Parts of this study were kindly supported by a grant from the Deutsche Forschungsgemeinschaft (SFB 1027, A6).

NOTE: An overview of the general experimental workflow is depicted in Figure 1.
Caution: RTA is highly toxic for humans. Safety lab permission S2 (biosafety level 2 equivalent) is needed. Please wear gloves during the entire experiment.
1. Heterologous Expression of His-tagged RTA in Escherichia coli
<ol>
	<li>Transform E. coli cells with the expression plasmid pET24-RTA(His)6 or the empty vector pET24a(+) using standard electroporation protocols9,10. Cells containing the empty plasmid serve as a negative control.</li>
	<li>After selection of positive clones on kanamycin-containing (100 &#181;g/mL) LB plates, inoculate cells containing the RTA expression plasmid or the empty vector in 5 mL LBkan medium (LB medium with 100 &#181;g/mL of kanamycin) and incubate at 37 &#176;C and 220 rpm for 24 h.</li>
	<li>Supplement 1 L LBkan medium with 5 mL pre-culture and incubate cells at 37 &#176;C and 220 rpm until cells have reached an OD600 of 0.8-1.0 (approximately 3-4 h). Thereafter, reduce culture temperature to 28 &#176;C and prepare a 1 M of isopropyl-&#946;-D-1-thiogalactopyranoside (IPTG) stock solution in H2O.</li>
	<li>Induce RTA expression of the E. coli by adding IPTG to a final concentration of 1 mM.</li>
	<li>After 3.5 h at 28 &#176;C and 220 rpm, harvest cells by centrifugation at 10,000 x g and 4 &#176;C for 10 min, wash the pellet two times with 5 mL of binding buffer (500 mM NaCl, 10 mM imidazole, and 20 mM KH2PO4, pH = 7.4) at 10,000 x g and 4 &#176;C for 10 min, and resuspend pellet in 5 mL binding buffer. 
	NOTE: Protocol can be paused at this stage and cells can be stored at 80 &#176;C for several days.</li>
</ol>
2. Purification of His-tagged RTA via Affinity Chromatography
<ol>
	<li>Sonicate cells on ice using the following protocol: 15 s pulse (20 microns), 30 s pause. Repeat this step five times.</li>
	<li>Centrifuge cell lysate at 21,000 x g and 4 &#176;C for 15 min and filter supernatant using a sterile syringe filter system (0.2 &#181;m pore size). 
	NOTE: Cell pellets of successfully sonicated samples show transparent borders.</li>
	<li>Use an automated purification system equipped with a 5 mL Ni2+-based affinity column to purify the His-tagged RTA fraction from the sterile-filtered E. coli supernatant. In general, use an elution speed of 1 mL/min and cool the whole purification system to prevent non-efficient toxin binding and loss of toxin activity. 
	NOTE: Parameters for efficient RTA purification are listed in Table 1. See also Becker et al. for further information9.
	<ol>
		<li>Briefly, equilibrate the affinity column with 20 mL of binding buffer to remove the storage buffer. Apply sterile-filtered supernatant onto the affinity column using a syringe.</li>
		<li>Wash the column with 25-35 mL of binding buffer to remove the unbound proteins from the column. Perform the washing step until UV absorbance at 280 nm is close to the initial UV value.</li>
		<li>Elute bound RTA fraction in 20-35 mL of elution buffer (500 mM imidazole, 500 mM NaCl, 20 mM KH2PO4, pH = 7.2) and keep the sample on ice (Figure 2A and Figure 2B). 
		NOTE: Elution of the RTA fraction is marked by an increase in UV absorption. Please note to exclusively collect this fraction to prevent contamination with nonspecific bound proteins.</li>
		<li>Use 20 &#181;L of eluted samples to perform Coomassie Blue staining11 or Western blot analysis12,13 (Optional). Use primary anti-His antibodies (1:1,000) and secondary anti-mouse-IgG-HRP (1:10,000) to detect RTA and verify sample purity (Figure 3).</li>
		<li>Desalt eluted RTA fraction on a 5 mL desalting column and equilibrate sample in 0.8 M sorbitol.
		<ol>
			<li>Replace the affinity column of the purification system by the desalting column and first equilibrate the column with 20 mL of 0.8 M sorbitol.</li>
			<li>Apply the eluted RTA fraction to the column via a syringe. Wash the column with 100 mL of 0.8 M sorbitol and elute the desalted RTA fraction in a 15 mL tube as soon as the UV absorption starts to increase (Figure 2C).</li>
			<li>Store the eluted RTA fraction at 4 &#176;C. 
			NOTE: Directly stop RTA fraction sampling when conductance increases to avoid salt contamination. Parameters for the desalting procedure are listed in Table 1.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Concentrate eluted RTA fraction at 10,000 x g and 4 &#176;C for 30-180 min using a 10 kDa cut-off spin column to a final volume of 1-2 mL and store sample at 4 &#176;C for 3-4 weeks. 
	Caution: Do not freeze the sample since freezing leads to a complete loss of RTA activity.</li>
	<li>Determine protein concentration using a conventional protein determination kit. Protein concentration should be in the range of 1-1.5 mg/mL. 
	NOTE: RTA tends to precipitate if the protein concentration is too high (&#62;5 mg/mL).</li>
</ol>
3. Yeast Transformation and Cell Wall Removal
<ol>
	<li>Transform wild-type or selected yeast deletion mutants with the GFP reporter plasmid pRS315-K28SP-GFP6 using standard lithium acetate transformation methods14. Incubate cells on leucine drop-out (d/o) glucose plates (2% glucose, 1.5% agar, 0.5% ammonium sulfate, 0.17% Yeast Nitrogen Base (YNB), and 0.087% d/o mix without leucine) at 30 &#176;C for 2-3 days for positive clone selection.</li>
	<li>Pick 3 different yeast clones from each plate and inoculate the clones in 100 mL of leucine d/o raffinose medium (2% raffinose, 0.5% ammonium sulfate, 0.17% YNB, and 0.087% d/o mix without leucine) at 220 rpm and 30 &#176;C to OD600 = 1.0-2.0 (2-4 x 107 cells/mL). 
	NOTE: Cell growth of the different yeast deletion strains is different. Monitor OD600 via a spectrophotometer.</li>
	<li>To calculate OD600 values, dilute samples to OD600 = 0.1-0.3 (1:5 to 1:10 dilutions) and measure OD600 in a spectrophotometer. As reference, use H2O supplemented with the corresponding amount of leucine d/o raffionose medium. Finally, resuspend OD600 = 125 in 5 mL of sterile water (5 x 108 cells/mL).</li>
	<li>Centrifuge cells at 25 &#176;C for 5 min at 10,000 x g, wash cells twice with 5 mL sterile water, and resuspend cells in 50 mL spheroplasting buffer (0.8 M sorbitol, 10 mM Tris-HCl (pH 7.5), 10 mM CaCl2, 2 mM dithiothreitol (DTT), and 200 &#181;g/mL lytic enzyme).</li>
	<li>Incubate the 50 mL culture at 100 rpm and 30 &#176;C for 90 min. Optional: Monitor spheroplast formation every 15 min by phase contrast microscopy. Under the microscope, cells should have a dark translucent gray color. Bright (refractile) cells are insufficiently digested, whereas ghosts (pale gray cells with little, if any, internal structure) are over-digested.</li>
	<li>Check spheroplast preparation efficacy for each sample.
	<ol>
		<li>After finishing step 3.5, mix 4 &#181;L of the spheroplasted 50 mL culture with 496 &#181;L spheroplasting buffer (approximately 2 &#215; 106 spheroplasts) and centrifuge for 10 min at 400 x g.</li>
		<li>Resuspend pellet in 10 mL H2O distilled, vortex sample for 30 s, and plate out 10 &#181;L of the sample on leucine d/o glucose plates (2% glucose, 1.5% agar, 0.5% ammonium sulfate, 0.17% YNB, and 0.087% d/o mix without leucine).</li>
		<li>Incubate cells for 3 days at 30 &#176;C. Count the total number of grown cell colonies on the plate. For data evaluation, use only samples with efficiency higher than 98% (total cell colony number &#60;40 colonies/plate).</li>
	</ol>
	</li>
	<li>Centrifuge cells from step 3.5 at 400 x g and 4 &#176;C for 10 min and wash cells twice with 5 mL stabilized leucine d/o raffinose medium (0.8 M sorbitol, 2% raffinose, 0.5% ammonium sulfate, 0.17% YNB, and 0.087% drop-out mix without leucine). Resuspend cells in 5 mL stabilized leucine d/o raffinose medium (1 x 108 cells/mL) and directly use cells for the GFP reporter assay measurements (Section 4).</li>
</ol>
4. GFP Reporter Assay Measurement in 96-Well Plates
<ol>
	<li>Seed out the yeast cell spheroplasts obtained in step 3.7 into 96-well plates (200 &#181;L/well).</li>
	<li>Add 70 &#181;L stabilized leucine d/o raffinose medium containing negative control (eluate of a Ni2+-affinity purified cell lysate from IPTG-induced E. coli cells expressing the empty vector) or purified RTA in a final RTA concentration of 5 &#181;M (corresponding to 160 g/L RTA) in each well.</li>
	<li>Immediately add 30 &#181;L stabilized galactose solution (30% galactose, 0.8 M sorbitol) to induce GFP expression and subsequently start the measurement. 
	NOTE: Perform at least 3 technical replicates per experiment and 3 biological replicates per yeast strain.</li>
	<li>After finishing sample preparation (steps 4.1-4.3), put the 96-well plate in a fluorescence reader and start the measurement. Use the 475/509 nm filter set required for GFP fluorescence detection. Perform measurements at 30 &#176;C, 120 rpm, and with a shaking diameter of 1 mm over a time window of 20 h (measuring intervals of 10 min). 
	NOTE: The GFP filter set is normally available in all reader systems. Temperature, time window, measuring intervals, and RTA concentration can be adjusted for own needs. Representative results are shown in Figure 4.</li>
	<li>Optional: Use additional internal controls in the measurement for quality control. Prepare a negative control by adding 30 &#181;L stabilized leucine d/o raffinose medium instead of 30% galactose (no GFP induction). In addition, add 70 &#181;L of 0.8 M sorbitol stabilized G418 solution (300 &#181;g/mL). The protein translation inhibitor G418 serves as positive control for protein inhibition as it prevents GFP expression in yeast.</li>
	<li>Calculate relative GFP fluorescence in percent for the 20 h time point according to the following equation (see Figure 4A):&#160; <img alt="Equation" src="/files/ftp_upload/56588/56588eq1.jpg" />&#160;&#160; Where NC is the negative control
	<ol>
		<li>Alternatively, determine the relative GFP fluorescence for each measuring point (in this case 10 min, see also step 4.4) using the above equation. As shown in Figure 4B, create a graph by blotting the GFP fluorescence intensities (y-axis) over time (x-axis).</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

When performing the above protocol, we recommend the following suggestions to achieve a successful outcome of the experiment.
For heterologous protein expression, it is important to not exceed the IPTG concentration of 1 mM. IPTG concentrations &#62;1 mM inhibit promoter-induced RTA expression and lead to lower toxin yields. Furthermore, cells should not be cultivated at temperatures higher than 28 &#176;C to prevent inclusion body formation, inefficient folding, and toxin inactivation. RTA expression at lower temperature (e.g., 20-28 &#176;C) is possible and also leads to the production of biological active toxin. However, the additional temperature reduction does not significantly improve RTA activity/folding and results in a lower total toxin yield compared to 28 &#176;C (data not shown).
Purification parameters of the affinity chromatography (Table 1) are optimized for the use of an automated purification system equipped with 5 mL Ni2+ affinity column and should be adjusted to other home-made or commercial column systems if RTA purity and yield are sub-optimal. In case of sub-optimal RTA purity (indicated by the appearance of additional proteins in Coomassie-stained SDS-gels), an increase in the imidazole concentration of the binding buffer normally reduces unspecific binding of positively charged proteins to the column, thereby increasing overall RTA purity. In case of sub-optimal RTA yields, introduction of additional sonification steps (4-8 pulses), longer incubation times (5 h) or larger culture volumes (&#62;1 L) can help to improve the overall toxin yield. However, there is also the opportunity to successfully purify RTA via spin column based systems (data not shown) if an automated system is not available in the lab.
During the desalting step, it is important to skip the sampling procedure as soon as the conductance starts to rise. Contamination with salt, especially imidazole, influences the protein concentration measurement, resulting in inaccurate RTA concentration determination which directly affects the RTA content used in the GFP reporter assay.
It is essential to store purified RTA at 4 &#176;C and avoid freezing. Frozen samples show a reduced activity of RTA in XTT-based cell vitality assays on HeLa cells (data not shown). Purified samples can be kept for around 3-4 weeks before their biological activity is continuously diminished. In addition, high RTA concentrations (&#8805;5 mg/mL) after spin column concentration are critical because RTA shows a tendency to precipitate in 0.8 M sorbitol.
A drawback of the method is the fact that RTA is normally not taken up by intact yeast cells. To achieve reliable results, the yeast cell wall needs to be completely removed by enzymatic treatment. Therefore, it is essential to check spheroplast formation efficiency of each yeast strain by the methods described in steps 3.4-3.5. In case of sub-optimal spheroplast preparation, the incubation time and lytic enzyme concentration should be optimized and monitored via phase contrast microscopy. Overdigestion (ghost formation) can be prevented by reducing lytic enzyme concentration and/or incubation time whereas the opposite approach is needed in case of insufficient digestion. Data from strains with insufficient cell wall removal should be excluded from the data evaluation.
It should also be mentioned that the experimental setup does not completely mimic the natural intoxication process of ricin. The cell binding B subunit of ricin (RTB) is missing which is normally responsible for efficient toxin binding to galactose residues at the cell surface of mammalian cells15. Theoretically, this limitation can be overcome by performing the experiment with purified ricin holotoxin containing its A and B subunit. Nevertheless, yeast cells do not possess galactose residues on their cell surface which could mediate binding of RTB8; this fact also explains why intact yeast cells are not sensitive against ricin. Thus, it is unclear if RTB would act as cell binding component in the yeast model. Interestingly, former studies already demonstrated that RTA is able to reach the cytosol of mammalian cells without its cell binding B subunit16. To understand this phenomenon, we decided to use only RTA in the experimental setup to identify cellular components in yeast which facilitate toxin trafficking from the plasma membrane into the cytosol. Surprisingly, the components involved in RTA trafficking in yeast show striking similarities to the components required for efficient ricin holotoxin transport in mammalian cells6. Based on these results, it can be speculated that RTB plays a minor role in intracellular ricin transport than previously supposed and is only important for the initial contact to the mammalian cell surface.
In contrast, siRNA-based systems in mammalian cells perfectly reflect the natural toxin situation, but are time-consuming and expensive3. Furthermore, the use of yeast deletion mutants bears the important advantage that the mutants show a complete knock-out of the protein of interest, whereas siRNA-based systems generally lead to a reduction of the protein level. In some cases, residual protein levels can be sufficient for toxin transport whereby no altered phenotype is observed and, thus, the involvement of the specific protein is not visible in siRNA-based systems.
Although only non-essential yeast deletion strains were used in the original study6, temperature-sensitive strains as well as decreased abundance by mRNA perturbation (DAmP) collection strains17 (reduced expression level of essential proteins) are likewise suitable for this kind of method in the future. In general, the role of essential proteins cannot be investigated with this assay because of their lack of viability, therefore represent a limitation of the assay system.
However, the new reporter assay represents an elegant and alternative strategy to existing methods which try to analyze RTA transport in the model organism S. cerevisiae. The existing studies used intracellular plasmid-driven RTA expression systems and are restricted to the analysis of the retro-translocation from the ER into the cytosol1,7. Successful implementation of the extracellular RTA application now opens the possibility to not only study ER retrotranslocation but also to indirectly analyze RTA transport from the PM though the Golgi to the ER. As the cellular function and localization of the majority of yeast genes is nowadays discovered and available in databases, the identified candidate proteins can be directly allocated to specific compartments and/or transport pathways.
Overall, the introduced reporter assay method represents an easy and robust approach to identify candidate proteins involved in intracellular trafficking of toxins or toxin subunits (e.g., RTA) which block protein translation in vivo. Relatively low standard deviations (1-10%) as well as the high data reproducibility in the original study confirms these statements. Due to the 96-well plate format, a fast screening of various yeast mutants is possible within a day after toxin purification.
In the future, there is the possibility to use the method for high-throughput screening of yeast deletion libraries. In the current setup, the applied RTA concentration (5 &#181;M) is able to decrease the relative GFP fluorescence to 50%. Under these conditions, the method offers the advantage to identify deletion mutants with negative (increased GFP fluorescence) and positive (reduced GFP fluorescence) effects on RTA transport. For high throughput screenings, a stronger difference between treated and non-treated samples is sometimes beneficial. By using higher RTA concentrations and/or increasing the measure interval (e.g., 48 h), an even more pronounced block of GFP expression should be achievable.
It is to be emphasized that this method is not only restricted to analyze the intoxication process of extracellular applied RTA in yeast, but there is also the opportunity to analyze the ER import of RTA by expressing RTA via a plasmid in this GFP reporter strain (data not shown). Furthermore, the screening approach can likewise be adapted to study intracellular transport pathways of other protein biosynthesis inhibiting proteins such as zymocin18 in the future. In contrast to the well characterized killing mode of zymocin, relatively little is known about the trafficking pathways responsible for efficient toxin transport into the cytosol19,20. Thereby, zymocin represents an optimal candidate for future studies as zymocin is naturally taken up by yeast cells. Thus, the removal of the cell wall can be omitted which improves the overall performance of the GFP reporter assay (with respect to time and costs). With the help of this powerful reporter assay, it would be feasible to dissect the intracellular transport route(s) of zymocin in yeast.

Patients suffering from infections by toxin producing bacteria represent a severe medical and financial burden for each social health care system, in particular since efficient therapeutic treatments are still largely missing. To develop new therapeutic strategies, the complex intoxication mechanisms of medically relevant A/B toxins such as cholera toxin, Shiga toxin, or ricin need to be fully understood at the molecular level based on novel powerful assays that have to be implemented.
In recent years, several studies attempted to analyze A/B toxin transport in yeast and mammalian cells by using time-consuming and cost-intensive methods such as radioactive toxin labeling1,2 as well as siRNA-based screening approaches3. In some cases, toxin trafficking has been visualized in vivo by fluorescence microscopy after chemical and/or genetic coupling of individual toxin subunits with fluorophores, quantum dots, or fluorescent proteins4,5. Unfortunately, such modifications often lead to inactive and/or altered natural properties of the toxins. Another elegant way to indirectly answer a wide variety of scientific questions is the use of reporter systems based on enzymes such as lacZ, luciferase, or fluorescent proteins (e.g. GFP or Discosoma sp. red fluorescent protein (dsRed)).
In this manuscript, a simple protocol is described which identifies cellular components required for the intracellular transport of extracellularly applied RTA in S. cerevisiae. Thereby, a fluorescence-reporter plasmid containing an N-terminal ER-import signal followed by GFP acts as a protein biosynthesis sensor, which indirectly measures RTA-mediated protein translation inhibition by GFP fluorescence emission after in vivo translation6. In case that RTA endocytosis and/or intracellular trafficking is negatively (or positively) affected in a particular yeast deletion mutant compared to wild-type, this can be detected through an increase (or decrease) in GFP fluorescence emission6.
So far, all methods analyzing RTA transport in yeast cells were restricted to the ER-to-cytosol retro-translocation process of RTA. In such an artificial system, RTA containing an ER import signal is expressed from an inducible promoter resulting in a suicidal phenotype1,7. Although the cell binding B-subunit of ricin is likewise missing in the experimental setup described in this manuscript and, thus, does not fully represent the natural situation of ricin holotoxin intoxication8, toxin transport from the plasma membrane through the Golgi apparatus to the ER can be closely mimicked with this novel assay. Interestingly, the preliminary results obtained in the pilot study indicate that the trafficking pathways used by RTA reveal striking similarities with the intoxication route of ricin holotoxin.
In summary, the described method can be used to determine the specific role of selected cellular proteins in RTA endocytosis and trafficking in yeast. Furthermore, this experimental setup might be easily adapted to other ribosome inactivating toxins produced and secreted from various yeast and bacterial species such as zymocin or Shiga toxin.

<table><tbody><tr><td>&lt;strong&gt;Bacterial and yeast strains&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>&lt;em&gt;E. coli&lt;/em&gt; BL21 DE3(Gold)</td><td>Aligent Technologies</td><td>230130</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742</td><td>Euroscarf</td><td>Y10000</td><td></td></tr><tr><td>&lt;em&gt;S. cerevisiae&lt;/em&gt; BY4742 deletion mutants</td><td>Dharmacon</td><td>YSC1054</td><td>whole collection</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Plasmids used in this protocol&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>pET24a&lt;sup&gt;(+) &lt;/sup&gt;(Novagen)</td><td>Millipore</td><td>69772-3</td><td></td></tr><tr><td>pET-RTA&lt;sub&gt;(His6)&lt;/sub&gt;</td><td>Becker &lt;em&gt;et al.&lt;/em&gt; (2016)&lt;sup&gt;3&lt;/sup&gt;</td><td></td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Reagents&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Zymolyase 20T</td><td>USBio</td><td>Z1000.250</td><td>lytic enzyme for cell wall removal</td></tr><tr><td>LB broth medium</td><td>Thermo Scientific</td><td>10855021</td><td>15 g agar was added for plate production</td></tr><tr><td>YNB</td><td>Thermo Scientific</td><td>DF0335-15-9</td><td></td></tr><tr><td>Ammonium sulfate</td><td>Sigma-Aldrich</td><td>A4418-100G</td><td></td></tr><tr><td>Yeast drop-out mix supplemts without leucine</td><td>Sigma-Aldrich</td><td>Y1376-20G</td><td></td></tr><tr><td>Agar</td><td>Sigma-Aldrich</td><td>05040-100G</td><td></td></tr><tr><td>D-glucose</td><td>Sigma-Aldrich</td><td>G8270-100G</td><td></td></tr><tr><td>DTT</td><td>Sigma-Aldrich</td><td>10197777001</td><td></td></tr><tr><td>D-raffinose</td><td>Sigma-Aldrich</td><td>83400-25G</td><td></td></tr><tr><td>D-sorbitol</td><td>Sigma-Aldrich</td><td>S1876-1KG</td><td></td></tr><tr><td>D-galactose</td><td>Sigma-Aldrich</td><td>G0750-10MG</td><td></td></tr><tr><td>G418</td><td>Thermo Scientific</td><td>11811031</td><td></td></tr><tr><td>IPTG</td><td>Sigma-Aldrich</td><td>I6758-1G</td><td></td></tr><tr><td>Imidazole</td><td>Roth</td><td>3899.1</td><td></td></tr><tr><td>PAGE ruler prestained</td><td>Fermentas</td><td>26616</td><td>protein ladder used for Western analysis</td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Material for RTA purification, desalting and reader measurements&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Spectrophotometer Ultrospec 2100 pro</td><td>Amersham</td><td></td><td></td></tr><tr><td>Soniprep 150</td><td>MSE</td><td></td><td>old model, other models available</td></tr><tr><td>Fluoroskan Ascent</td><td>Thermo Scientific</td><td>5210470</td><td>old model, not available anymore</td></tr><tr><td>&amp;Auml;KTAPurifier</td><td>Thermo Scientific</td><td>28406266</td><td>Product is discontinued and replaced</td></tr><tr><td>HisTRAP HP column</td><td>GE Healthcare</td><td>17-5248-02</td><td></td></tr><tr><td>HiTRAP desalting column</td><td>GE Healthcare</td><td>11-0003-29</td><td></td></tr><tr><td>Midisart sterile filter</td><td>Sartorius</td><td>16534K</td><td>0.2 &amp;micro;m pore size</td></tr><tr><td>BCA protein assay kit</td><td>Pierce</td><td>23225</td><td></td></tr><tr><td>660 nm assay kit</td><td>Thermo Scientific</td><td>22660</td><td></td></tr><tr><td>96 well plates</td><td>Thermo Scientific</td><td>260860</td><td></td></tr><tr><td>&lt;strong&gt;Name&lt;/strong&gt;</td><td>&lt;strong&gt;Company&lt;/strong&gt;</td><td>&lt;strong&gt;Catalog Number&lt;/strong&gt;</td><td>&lt;strong&gt;Comments&lt;/strong&gt;</td></tr><tr><td>&lt;strong&gt;Antibodies (optional)&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Anti-Tetra-His</td><td>Qiagen</td><td>34670</td><td>primary antibody; 1:1,000 dilution</td></tr><tr><td>Anti-mouse-HRP</td><td>Sigma-Aldrich</td><td>A9044-2ML</td><td>secondary antibody, 1:10,000 dilution</td></tr></tbody></table>

a simple fluorescence-based reporter assay to identify cellular components required for ricin toxin a chain (rta) trafficking in yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ultimately kill. Such A/B toxins generally consist of an enzymatically active Asubunit (e.g., ricin toxin A (RTA)) and one or more cell binding Bsubunit(s), which are responsible for toxin binding to specific cell surface receptors. Our current knowledge of how A/B toxins are capable of efficiently intoxicating cells helped scientists to understand fundamental cellular mechanisms, like endocytosis and intracellular protein sorting in higher eukaryotic cells. From a medical point of view, it is likewise important to identify the major toxin trafficking routes to find adequate treatment solutions for patients or to eventually develop therapeutic toxin-based applications for cancer therapy.
Since genome-wide analyses of A/B toxin trafficking in mammalian cells is complex, time-consuming, and expensive, several studies on A/B toxin transport have been performed in the yeast model organism Saccharomyces cerevisiae. Despite being less complex, fundamental cellular processes in yeast and higher eukaryotic cells are similar and very often results obtained in yeast can be transferred to the mammalian situation.
Here, we describe a fast and easy to use reporter assay to analyze the intracellular trafficking of RTA in yeast. An essential advantage of the new assay is the opportunity to investigate not only RTA retro-translocation from the endoplasmic reticulum (ER) into the cytosol, but rather endocytosis and retrograde toxin transport from the plasma membrane into the ER. The assay makes use of a reporter plasmid that allows indirect measurement of RTA toxicity through fluorescence emission of the green fluorescent protein (GFP) after in vivo translation. Since RTA efficiently prevents the initiation of protein biosynthesis by 28S rRNA depurination, this assay allows the identification of host cell proteins involved in intracellular RTA transport through the detection of changes in fluorescence emission.

The general workflow of the protocol described in this manuscript is illustrated in Figure 1, roughly summarizing the single steps for successful RTA purification and the subsequent GFP reporter assay experiment. A more detailed description of each individual step can be found in the protocol. Figure 2 illustrates the expected result of a successful RTA purification by affinity chromatography (Figure 2A) and empty vector control (Figure 2B). Figure 2C shows a representative outcome of a desalting experiment illustrating the ideal separation of the protein peak (blue) and the salt-eluted peak (brown). An effective RTA purification is presented in form of a Coomassie-stained SDS-gel in Figure 3. Finally, Figure 4 summarizes some original results obtained by this simple and robust GFP-based reporter assay method6. Figure 4A illustrates that the established GFP reporter assay is able to produce reliable results and schematically depicts the GFP reporter plasmid used in this study. In the graph, the relative GFP fluorescence of yeast spheroplasts under RTA-treated and non-treated conditions is compared with each other. As expected, non-induced as well as G418-treated cells show almost no GFP fluorescence. In non-treated and negative control-treated (empty vector) cells, GFP expression is not affected and significant differences between both samples were not observed. In contrast, RTA treatment leads to the expected GFP fluorescence reduction mediated by its ribosome inhibiting activity. In Figure 4B, a time curve of RTA-induced GFP fluorescence inhibition is displayed. This kind of data presentation contains additional information about the timing of GFP expression (90 min after galactose induction) and the starting point of the translational inhibition by RTA (210 min after application in wild-type cells). The delay in RTA-mediated fluorescence decrease most likely reflects the uptake kinetics and intracellular transport of RTA to the cytosol. Figure 4C illustrates the relative GFP fluorescence obtained from selected yeast mutants after a 20 h RTA-treatment and compared to RTA-treatment and compared to RTA-treated wild-type cells. The ERAD components Hrd1p and Der1p were chosen as suitable positive controls because earlier studies already demonstrated that both proteins are involved in ER-to-cytosol retrotranslocation of RTA in yeast1. Therefore, both mutants show the expected increase in GFP fluorescence compared to wild-type cells. In contrast, relative GFP fluorescence values in &#916;yos9 and &#916;nup120 cells are not significantly different. It has already been described that neither a lack of the ER quality control lectin Yos9p nor a lack in the nuclear pore protein Nup20p affect RTA toxicity and transport1. A list of the specific affinity chromatography and desalting parameters can be found in Table 1.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56588/56588fig1.jpg" /> 
Figure 1: General workflow of RTA purification and GFP-based reporter assay. The RTA purification (left) starts with transformation of E. coli with the expression or empty vector followed by cultivation and IPTG-induced RTA expression. After cell lysis and sterile-filtration, the supernatant is purified via Ni2+ affinity chromatography and sample purity is verified via Coomassie staining or Western blot. Before the eluted RTA fractions are ready to use in the GFP reporter assay experiment, fractions need to be desalted and concentrated, and protein concentration must be determined. The GFP reporter assay experiment (right) starts with the transformation of wild-type and/or selected yeast deletion mutants with the GFP expression construct. After cultivation, the cell wall is removed by enzymatic treatment to allow RTA in vivo uptake. Then, GFP fluorescence of yeast cell spheroplasts is measured under toxin-treated and non-treated conditions over time in a fluorescence reader (475/509 nm). Finally, relative GFP fluorescence is determined using the equation shown in step 4.6 and is displayed as illustrated in Figure 4. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56588/56588fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56588/56588fig2.jpg" /> 
Figure 2: Representative affinity chromatography and desalting results of E. coli cells containing pET-RTA(His)6 or the empty vector control. (A) Typical purification graph of an E. coli cell lysate sample expressing RTA(His)6 from vector pET24-RTA(His)6. Blue curve represents the UV absorption at 280 nm caused by the aromatic rings of the amino acids Trp, Tyr, and Cys and serves as protein indicator. During column loading, unbound proteins are detected in the flow-through (0-10 mL). After removing unbound proteins by washing with binding buffer, elution buffer concentration is increased from 0 to 100% (red curve) and bound proteins are immediately eluted from the column. The peak of the blue curve between 25 and 30 mL represents the fraction of bound His-tagged RTA (black box). (B) Typical purification graph of an E. coli lysate from cells expressing empty vector control. Same purification protocol as in (A). As shown in the black box, no proteins are eluted from the column in the negative control. (C) Desalting diagram of an E. coli pET24-RTA(His)6 sample after affinity purification. The diagram shows the separation of the RTA protein fraction (peak of 280 nm absorption between 20-30 mL) and the salt fraction (increasing conductance peak after 30 mL). <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56588/56588fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56588/56588fig3.jpg" /> 
Figure 3: Representative Western analysis of different samples before and after affinity purification. 20 &#181;L cell lysate (lanes 2-4) or purified sample (lanes 5-7) of different RTA variants (only unmodified RTA was used in this study) analyzed by SDS-PAGE and Coomassie blue staining. Lane 1, protein ladder; lane 2, unmodified RTA; lane 3, RTAHDEL; lane 4, RTAKDEL; lane 5, unmodified RTA; lane 6, RTAHDEL; lane 7, RTAKDEL. <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56588/56588fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/56588/56588fig4.jpg" /> 
Figure 4: Representative results obtained by the reporter assay for RTA-treated wild-type and selected yeast deletion mutants. (A) Relative GFP fluorescence of wild-type yeast spheroplasts containing the reporter plasmid pRS315-K28SP-GFP (right) under induced (3% galactose, GFPind.) and non-induced (2% raffinose, GFPrepr.) conditions after 20 h GFP induction. Relative GFP fluorescence was also analyzed in the presence of RTA (5 &#181;M), G418 (300 &#181;g/mL) or the negative control (NC). The protein translation inhibitor G418 serves as positive control and prevents GFP expression in yeast. Mean values and standard deviation (SD) are indicated. (B) Relative GFP fluorescence from (A) displayed as time course over 20 h. This diagram provides additional information about the timing of GFP expression and translation inhibition by RTA; the curve behavior indirectly reflects the kinetics of RTA uptake and intracellular transport to the cytosol. (C) Representative result of selected yeast mutants defective in cellular proteins that are known to be involved (Hrd1p and Der1p) or not involved (Yos9p and Nup120p) in RTA trafficking in yeast1,6. The dotted line represents a threshold of significance which is based on the fluorescence emission obtained by the positive control strains (for further explanation see reference6). Mean values and standard deviation (SD) are indicated. The figure was modified from Becker et al.6 <a href="https://cloudflare-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56588/56588fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Affinity chromotography parameter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring parameters</td>
			<td>UV (280 nm), Elution buffer concentration, Conductance</td>
			<td></td>
		</tr>
		<tr>
			<td>Settings</td>
			<td>Variable/Unit</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Flow rate</td>
			<td>mL/min</td>
			<td>1</td>
		</tr>
		<tr>
			<td>Maximum column pressure</td>
			<td>MPa</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>Wavelength</td>
			<td>nm</td>
			<td>280 nm</td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td>Pump position</td>
			<td>A</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td>Pump position</td>
			<td>B</td>
		</tr>
		<tr>
			<td>Starting concentration elution buffer</td>
			<td>%</td>
			<td>0</td>
		</tr>
		<tr>
			<td>System volume compensation</td>
			<td>mL</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Column equilibration</td>
			<td>mL</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Sample injection over superloop/syringe</td>
			<td>mL</td>
			<td>cell lysate volume</td>
		</tr>
		<tr>
			<td>Washing step</td>
			<td>mL</td>
			<td>20-35</td>
		</tr>
		<tr>
			<td>Elution step</td>
			<td>mL</td>
			<td>20-35</td>
		</tr>
		<tr>
			<td>Concentration elution buffer during elution</td>
			<td>%</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Recalibration of column with 20% EtOH</td>
			<td>mL</td>
			<td>50</td>
		</tr>
		<tr>
			<td>Desalting parameter</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Measuring parameters</td>
			<td>UV (280 nm), Conductance</td>
			<td></td>
		</tr>
		<tr>
			<td>Settings</td>
			<td>Variable/Unit</td>
			<td>Value</td>
		</tr>
		<tr>
			<td>Flow rate</td>
			<td>mL/min</td>
			<td>4</td>
		</tr>
		<tr>
			<td>Maximum column pressure</td>
			<td>MPa</td>
			<td>0.35</td>
		</tr>
		<tr>
			<td>Wavelength</td>
			<td>nm</td>
			<td>280 nm</td>
		</tr>
		<tr>
			<td>Binding buffer</td>
			<td>Pump position</td>
			<td>A</td>
		</tr>
		<tr>
			<td>Elution buffer</td>
			<td>Pump position</td>
			<td>B</td>
		</tr>
		<tr>
			<td>Starting concentration elution buffer</td>
			<td>%</td>
			<td>0</td>
		</tr>
		<tr>
			<td>System volume compensation</td>
			<td>mL</td>
			<td>10</td>
		</tr>
		<tr>
			<td>Column equilibration</td>
			<td>mL</td>
			<td>20</td>
		</tr>
		<tr>
			<td>Sample injection over superloop/syringe</td>
			<td>mL</td>
			<td>cell lysate volume</td>
		</tr>
		<tr>
			<td>Desalting buffer injection (0.8 M sorbitol)</td>
			<td>mL</td>
			<td>100</td>
		</tr>
		<tr>
			<td>Recalibration of column with 20% EtOH</td>
			<td>mL</td>
			<td>50</td>
		</tr>
	</tbody>
</table>
Table 1: Parameters used for column-based affinity purification and desalting. List of all relevant column settings (e.g., flow rate, elution buffer concentration, equilibration and washing step duration) and measured parameters (e.g., conductance or UV absorption).

Watch this Scientific Journal Video about A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast at JoVE.com

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

Bacterial and plant A/B toxins exploit the natural trafficking pathways in eukaryotic cells to reach their intracellular target(s) in the cytosol and to ...

a-simple-fluorescence-based-reporter-assay-to-identify-cellular

Nanyang Technological University

Research

JoVE Journal

Biology

7.9K Views.  Saarland University. In the manuscript, we describe the use of a yeast-based fluorescence reporter assay to identify cellular components involved in the trafficking and killing processes of the cytotoxic A subunit of the plant toxin ricin (RTA).

Video: A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain RTA Trafficking in Yeast

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence microscopy. The technique is equally suited for visualization of static yeast populations, or time courses experiments up to ten hours in length. My microscopy investigates epigenetic inheritance at the silent mating loci in S. cerevisiae. There are two silent mating loci, HML and HMR, which are normally not expressed as they are packaged in heterochromatin. In the sir1 mutant background silencing is weakened such that each locus can either be in the expressed or silenced epigenetic state, so in the population as a whole there is a mix of cells of different epigenetic states for both HML and HMR. My microscopy demonstrated that there is no relationship between the epigenetic state of HML and HMR in an individual cell. sir1 cells stochastically switch epigenetic states, establishing silencing at a previously expressed locus or expressing a previously silenced locus. My time course microscopy tracked individual sir1 cells and their offspring to score the frequency of each of the four possible epigenetic switches, and thus the stability of each of the epigenetic states in sir1 cells. See also  Xu et al., Mol. Cell 2006.

A rapid technique for the visualization of growing immobilized yeast cells, here applied to fluorescent reporters at the silent mating loci HML and HMR

We present here a simple, rapid, and extremely flexible technique for the immobilization and visualization of growing yeast cells by epifluorescence ...

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is particularly relevant for survival in fluctuating environments such as those found in beer fermentation. However, sugar transitions are an environmental variable that is challenging to predict and detect, impacting the outcome of beer fermentations. This protocol describes an in vivo system to monitor transcriptional activation associated with the glucose-to-maltose metabolic shift in Saccharomyces eubayanus that applies to different wild Saccharomyces yeast strains. 
The system employs an episomal bioluminescent transcriptional reporter for maltose metabolism, focusing on MAL32, since it provides a good readout for metabolic shifts, as studied in S. cerevisiae. For this, yeast strains were transformed with plasmids containing the MAL32 regulatory region from S. eubayanus, controlling the expression of a gene encoding for a destabilized version of firefly luciferase1, and a hygromycin resistance gene used exclusively during transformation to ensure plasmid acquisition. Following selection, transformed yeast cells can be cultured under non-selective conditions, as the episomal plasmid remains stable in culture conditions for up to 7 days. 
This system was validated under a complex sugar environment in microfermentation assays, confirming the effectiveness of the luciferase reporter in informing metabolic transitions. Samples were collected regularly and analyzed with a luminometer, providing continuous insights into yeast responses. While broadly applicable, this protocol is particularly valuable for assessing yeast performance under fermentation conditions, where metabolic changes pose a significant challenge. Additionally, this methodology can be adapted by selecting alternative promoters to explore a broader range of responses to environmental changes, allowing characterization as well as optimization of wild yeast strains for diverse industrial applications.

This protocol employs a bioluminescent reporter, allowing measurements of transcriptional activity in Saccharomyces eubayanus to monitor the glucose-to-maltose transition, enabling real-time analysis of metabolic adaptations and supporting strain optimization for industrial fermentation under diverse conditions.

Sequential sugar consumption, from a preferred sugar source to a less preferred one, represents a critical metabolic adaptation in yeast, which is ...

Genetics

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target cells. Inside the host cell the effector proteins manipulate cellular functions to the benefit of the bacteria. To better understand the control of type III secretion during host cell interaction, sensitive and accurate assays to measure translocation are required. We here describe the application of an assay based on the fusion of a Yersinia enterocolitica effector protein fragment (Yersinia outer protein; YopE) with TEM-1 beta-lactamase for quantitative analysis of translocation. The assay relies on cleavage of a cell permeant FRET dye (CCF4/AM) by translocated beta-lactamase fusion. After cleavage of the cephalosporin core of CCF4 by the beta-lactamase, FRET from coumarin to fluorescein is disrupted and excitation of the coumarin moiety leads to blue fluorescence emission. Different applications of this method have been described in the literature highlighting its versatility. The method allows for analysis of translocation in vitro and also in in vivo, e.g., in a mouse model. Detection of the fluorescence signals can be performed using plate readers, FACS analysis or fluorescence microscopy. In the setup described here, in vitro translocation of effector fusions into HeLa cells by different Yersinia mutants is monitored by laser scanning microscopy. Recording intracellular conversion of the FRET reporter by the beta-lactamase effector fusion in real-time provides robust quantitative results. We here show exemplary data, demonstrating increased translocation by a Y. enterocolitica YopE mutant compared to the wild type strain.

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

Effector translocation into host cells via a type III secretion system is a common virulence strategy among gram-negative bacteria. A beta-lactamase effector fusion based assay for quantitative analysis of translocation was applied. In Yersinia infected cells, conversion of a FRET reporter by the beta-lactamase is monitored using laser scanning microscopy.

Many gram-negative bacteria including pathogenic Yersinia spp. employ type III secretion systems to translocate effector proteins into eukaryotic target ...

Immunology and Infection

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a quality control mechanism that can target and selectively degrade cellular material including organelles1-4. For example, damaged or redundant mitochondria (Fig. 1), not disposed of by autophagy, can represent a threat to cellular homeostasis and cell survival. In the yeast, Saccharomyces cerevisiae, nutrient deprivation (e.g., nitrogen starvation) or damage can promote selective turnover of mitochondria by autophagy in a process termed mitophagy 5-9.
We describe a simple fluorescence microscopy approach to assess autophagy. For clarity we restrict our description here to show how the approach can be used to monitor mitophagy in yeast cells. The assay makes use of a fluorescent reporter, Rosella, which is a dual-emission biosensor comprising a relatively pH-stable red fluorescent protein linked to a pH-sensitive green fluorescent protein. The operation of this reporter relies on differences in pH between the vacuole (pH ~ 5.0-5.5) and mitochondria (pH ~ 8.2) in living cells. Under growing conditions, wild type cells exhibit both red and green fluorescence distributed in a manner characteristic of the mitochondria. Fluorescence emission is not associated with the vacuole. When subjected to nitrogen starvation, a condition which induces mitophagy, in addition to red and green fluorescence labeling the mitochondria, cells exhibit the accumulation of red, but not green fluorescence, in the acidic vacuolar lumen representing the delivery of mitochondria to the vacuole. Scoring cells with red, but not green fluorescent vacuoles can be used as a measure of mitophagic activity in cells5,10-12.

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

A robust approach to monitor the delivery of organelles to the acidic lumen of the yeast vacuole for degradation and recycling is described. The method relies on the specific labeling of target organelles with a genetically encoded dual-emission fluorescence pH-biosensor, and visualization of individual cells using fluorescence microscopy.

Autophagy is important for turnover of cellular components under a range of different conditions. It serves an essential homeostatic function as well as a ...

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using fluorochromes like Green Fluorescent Protein (GFP) directly attached to the protein of interest has become increasingly popular 1. Using GFP and similar fluorochromes the subcellular localisations and movements of proteins can be detected in a fluorescent microscope. Moreover, also the subnuclear localisation of a certain region of a chromosome can be studied using this technique. GFP is fused to the Lac Repressor protein (LacR) and ectopically expressed in the cell where tandem repeats of the lacO sequence has been inserted into the region of interest on the chromosome2. The LacR-GFP will bind to the lacO repeats and that area of the genome will be visible as a green dot in the fluorescence microscope. Yeast is especially suited for this type of manipulation since homologous recombination is very efficient and thereby enables targeted integration of the lacO repeats and engineered fusion proteins with GFP 3. Here we describe a quantitative method for live cell analysis of fission yeast. Additional protocols for live cell analysis of fission yeast can be found, for example on how to make a movie of the meiotic chromosomal behaviour 4. In this particular experiment we focus on subnuclear organisation and how it is affected during gene induction. We have labelled a gene cluster, named Chr1, by the introduction of lacO binding sites in the vicinity of the genes. The gene cluster is enriched for genes that are induced early during nitrogen starvation of fission yeast 5. In the strain the nuclear membrane (NM) is labelled by the attachment of mCherry to the NM protein Cut11 giving rise to a red fluorescent signal. The Spindle Pole body (SPB) compound Sid4 is fused to Red Fluorescent Protein (Sid4-mRFP) 6. In vegetatively growing yeast cells the centromeres are always attached to the SPB that is embedded in the NM 7. The SPB is identified as a large round structure in the NM. By imaging before and 20 minutes after depletion of the nitrogen source we can determine the distance between the gene cluster (GFP) and the NM/SPB. The mean or median distances before and after nitrogen depletion are compared and we can thus quantify whether or not there is a shift in subcellular localisation of the gene cluster after nitrogen depletion.

The fission yeast, Schizosaccharomyces pombe, is a good model system to study basic cellular processes. Here we describe a method to perform quantitative live cell analysis of fission yeast. In this particular experiment we focus on organisation of the genome within the cell nucleus, but the method can also be used to study cytosolic factors.

Several microscopy techniques are available today that can detect a specific protein within the cell. During the last decade live cell imaging using ...

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

The quantification of gene expression at the single cell level uncovers novel regulatory mechanisms obscured in measurements performed at the population level. Two methods based on microscopy and flow cytometry are presented to demonstrate how such data can be acquired. The expression of a fluorescent reporter induced upon activation of the high osmolarity glycerol MAPK pathway in yeast is used as an example. The specific advantages of each method are highlighted. Flow cytometry measures a large number of cells (10,000) and provides a direct measure of the dynamics of protein expression independent of the slow maturation kinetics of the fluorescent protein. Imaging of living cells by microscopy is by contrast limited to the measurement of the matured form of the reporter in fewer cells. However, the data sets generated by this technique can be extremely rich thanks to the combinations of multiple reporters and to the spatial and temporal information obtained from individual cells. The combination of these two measurement methods can deliver new insights on the regulation of protein expression by signaling pathways.

Two complementary methods based on flow cytometry and microscopy are presented which enable the quantification, at the single cell level, of the dynamics of gene expression induced by the activation of a MAPK pathway in yeast.

The quantification of gene expression at the  single cell level uncovers novel regulatory mechanisms obscured in measurements  performed at the population ...

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 ...

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal remodeling and vesicular trafficking in living cells. Quantitative methodologies using chimeric GFP fusions have been developed for many applications; however, GFP is somewhat resistant to proteolysis, thus its fluorescence persists in the lysosome/vacuole, which can impede quantification of cargo trafficking in the endocytic pathway. An alternative method for quantifying endocytosis and post-endocytic trafficking events makes use of superecliptic pHluorin, a pH-sensitive variant of GFP that is quenched in acidic environments. Chimeric fusion of pHluorin to the cytoplasmic tail of transmembrane cargo proteins results in a dampening of fluorescence upon incorporation of the cargo into multivesicular bodies (MVBs) and delivery to the lysosome/vacuole lumen. Thus, quenching of vacuolar fluorescence facilitates quantification of endocytosis and early events in the endocytic pathway. This paper describes methods using pHluorin-tagged cargos for quantification of endocytosis via fluorescence microscopy, as well as population-based assays using flow cytometry.

Accurate quantification of vesicular trafficking events often provides key insights into roles for specific proteins and the effects of mutations. This paper presents methods for using superecliptic pHluorin, a pH-sensitive GFP variant, as a tool for quantification of endocytic events in living cells using quantitative fluorescence microscopy and flow cytometry.

Green fluorescent protein (GFP) and its variants are widely used tools for studying protein localization and dynamics of events such as cytoskeletal ...

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a fluorescent reporter. The constantly evolving list of genetically encoded fluorescent proteins (FPs) presents users with several alternatives when it comes to fluorescent fusion design. Each FP has specific optical and biophysical properties that can affect the biochemical, cellular, and functional properties of the resulting fluorescent fusions. For instance, several FPs tend to form nonspecific oligomers that are susceptible to impede on the function of the fusion partner. Unfortunately, only a few methods exist to test the impact of FPs on the behavior of the fluorescent reporter. Here, we describe a simple method that enables the rapid assessment of the impact of FPs using polyglutamine (polyQ) toxicity assays in the budding yeast Saccharomyces cerevisiae. PolyQ-expanded huntingtin proteins are associated with the onset of Huntington&#39;s disease (HD), where the expanded huntingtin aggregates into toxic oligomers and inclusion bodies. The aggregation and toxicity of polyQ expansions in yeast are highly dependent on the sequences flanking the polyQ region, including the presence of fluorescent tags, thus providing an ideal experimental platform to study the impact of FPs on the behavior of their fusion partner.

This article describes protocols to assess the effect of fluorescent proteins on the aggregation and toxicity of misfolded polyglutamine expansion for the rapid evaluation of a newly uncharacterized fluorescent protein in the context of fluorescent reporters.

For the investigation of protein localization and trafficking using live cell imaging, researchers often rely on fusing their protein of interest to a ...

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry

Protein self-assembly governs protein function and compartmentalizes cellular processes in space and time. Current methods to study it suffer from low-sensitivity, indirect read-outs, limited throughput, and/or population-level rather than single-cell resolution. We designed a flow cytometry-based single methodology that addresses all of these limitations: Distributed Amphifluoric FRET or DAmFRET. DAmFRET detects and quantifies protein self-assemblies by sensitized emission FRET in vivo, enables deployment across model systems-from yeast to human cells-and achieves sensitive, single-cell, high-throughput read-outs irrespective of protein localization or solubility.

This article describes a FRET-based flow cytometry protocol to quantify protein self-assembly in both S. cerevisiae and HEK293T cells.

A Simple Fluorescence-based Reporter Assay to Identify Cellular Components Required for Ricin Toxin A Chain (RTA) Trafficking in Yeast

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Chapters in this video

A Rapid Technique for the Visualization of Live Immobilized Yeast Cells

In Vivo Monitoring of Transcriptional Activity During Metabolic Transition Using a Bioluminescent Reporter in Yeast

Analysis of Yersinia enterocolitica Effector Translocation into Host Cells Using Beta-lactamase Effector Fusions

A Fluorescence Microscopy Assay for Monitoring Mitophagy in the Yeast Saccharomyces cerevisiae

Quantitative Live Cell Fluorescence-microscopy Analysis of Fission Yeast

Temporal Quantification of MAPK Induced Expression in Single Yeast Cells

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

Applications of pHluorin for Quantitative, Kinetic and High-throughput Analysis of Endocytosis in Budding Yeast

Effect of Fluorescent Proteins on Fusion Partners Using Polyglutamine Toxicity Assays in Yeast

Detecting and Characterizing Protein Self-Assembly In Vivo by Flow Cytometry