Uptake Rate Measurement of Amino Acids in Normal and Treated Yeast Cells

1. Introduction

Yeast (Saccharomyces cerevisiae) is a widely used model organism for studying cellular transport processes. Quantifying the uptake of amino acids provides insight into metabolic regulation, membrane transporters, and the effect of xenobiotic compounds that may interfere with normal nutrient acquisition.

This page outlines a standard protocol for measuring the uptake rates of selected amino acids using ^14Clabelled substrates. The method is applicable to both untreated (control) yeast cultures and cultures exposed to xenobiotics (e.g., heavy metals, pharmaceuticals, or industrial solvents) that may alter transporter activity.

2. Principle of the Assay

The assay is based on the rapid incorporation of a radiolabeled amino acid into the cytoplasm of yeast cells. After a short incubation, the reaction is stopped by rapid filtration and washing, and the retained radioactivity on the filter (proportional to intracellular amino acid) is measured with a scintillation counter. The uptake rate (nmolminmg dry weight) is calculated from the amount of radioactivity, specific activity of the labelled substrate, and cell mass.

3. Materials and Reagents

• Yeast strain (wildtype or genetically defined mutant)
• Growth medium: YPD or defined minimal medium
• ^14Clabeled amino acid (e.g., L[U^14C]leucine, L[U^14C]alanine)
• Unlabeled amino acid (for competition studies)
• Xenobiotic compound(s) to be tested
• Icecold phosphatebuffered saline (PBS, pH 7.4)
• 0.45m nitrocellulose filters (prewet with PBS)
• Scintillation cocktail and liquid scintillation counter
• Microcentrifuge, vortex, water bath (30C), and a timer

4. Cell Preparation

1. Culture growth: Inoculate a single colony into 50mL YPD and incubate at 30C, 200rpm until midlog phase (OD0.60.8).
2. Treatment with xenobiotic: Split culture into two flasks. Add the xenobiotic to the test flask at the desired concentration (e.g., 100M CdCl) and incubate for 30min. Keep the control flask untreated.
3. Harvest cells: Chill both flasks on ice, centrifuge at 3000g for 5min at 4C, discard supernatant.
4. Wash: Resuspend pellets in 20mL icecold PBS, repeat centrifugation. Perform two washes to remove residual medium and xenobiotic.
5. Standardize cell concentration: Adjust each suspension to an OD of 1.0 (110cellsmL). Record the exact OD for later conversion to dry weight (1OD0.16mgdryweightmL).

5. Uptake Assay Procedure

5.1. Reaction Setup

1. Prewarm a water bath to 30C.
2. Prepare uptake buffer containing the unlabeled amino acid at the desired concentration (typically 10M). Add ^14Camino acid to give a final specific activity of 50cpmpmol.
3. Aliquot 500L of cell suspension into prewarmed microcentrifuge tubes (one tube per time point). Keep tubes on a rack inside the water bath.

5.2. Initiate Uptake

4. Start timing, then add 50L of the uptake mixture to each tube (final volume550L). Mix gently by vortexing for 5s.
5. Collect samples at defined times (e.g., 10, 20, 30, 60s). For each time point, have a duplicate tube.

5.3. Stop Reaction

7. At each time point, rapidly filter the entire reaction mixture through a prewet nitrocellulose filter using a vacuum manifold (<150mmHg).
8. Immediately wash the filter with 5mL icecold PBS to remove extracellular radioactivity.
9. Transfer the filter to a scintillation vial, add 5mL scintillation cocktail, and let the filter soak for at least 30min before counting.

5.4. Data Acquisition

Count each vial for 2min (or until a stable count rate is achieved). Record counts per minute (cpm) and subtract background counts obtained from blank filters processed identically but without cells.

6. Data Analysis and Calculations

For each time point:

1. Convert cpm to picomoles of amino acid taken up:
   pmol=(cpmbackground)(Specific Activity10)
   where Specific Activity = cpmpmol of the ^14C tracer.
2. Normalize to cell dry weight:
   Uptake (nmolminmg) = (pmolincubation time (min))(OD0.16mgmL)

Plot uptake (nmolminmg) versus time. The initial linear portion (first 3060s) gives the initial uptake velocity (V). Compare V between control and treated cells to assess xenobiotic impact.

Figure 1 Representative plot of aminoacid uptake in untreated (blue) and xenobiotictreated (red) yeast. Initial slopes correspond to V.

7. Interpretation of Results

• Decrease in V suggests inhibition of transporter activity, possible competition at the binding site, or membrane damage.
• Increase in V may indicate upregulation of transporters as a stress response.
• Perform kinetic analysis (MichaelisMenten) by varying unlabeled aminoacid concentration (02mM) and fitting V to obtain K_M and V_max. Shifts in K_M imply altered affinity; changes in V_max reflect changes in transporter number or activity.
• Correlate findings with independent assays (e.g., transcript levels of aminoacid permeases, membrane integrity tests).

8. Troubleshooting and Tips

9. References

1. Rothstein, S. J., & Bruening, G. (2013). Radiolabeled substrate uptake in yeast. Methods in Enzymology, 531, 247261.
2. Alvarez, A., et al. (2019). Impact of cadmium on aminoacid transport in S. cerevisiae. Environmental Toxicology, 34(2), 112120.
3. ShacharHadar, L., & Dahan, O. (2020). Kinetic analysis of permease activity using ^14Clabelled amino acids. Biochimica et Biophysica Acta, 1862(7), 183245.