Yeast Trk1 Potassium Transporter Gradually Changes Its Affinity in Response to Both External and Internal Signals
<p>Changes in Trk1’s transport capacity, intracellular K<sup>+</sup> content and membrane potential upon shifts in external K<sup>+</sup> concentration. (<b>A</b>) Growth of strains BYT12 (trk1Δ trk2Δ), BY4741 wt and BYT2 (trk2Δ), transformed either with the YCp352 empty vector, pScTRK1 multicopy vector or pCScTRK1 centromeric vector, on YNB-F plates supplemented with indicated concentrations of K<sup>+</sup>. Pictures were captured after 3 days of incubation at 30 °C. (<b>B</b>) Kinetic parameters of rubidium uptake in potassium non-starved (NS) and starved (ST) cells. BYT12[pCScTRK1] cells were grown as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. ST-cells were further incubated in a YNB-F (15 μM K<sup>+</sup>, conditions of potassium starvation) for an additional 3 h. Rb<sup>+</sup> uptake was measured and kinetic parameters calculated as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>C</b>) Time course of the changes in kinetic parameters of rubidium uptake. BYT12[pCScTRK1] cells were grown as in (<b>B</b>) and starved of potassium in YNB-F for 1, 3 and 5 h. The estimation of kinetic parameters was the same as in (<b>B</b>). (<b>D</b>) Initial rates of 50 µM Rb<sup>+</sup> uptake in BYT12[pCScTRK1] cells within the first hour of K<sup>+</sup> starvation. Cells were grown overnight as in (<b>B</b>), transferred to a YNB-F, and the uptake of Rb<sup>+</sup> was measured immediately and after 5, 15, 30, 45 and 60 min. (<b>E</b>) Initial rates of 50 µM Rb<sup>+</sup> uptake in BYT12[pCScTRK1] (black columns) and BYT12[YCp352] (grey columns) cells incubated in media with various K<sup>+</sup> concentrations for 1 h. Cells were grown as in (<b>B</b>) and then incubated in a YNB-F supplemented with the indicated concentrations of K<sup>+</sup>. (<b>F</b>) Initial rates of 5 mM Rb<sup>+</sup> uptake in BYT12 [pCScTRK1] (black columns) and BYT12 [YCp352] (grey columns). The experiment was performed as in (<b>E</b>). (<b>G</b>) Intracellular K<sup>+</sup> content in BYT12[pCScTRK1] cells after incubation for 1 h in YNB-F media with various K<sup>+</sup> concentrations. Cells were incubated as in (<b>E</b>), 3 aliquots of cell suspension were withdrawn immediately, and the concentration of K<sup>+</sup> was estimated as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>H</b>) Relative membrane potential of BYT12[pCScTRK1] cells after incubation for 1 h in YNB-F media with various K<sup>+</sup> concentrations. Cells were grown overnight and incubated as in (<b>E</b>), then resuspended in an MES buffer, diS-C3(3) probe was added and the fluorescence emission spectra were measured. Staining curves are presented as the time-dependent spectral shift of the wavelength maximum (λ<sub>max</sub>) of diS-C3(3) fluorescence emission. Non-starved cells (black curve) are cells probed without 1-h incubation in a YNB-F.</p> "> Figure 2
<p>Effect of high external Na<sup>+</sup> concentration on Trk1-mediated Rb<sup>+</sup> uptake. BYT12 [pCScTRK1] cells were grown overnight as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>, and then incubated in a YNB-F supplemented with either 1 mM K<sup>+</sup> or 1 mM K<sup>+</sup> and 0.5 M Na<sup>+</sup> for 1 h. Initial rates of 50 µM (black columns) and 5 mM (grey columns) Rb<sup>+</sup> uptake were measured, as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>.</p> "> Figure 3
<p>Differences in kinetic parameters, intracellular K<sup>+</sup> content and membrane potential among strains. Cells expressing <span class="html-italic">TRK1</span> either from chromosome (BY4741[YCp352] and BYT2 (<span class="html-italic">trk2</span>Δ)[YCp352]) or from centromeric (BYT12[pCScTRK1]) and multicopy plasmids (BYT12[pScTRK1]), respectively, were grown as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>, and used directly (non-starved (NS) cells) or first incubated in a YNB-F (15 μM K<sup>+</sup>, conditions of potassium starvation) for 1 h (starved (ST) cells). (<b>A</b>) Differences in kinetic parameters of Rb<sup>+</sup> uptake. Initial uptake rates and kinetic parameters were estimated as in <a href="#jof-08-00432-f001" class="html-fig">Figure 1</a>B. (<b>B</b>) Intracellular K<sup>+</sup> content. Three aliquots of NS (black columns) or ST (grey columns) cell suspension were withdrawn and the concentration of K<sup>+</sup> in cells was estimated as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>C</b>) Relative membrane potential. The membrane-potential measurement was performed as in <a href="#jof-08-00432-f001" class="html-fig">Figure 1</a>G with NS (black columns) and ST (grey columns) cells. The λ<sub>max</sub> reached after 50 min of staining is shown for each strain. Significant differences in (<b>B</b>,<b>C</b>) are indicated with asterisks (* <span class="html-italic">p</span> < 0.05; ** <span class="html-italic">p</span> < 0.01; *** <span class="html-italic">p</span> < 0.001).</p> "> Figure 4
<p>Effects of Leu 949 substitution. (<b>A</b>) Localization of Leu949 within a 3D model of Trk1 protein. P-helix 3 is shown in green, the position of Leu949 in P3 is highlighted in red, and the part of the selectivity filter (SF) in blue. For better clarity, only MPM domains 1 and 3 are shown. (<b>B</b>) Growth of BYT12 cells without <span class="html-italic">TRK1</span> (BYT12[YCp352]) or with centromeric vector harbouring <span class="html-italic">TRK1</span> (native) and its mutated versions on YNB-F plates supplemented with K<sup>+</sup>, as indicated. Images were captured after 5 days of incubation at 30 °C. (<b>C</b>) Localization of GFP-tagged Trk1 and its mutated versions in BYT12 cells. Cells were grown and viewed as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>D</b>) Growth of cells on YNB-F plates supplemented with indicated concentrations of K<sup>+</sup>, Na<sup>+</sup> and Li<sup>+</sup>. The order of strains and length of incubation were the same as in (<b>B</b>). (<b>E</b>) Kinetic parameters of native Trk1 and its mutated versions after 3 h of K<sup>+</sup> starvation. Cells were grown as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. ST cells were incubated in a YNB-F for an additional 3 h. Rb<sup>+</sup> uptake was measured and kinetic parameters were estimated as described in Materials and Methods.</p> "> Figure 5
<p>Effects of introduction of proline into P-helices. (<b>A</b>) Sequence alignment of Trk1’s MPM domains 1–4. Leu949 and corresponding amino acids in MPM 1, 2 and 4 are indicated with red arrows. P-helices within MPM domains are underlined. (<b>B</b>) Positions of amino acids Leu81, Phe820, Leu949 and Leu1115 within 3D model of Trk1 protein. P-helix 1 is highlighted in blue, 2 in grey, 3 in green and 4 in orange. Studied amino-acid residues are shown in red. For better clarity, only two opposing MPM domains are shown. (<b>C</b>) Growth of BYT12 cells without <span class="html-italic">TRK1</span> (BYT12 [YCp352]) or with centromeric vector harbouring <span class="html-italic">TRK1</span> (native) and its mutated versions on YNB-F plates supplemented with K<sup>+</sup> as indicated. Images were captured after 5 days of incubation at 30 °C. (<b>D</b>) Localisation of GFP-tagged Trk1 and its mutated versions in BYT12 cells. Cells were grown and viewed as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>E</b>) Kinetic parameters of native Trk1 and its mutated versions after 3 h of K<sup>+</sup> starvation. Both non-starved (NS) and starved (ST) cells were grown as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. ST cells were incubated in a YNB-F for an additional 3 h. Rb<sup>+</sup> uptake was measured and kinetic parameters were estimated as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>.</p> "> Figure 6
<p>Intracellular K<sup>+</sup> content and relative membrane potential in BYT12 cells with native, L949P and F820P Trk1 versions. Cells were grown as described in Materials and Methods, and used directly (non-starved cells), or first incubated in a YNB-F for 1h (starved cells). (<b>A</b>) Intracellular K<sup>+</sup> content was estimated in non-starved (black columns) and starved (grey columns) cells as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>. (<b>B</b>) Relative membrane potential was estimated as described in <a href="#sec2-jof-08-00432" class="html-sec">Section 2</a>.</p> ">
Abstract
:1. Introduction
2. Materials and Methods
2.1. Strains and Growth Conditions
2.2. Plasmids
2.3. Growth Assay
2.4. Estimation of Kinetic Parameters for Rb+ Uptake
2.5. Measurements of K+ Content
2.6. Estimation of Relative Membrane Potential
2.7. Fluorescence Microscopy
2.8. Bioinformatics
2.9. Statistics
3. Results
3.1. Role of Extracellular and Intracellular K+ Content in Changes in Trk1 Affinity
3.2. Trk1 Affinity Is Directly Proportional to the Level of Plasma-Membrane Potential
3.3. Role of Short P-Helices in Trk1 Activity and Affinity Adjustment
4. Discussion
5. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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External K+ | Intracellular K+ | λmax | KT | Vmax |
---|---|---|---|---|
[mM] | [nmoles mg−1] | [nm] | [µM] | [nmoles mg−1 min−1] |
0.015 | 330 ± 9 | 574.43 | 288.7 | 51.6 |
0.05 | 339 ± 12 | 574.59 | 290.8 | 51.0 |
0.1 | 363 ± 3 | 574.33 | 287.9 | 50.5 |
0.2 | 418 ± 11 | 574.05 | 309.4 | 44.8 |
0.3 | 492 ± 32 | 573.61 | 317.6 | 42.9 |
0.5 | 589 ± 26 | 572.94 | 328.4 | 36.9 |
0.75 | 720 ± 5 | 571.95 | 407.3 | 30.4 |
1 | 796 ± 39 | 571.22 | 547.6 | 25.7 |
2 | 824 ± 19 | 571.03 | 860.1 | 26.1 |
10 | 919 ± 2 | 570.92 | 1590.6 | 19.7 |
100 | 958 ± 41 | 570.85 | 1439.4 | 18.1 |
Km [µM] | |||
NS | ST | KTNS/KTST | |
BYT12 [pCScTRK1] | 2488.0 | 181.1 | 13.7 |
BYT12 [pScTRK1] | 1968.3 | 38.2 | 51.5 |
BY4741 [YCp352] | 1045.7 | 210.9 | 5.0 |
BYT2 [YCp352] | 1069.0 | 203.7 | 5.2 |
λmax [nm] | |||
NS | ST | ∆λmaxST-NS [nm] | |
BYT12 [pCScTRK1] | 571.73 | 574.74 | 3.01 |
BYT12 [pScTRK1] | 571.89 | 576.22 | 4.33 |
BY4741 [YCp352] | 570.21 | 571.36 | 1.15 |
BYT2 [YCp352] | 571.27 | 572.35 | 1.08 |
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Masaryk, J.; Sychrová, H. Yeast Trk1 Potassium Transporter Gradually Changes Its Affinity in Response to Both External and Internal Signals. J. Fungi 2022, 8, 432. https://doi.org/10.3390/jof8050432
Masaryk J, Sychrová H. Yeast Trk1 Potassium Transporter Gradually Changes Its Affinity in Response to Both External and Internal Signals. Journal of Fungi. 2022; 8(5):432. https://doi.org/10.3390/jof8050432
Chicago/Turabian StyleMasaryk, Jakub, and Hana Sychrová. 2022. "Yeast Trk1 Potassium Transporter Gradually Changes Its Affinity in Response to Both External and Internal Signals" Journal of Fungi 8, no. 5: 432. https://doi.org/10.3390/jof8050432