and protein stability was subsequently determined by a CHX chase assay6. Anti-FLAG immunoblotting revealed a stable Deg1-VU protein band in ubc7 cells that was absent in wild type cells (Figure 3B). The absence of Deg1-VU in wild type cells is attributed to its continuous degradation that was abolished in ubc7 cells, indicating that Deg1-VU stability is strongly dependent on Ubc7.
Obviously, the degradation of Deg1-VU is rapid. However, the relative rate of degradation cannot be determined by a CHX assay since the protein is undetectable in wild type cells from the outset. Consequently, the newly-developed GiLS assay method was employed, in order to compare the relative differences in the kinetics of protein degradation between wild-type and various mutant strains (Figure 3C). Initially, wild type, ubc6, ubc7 or doa10 cells expressing Deg1-VU were grown on SD complete medium. To measure GiLS, cells were washed and incubated in SD-URA. MDT calculation was executed as exemplified in the protocol. Figures 3C, 3D show similar growth curves and calculated MDT (~2.5 hr) for all strains grown in SD complete, excluding the possibility that the deletion of any of the examined genes affects cell growth. In contrast, incubation in SD-URA resulted in poor growth of wild-type cells (MDT=6.34), whereas only a minor growth defect was observed in the mutant strains (MDT ~3 hr).
Examining the effect of different mutants of the E2 conjugating enzyme Ubc7 on the degradation of Deg1-VU
The fact that Ubc7 is absolutely required for Deg1-VU degradation enables accurate assessment of even partial effects of various E2 mutants, since the experimental range of the system is very wide. Accordingly, plasmids containing wild type or mutant UBC7 were integrated into Ubc7 cells expressing Deg1-VU and their contribution to degradation was determined by GiLS. As expected, fast growth kinetics were observed in cells expressing Ubc7 with the active-site mutants C89S and N81A22 (Figure 4). In addition, two mutants predicted to indirectly hinder Deg1-VU degradation (V25G and H94K) were tested. These mutations also enhanced cell growth, compared to wild-type Ubc7, albeit to a lesser degree than the active-site mutants (average MDTs of 3.4 hr for V25G and H94K compared to average MDTs of 2.7 hr for C89S and N81K) (Figure 4). Thus, the GiLS method can be used for accurate measurement of the relative contribution of various degradation factors to the stability of a reporter substrate.
Isolation and evaluation of novel degrons 
The GiLS system was also employed in a high throughput screen format to identify novel degrons and quantify their relative potency, i.e., the degree by which they induce degradation (Figure 5). To optimize the identification of bona fide degrons, an additional selection step, which is growth in the presence of Fluoroorotic Acid (5-FOA), was added. Ura3 efficiently converts 5-FOA into a toxic compound (5-fluorouracil) that can serve as a positive selection for the isolation of yeast strains where Ura3 is destabilized16,23. To identify novel degrons, a library of reporter plasmids was generated by fusing random fragments derived from a yeast cDNA library to Ura3-GFP plasmid24 (Figure 5A). The GFP moiety was added in order to enable a secondary screen and provide information about the localization of the fusion degron (see discussion). The library was transformed into yeast, then selected in the presence of 5-FOA (Figure 5B) and positive clones were isolated and re-seeded on agar plates in a 96-well plate format. Each colony was transferred into 96-well plate, incubated O/N, and diluted in a new 96-well plate as described in section 2.2. It is expected that growth of cells in SD-URA medium will correlate with the degree of Ura3 expression, which is the outcome of the degron&#39;s potency to induce degradation. This was subsequently confirmed by applying GiLS (Figure 5C). We confirmed that all randomly-picked colonies that were pre-selected with 5-FOA showed similar MDT values, on SD-LEU medium (Figure 5C, empty bars). In contrast, all clones showed variable growth rates on SD-URA, which were significantly slower than cells expressing control Ura3-GFP (Data not shown). As indicated, there is an inverse relationship between growth rate on SD-URA and MDT. Thus, the higher the MDT, the more potent is the degron. Indeed, all MDTs derived from positively selected clones were higher than that of the control (above the red line) (Figure 5C, filled bars).
<img alt="Table 1" fo:content-width="5in" src="/files/ftp_upload/52021/52021table1.jpg" width="500" /> 
Table 1. Illustration of the calculation of yeast MDT. Time units are hours (hr). Blank and sample are from OD600 reads. Transformation (Trans.) is the OD600 value after blank subtraction and path-length correction. Log2 is calculated from the transformed data. Slope is the Log2 (OD600) values within consecutive of intervals of 10 time points divided by time. 1/Slope is the time required for the cell population to duplicate itself. Black rectangle marks the MDT value. Red rectangle marks the time interval from which the MDT was calculated.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Yeast</td>
			<td>Genotype</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>TRy467</td>
			<td>&#945;, his3&#8710;1, lys2&#8710;0, ura3&#8710;0, leu2&#8710;0</td>
			<td>Euroscarf</td>
		</tr>
		<tr>
			<td>TRy508</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7-3HA::HIS,&#160; leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy528</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7[N81A]-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy530</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7[C89S]-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy532</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7[H94K]-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy556</td>
			<td>&#945;, his3-&#8710;200::pRS303::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy563</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2, ubc6&#8710;::KanMX&#160;</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy633</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7[V25A]-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7&#8710;::LEU2</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>TRy786</td>
			<td>&#945;, his3-&#8710;200::pRS303/Ubc7prom-Ubc7-3HA::HIS, leu2-3,112,&#160; ura3-5, lys2-801::Met25-Deg1-Flag-Vma12-Ura3::LYS,&#160; trp1-1, ubc7::LEU, doa10&#8710;::HIS3</td>
			<td>This study</td>
		</tr>
	</tbody>
</table>
Table 2. List of yeast strains used in this study.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td colspan="3">Plasmids used in this study</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td>Relevant markers</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>pTR717</td>
			<td>YDpK-MET25p-Deg1-Flag-Vma12-Ura3 (integrative plasmid containing LYS1)</td>
			<td>This study</td>
		</tr>
		<tr>
			<td>pTR1412</td>
			<td>pRS315-CUP1p-URA3-HA-GFP (LEU)</td>
			<td>This study</td>
		</tr>
		<tr>
			<td colspan="3">Additional suitable reporter plasmids</td>
		</tr>
		<tr>
			<td>Plasmid</td>
			<td>Relevant markers</td>
			<td>Source</td>
		</tr>
		<tr>
			<td>pOC9-CL1</td>
			<td>pOC9-CUP1p-HA-URA3 (TRP1)</td>
			<td>(17)</td>
		</tr>
		<tr>
			<td>URA3-2(GFP)</td>
			<td>pPS414-TRP1p-MYC-ura3-2-GFP</td>
			<td>Lewis and Pelham</td>
		</tr>
	</tbody>
</table>
Table 3. List of plasmids used in this study. Reference: Lewis, M. J. &#38; Pelham, H. R. Inefficient quality control of thermosensitive proteins on the plasma membrane. PLoS One 4, e5038, doi:10.1371/journal.pone.0005038 (2009).
<img alt="Figure 1" fo:content-width="6in" fo:src="/files/ftp_upload/52021/52021fig1highres.jpg" src="/files/ftp_upload/52021/52021fig1highres.jpg" width="500px" /> 
Figure 1. Principles of calculation of MDT. Try467 yeast cells at OD600 0.22, were incubated in SD complete media for the indicated time period. OD600 measurements were taken every ~15 min and the data was collected in a spreadsheet. (A) Transformed OD600 values from Table 1 plotted against time. (B) Log2 of OD600 plotted against time. Red line: Marks the time interval from which the MDT was calculated (D). (C) Yeast growth rate (slope) was calculated by measuring changes in OD600 of consecutive 10 time points (assuming linearity) over time. Start time: The time from which calculation was initiated. (D) The time required for yeast to double is the inverse of the slope from (C) (1/Slope). MDT: The calculated Minimal Doubling time (in hr).
<img alt="Figure 2" fo:content-width="6in" fo:src="/files/ftp_upload/52021/52021fig2highres.jpg" src="/files/ftp_upload/52021/52021fig2highres.jpg" width="500px" /> 
Figure 2. MDTcalc start screen: Protocol section 5.1. Typical input values to be entered are shown.
<img alt="Figure 3" fo:content-width="6in" fo:src="/files/ftp_upload/52021/52021fig3highres.jpg" src="/files/ftp_upload/52021/52021fig3highres.jpg" width="500px" /> 
Figure 3. Measuring the degradation of Deg1-VU.&#160;(A) Schematic presentation of the different protein elements of Deg1-VU and its organization at the ER membrane.(B) Determination of the degradation of Deg1-VU by the CHX assay in wild type and ubc7 cells. Cells collected at time zero or after 30 min with CHX were lysed and proteins were separated by 5-15% SDS PAGE. Deg1-VU was visualized by immunoblotting using anti FLAG antibodies. G6PD staining served as a loading control. (C) The indicated strains, expressing Deg1-VU were incubated in SD-complete or SD-URA media for 20 hr and OD600 was measured every 15 min. OD600 are the transformed values. (D) The MDT for each strain was calculated using MDTcalc.
<img alt="Figure 4" fo:content-width="6in" fo:src="/files/ftp_upload/52021/52021fig4highres.jpg" src="/files/ftp_upload/52021/52021fig4highres.jpg" width="500px" /> 
Figure 4. Measuring relative degradation rates of Deg1-VU in Ubc7 mutant strains. Left: Plot of the transformed OD600 values, measured during the replication of wild-type, ubc7&#916; and the indicated Ubc7 mutants, versus time. Cells expressing Deg1-VU were grown on SD-URA medium and OD600 was measured every ~15 min. Right: The MDT for each strain was calculated using MDTcalc.
<img alt="Figure 5" fo:content-width="6in" fo:src="/files/ftp_upload/52021/52021fig5highres.jpg" src="/files/ftp_upload/52021/52021fig5highres.jpg" width="500px" /> 
Figure 5. A high throughput assay for identification and isolation of novel degrons. (A) Schematic presentation of the features of the Ura3-GFP-cDNA reporter. (B) A flow chart describing the experimental steps for the isolation of yeast strains carrying a degron. The plasmid library described in A was transformed into Try467 cells, followed by selection on SD-LEU plates. Colonies were replicated to SD-LEU plates containing 5-FOA and fast-growing colonies were collected and organized on plates in an ordered array. (C) Colonies expressing Ura3-GFP-cDNA that grew on plates containing 5-FOA were incubated in either SD-LEU or SD-URA media for 20 hr. OD600 was measured every ~15 min and the minimal yeast doubling time (MDT) was calculated using the MDTcalc software. No degron: a plasmid expressing Ura3-GFP without a degron, serves as a positive control for growth. Red line: threshold determined by the MDT value of the control.

Watch this Scientific Journal Video about Reporter-based Growth Assay for Systematic Analysis of Protein Degradation at JoVE.com

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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.

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

reporter-based-growth-assay-for-systematic-analysis-protein

Universidad San Sebastian

Research

JoVE Journal

Biology

10.6K Views.  The Hebrew University of Jerusalem. 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.

Video: Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Developmental Biology

High-Throughput Cellular Profiling of Targeted Protein Degradation Compounds Using HiBiT CRISPR Cell Lines

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small molecule drug discovery. This class of compounds induces protein degradation by bringing into proximity the target protein and the E3 ligase machinery proteins required to ubiquitinate and ultimately degrade the target protein through the ubiquitin-proteasomal pathway (UPP). Profiling of target protein degradation in a high-throughput fashion, however, remains highly challenging given the complexity of cellular pathways required to achieve degradation. Here we present a protocol and screening strategy based on the use of CRISPR/Cas9 endogenous tagging of target proteins with the 11 amino acid HiBiT tag which complements with high affinity to the LgBiT protein, to produce a luminescent protein. These CRISPR targeted cell lines with endogenous tags can be used to measure compound induced degradation in either real-time, kinetic live cell or endpoint lytic modes by monitoring luminescent signal using a luminescent plate-based reader. Here we outline the recommended screening protocols for the different formats, and&#160;also describe the calculation of key degradation parameters of rate, Dmax, DC50, Dmax50, as well as multiplexing with cell viability assays. These approaches enable rapid discovery and triaging of early stage compounds while maintaining endogenous expression and regulation of target proteins in relevant cellular backgrounds, allowing for efficient optimization of lead therapeutic compounds.

This protocol describes the quantitative luminescent detection of protein degradation kinetics in living cells that have been engineered using CRISPR/Cas9 to express antibody free endogenous protein detection tag fused to a target protein. Detailed instructions for calculating and obtaining quantitative degradation parameters, rate, Dmax, DC50, and Dmax50 are included.

Targeted protein degradation compounds, including molecular glues or proteolysis targeting chimeras, are an exciting new therapeutic modality in small ...

Cancer Research

Measurement of Protein Turnover Rates in Senescent and Non-Dividing Cultured Cells with Metabolic Labeling and Mass Spectrometry

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as Alzheimer&#39;s and Parkinson&#39;s diseases. Cellular senescence is a state of permanent cell cycle arrest that typically occurs in response to exposure to sub-lethal stresses. However, like other non-dividing cells, senescent cells remain metabolically active and carry out many functions that require unique transcriptional and translational demands and widespread changes in the intracellular and secreted proteomes. Understanding how protein synthesis and decay rates change during senescence can illuminate the underlying mechanisms of cellular senescence and find potential therapeutic avenues for diseases exacerbated by senescent cells. This paper describes a method for proteome-scale evaluation of protein half-lives in non-dividing cells using pulsed stable isotope labeling by amino acids in cell culture (pSILAC) in combination with mass spectrometry. pSILAC involves metabolic labeling of cells with stable heavy isotope-containing versions of amino acids. Coupled with modern mass spectrometry approaches, pSILAC enables the measurement of protein turnover of hundreds or thousands of proteins in complex mixtures. After metabolic labeling, the turnover dynamics of proteins can be determined based on the relative enrichment of heavy isotopes in peptides detected by mass spectrometry. In this protocol, a workflow is described for the generation of senescent fibroblast cultures and similarly arrested quiescent fibroblasts, as well as a simplified, single-time point pSILAC labeling time-course that maximizes coverage of anticipated protein turnover rates. Further, a pipeline is presented for the analysis of pSILAC mass spectrometry data and user-friendly calculation of protein degradation rates using spreadsheets. The application of this protocol can be extended beyond senescent cells to any non-dividing cultured cells such as neurons.

This protocol describes the workflow for metabolic labeling of senescent and non-dividing cells with pulsed SILAC, untargeted mass spectrometry analysis, and a streamlined calculation of protein half-lives.

Mounting evidence has shown that the accumulation of senescent cells in the central nervous system contributes to neurodegenerative disorders such as ...

Biochemistry

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron (dgn)-destabilized Green Fluorescent Protein (GFP)-based Reporter Protein

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. Maintenance of proteasome activity was implicated in many key cellular processes, like cell's stress response 3, cell cycle regulation and cellular differentiation 4 or in immune system response 5. The dysfunction of the ubiquitin-proteasome system has been related to the development of tumors and neurodegenerative diseases 4, 6. Additionally, a decrease in proteasome activity was found as a feature of cellular senescence and organismal aging 7, 8, 9, 10. Here, we present a method to measure ubiquitin-proteasome activity in living cells using a GFP-dgn fusion protein. To be able to monitor ubiquitin-proteasome activity in living primary cells, complementary DNA constructs coding for a green fluorescent protein (GFP)&#x2013;dgn fusion protein (GFP&#x2013;dgn, unstable) and a variant carrying a frameshift mutation (GFP&#x2013;dgnFS, stable 11) are inserted in lentiviral expression vectors. We prefer this technique over traditional transfection techniques because it guarantees a very high transfection efficiency independent of the cell type or the age of the donor. The difference between fluorescence displayed by the GFP&#x2013;dgnFS (stable) protein and the destabilized protein (GFP-dgn) in the absence or presence of proteasome inhibitor can be used to estimate ubiquitin-proteasome activity in each particular cell strain. These differences can be monitored by epifluorescence microscopy or can be measured by flow cytometry.

Monitoring of Ubiquitin-proteasome Activity in Living Cells Using a Degron dgn-destabilized Green Fluorescent Protein GFP-based Reporter Protein

A method to monitor ubiquitin-proteasome activity in living cells is described. A degron-destabilized GFP- (GFP-dgn) and a stable GFP-dgnFS fusion protein are generated and transduced into the cell using a lentiviral expression vector. This technique allows to generate a stable GFP-dgn/GFP-dgnFS expressing cell line in which ubiquitin-proteasome activity can be easily assessed using epifluorescence or flow cytometry.

Proteasome is the main intracellular organelle involved in the proteolytic degradation of abnormal, misfolded, damaged or oxidized proteins 1, 2. ...

Assaying Proteasomal Degradation in a Cell-free System in Plants

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular functions in virtually all eukaryotic organisms. Specifically, in plants, the ubiquitin/26S proteasome system (UPS) regulates protein degradation and contributes significantly to development of a wide range of processes, including immune response, development and programmed cell death. Moreover, increasing evidence suggests that numerous plant pathogens, such as Agrobacterium, exploit the host UPS for efficient infection, emphasizing the importance of UPS in plant-pathogen interactions.
The substrate specificity of UPS is achieved by the E3 ubiquitin ligase that acts in concert with the E1 and E2 ligases to recognize and mark specific protein molecules destined for degradation by attaching to them chains of ubiquitin molecules. One class of the E3 ligases is the SCF (Skp1/Cullin/F-box protein) complex, which specifically recognizes the UPS substrates and targets them for ubiquitination via its F-box protein component. To investigate a potential role of UPS in a biological process of interest, it is important to devise a simple and reliable assay for UPS-mediated protein degradation. Here, we describe one such assay using a plant cell-free system. This assay can be adapted for studies of the roles of regulated protein degradation in diverse cellular processes, with a special focus on the F-box protein-substrate interactions.

Targeted protein degradation represents a major regulatory mechanism for cell function. It occurs via a conserved ubiquitin-proteasome pathway, which attaches polyubiquitin chains to the target protein that then serve as molecular &#8220;tags&#8221; for the 26S proteasome. Here, we describe a simple and reliable cell-free assay for proteasomal degradation of proteins.

The ubiquitin-proteasome pathway for protein degradation has emerged as one of the most important mechanisms for regulation of a wide spectrum of cellular ...

Massively Parallel Reporter Assays in Cultured Mammalian Cells

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. The potential throughput of this assay has, however, been limited by the need to individually clone and assay the activity of each sequence on interest using protein fluorescence or enzymatic activity as a proxy for regulatory activity. Advances in high-throughput DNA synthesis and sequencing technologies have recently made it possible to overcome these limitations by multiplexing the construction and interrogation of large libraries of reporter constructs. This protocol describes implementation of a Massively Parallel Reporter Assay (MPRA) that allows direct comparison of hundreds of thousands of putative regulatory sequences in a single cell culture dish.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory activities. Coupling candidate regulatory elements to reporter genes that carry identifying sequence tags enables massive parallelization of these assays.

The genetic reporter assay is a well-established and powerful tool for dissecting the relationship between DNA sequences and their gene regulatory ...

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.

Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

This article describes a yeast growth-based assay for the determination of genetic requirements for protein degradation. It also demonstrates a method for rapid extraction of yeast proteins, suitable for western blotting to biochemically confirm degradation requirements. These techniques can be adapted to monitor degradation of a variety of proteins.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic ...

Assays for the Degradation of Misfolded Proteins in Cells

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded proteins may serve as potential therapeutic targets. To distinguish degradation of misfolding-prone proteins from other mechanisms that regulate their levels, one important method is to measure protein half-life in cells. However, this can be challenging because misfolding-prone proteins may exist in different forms, including the native form and misfolded forms of distinct characteristics. Here we describe assays to examine the half-life of misfolded proteins in mammalian cells using a highly aggregation-prone protein, Ataxin-1 with an extended polyglutamine (polyQ) stretch, and a conformationally unstable luciferase mutant as models. Cycloheximide chase is combined with cell fractionation to examine the turnover rate of misfolding-prone proteins in various cellular fractions. We further depict a fluorescence-based assay using an enhanced green fluorescence protein (EGFP)-fusion of the luciferase mutant, which can be adapted for high throughput screening on a microplate-reader.

This report describes protocols for measuring degradation rates of misfolded proteins by either western blot or fluorescence-based assays. The methods can be applied to analysis of other misfolded proteins and for high throughput screening.

Protein misfolding and aggregation are associated with various neurodegenerative diseases. Cellular mechanisms that recognize and degrade misfolded ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

Misfolding-Prone Protein Degradation Assay: A Technique to Monitor Misfolded Protein Degradation Using Cycloheximide Treatment and Detergent Fractionation

Source: Guo, L. et al., <a href="https://app-jove-com.bdigitaluss.remotexs.co/v/54266">Assays for the Degradation of Misfolded Proteins in Cells.</a> J. Vis. Exp. (2016).
This video describes a degradation assay for misfolded proteins using cycloheximide treatment along with detergent fractionation. This method aids in studying the dynamics of misfolded proteins and uncovering the in-depth mechanisms of protein turnover.

Test degradacji białek podatnych na nieprawidłowe fałdowanie: technika monitorowania nieprawidłowo sfałdowanej degradacji białek przy użyciu obróbki cykloheksymidem i frakcjonowania detergentem

Source: Guo, L. et al., Assays for the Degradation of Misfolded Proteins in Cells. J. Vis. Exp. (2016).
This video describes a degradation assay for ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

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Growth-based Determination and Biochemical Confirmation of Genetic Requirements for Protein Degradation in Saccharomyces cerevisiae

Assays for the Degradation of Misfolded Proteins in Cells

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Test degradacji białek podatnych na nieprawidłowe fałdowanie: technika monitorowania nieprawidłowo sfałdowanej degradacji białek przy użyciu obróbki cykloheksymidem i frakcjonowania detergentem