Yeasts need a high intracellular concentration of potassium to grow. The main K+ uptake system in Saccharomyces cerevisiae is the Trk1 transporter, a complex protein with four MPM helical membrane motifs. Trk1 has been shown to exist in low- or high-affinity modes, which reflect the availability of potassium in the environment. However, when and how the affinity changes, and whether the potassium availability is the only signal for the affinity switch, remains unknown. Here, we characterize the Trk1 kinetic parameters under various conditions and find that Trk1's KT and Vmax change gradually. This gliding adjustment is rapid and precisely reflects the changes in the intracellular potassium content and membrane potential. A detailed characterization of the specific mutations in the P-helices of the MPM segments reveals that the presence of proline in the P-helix of the second and third MPM domain (F820P and L949P) does not affect the function of Trk1 in general, but rather specifically prevents the transporter's transition to a high-affinity state. The analogous mutations in the two remaining MPM domains (L81P and L1115P) result in a mislocalized and inactive protein, highlighting the importance of the first and fourth P-helices in proper Trk1 folding and activity at the plasma membrane.

Laboratory of Membrane Transport Institute of Physiology Czech Academy of Sciences 142 20 Prague Czech Republic

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Arino J., Ramos J., Sychrova H. Alkali metal cation transport and homeostasis in yeasts. Microbiol. Mol. Biol. Rev. 2010;74:95–120. doi: 10.1128/MMBR.00042-09. PubMed DOI PMC

Arino J., Ramos J., Sychrova H. Monovalent cation transporters at the plasma membrane in yeasts. Yeast. 2019;36:177–193. doi: 10.1002/yea.3355. PubMed DOI

Gaber R., Styles C., Fink G. TRK1 encodes a plasma membrane protein required for high-affinity potassium transport in Saccharomyces cerevisiae. Mol. Cell. Biol. 1988;8:2848–2859. doi: 10.1128/mcb.8.7.2848-2859.1988. PubMed DOI PMC

Ko C., Gaber R. TRK1 and TRK2 encode structurally related K+ transporters in Saccharomyces cerevisiae. Mol. Cell. Biol. 1991;11:4266–4273. doi: 10.1128/mcb.11.8.4266. PubMed DOI PMC

Bertl A., Ramos J., Ludwig J., Lichtenberg-Frate H., Reid J., Bihler H., Calero F., Martinez P., Ljungdahl P. Characterization of potassium transport in wild-type and isogenic yeast strains carrying all combinations of trk1, trk2 and tok1 null mutations. Mol. Microbiol. 2003;47:767–780. doi: 10.1046/j.1365-2958.2003.03335.x. PubMed DOI

Durell S., Hao Y., Nakamura T., Bakker E., Guy H. Evolutionary relationship between K+ channels and symporters. Biophys. J. 1999;77:775–788. doi: 10.1016/S0006-3495(99)76931-6. PubMed DOI PMC

Diskowski M., Mikusevic V., Stock C., Hanelt I. Functional diversity of the superfamily of K+ transporters to meet various requirements. Biol. Chem. 2015;396:1003–1014. doi: 10.1515/hsz-2015-0123. PubMed DOI

Rodriguez-Navarro A. Potassium transport in fungi and plants. Biochim. Biophys. Acta. 2000;1469:1–30. doi: 10.1016/S0304-4157(99)00013-1. PubMed DOI

Rivetta A., Slayman C., Kuroda T. Quantitative Modeling of chloride conductance in yeast TRK potassium transporters. Biophys. J. 2005;89:2412–2426. doi: 10.1529/biophysj.105.066712. PubMed DOI PMC

Durell S., Guy H. Structural models of the KtrB, TrkH, and Trk1,2 symporters based on the structure of the KcsA K+ channel. Biophys. J. 1999;77:789–807. doi: 10.1016/S0006-3495(99)76932-8. PubMed DOI PMC

Zayats V., Stockner T., Pandey S., Wortz K., Ettrich R., Ludwig J. A refined atomic scale model of the Saccharomyces cerevisiae K+-translocation protein Trk1p combined with experimental evidence confirms the role of selectivity filter glycines and other key residues. Biochim. Biophys. Acta. 2015;1848:1183–1195. doi: 10.1016/j.bbamem.2015.02.007. PubMed DOI

Doyle D., Morais C., Pfuetzner R., Kuo A., Gulbis J., Cohen S., Chait B., MacKinnon R. The structure of the potassium channel: Molecular basis of K+ conduction and selectivity. Science. 1998;280:69–77. doi: 10.1126/science.280.5360.69. PubMed DOI

Rodriguez-Navarro A., Ramos J. Dual system for potassium transport in Saccharomyces cerevisiae. J. Bacteriol. 1984;159:940–945. doi: 10.1128/jb.159.3.940-945.1984. PubMed DOI PMC

Ramos J., Contreras P., Rodriguez-Navarro A. A potassium transport mutant of Saccharomyces cerevisiae. Arch. Microbiol. 1985;143:88–93. doi: 10.1007/BF00414774. DOI

Ramos J., Rodriguez-Navarro A. Regulation and interconversion of the potassium transport systems of Saccharomyces cerevisiae as revealed by rubidium transport. Eur. J. Biochem. 1986;154:307–311. doi: 10.1111/j.1432-1033.1986.tb09398.x. PubMed DOI

Ramos J., Haro R., Rodriguez-Navarro A. Regulation of potassium fluxes in Saccharomyces cerevisiae. Biochim. Biophys. Acta. 1990;1029:211–217. doi: 10.1016/0005-2736(90)90156-I. PubMed DOI

Haro R., Rodriguez-Navarro A. Molecular analysis of the mechanism of potassium uptake through the Trk1 transporter of Saccharomyces cerevisiae. Biochim. Biophys. Acta. 2002;1564:114–122. doi: 10.1016/S0005-2736(02)00408-X. PubMed DOI

Cyert M., Philpott C. Regulation of cation balance in Saccharomyces cerevisiae. Genetics. 2013;193:677–713. doi: 10.1534/genetics.112.147207. PubMed DOI PMC

Zhao P., Zhao C., Chen D., Yun C., Li H., Bai L. Structure and activation mechanism of the hexameric plasma membrane H+-ATPase. Nat. Commun. 2021;12:6439. doi: 10.1038/s41467-021-26782-y. PubMed DOI PMC

Yenush L., Mulet J., Arino J., Serrano R. The Ppz protein phosphatases are key regulators of K+ and pH homeostasis: Implications for salt tolerance, cell wall integrity and cell cycle progression. EMBO J. 2002;21:920–929. doi: 10.1093/emboj/21.5.920. PubMed DOI PMC

Yenush L., Merchan S., Holmes J., Serrano R. pH-Responsive, Posttranslational Regulation of the Trk1 Potassium Transporter by the Type 1-Related Ppz1 Phosphatase. Mol. Cell. Biol. 2005;25:8683–8692. doi: 10.1128/MCB.25.19.8683-8692.2005. PubMed DOI PMC

Martinez-Munoz G., Kane P. Vacuolar and plasma membrane proton pumps collaborate to achieve cytosolic pH homeostasis in yeast. J. Biol. Chem. 2008;283:20309–20319. doi: 10.1074/jbc.M710470200. PubMed DOI PMC

Navarrete C., Petrezselyova S., Barreto L., Martinez J., Zahradka J., Arino J., Sychrova H., Ramos J. Lack of main K+ uptake systems in Saccharomyces cerevisiae cells affects yeast performance in both potassium-sufficient and potassium-limiting conditions. FEMS Yeast Res. 2010;10:508–517. doi: 10.1111/j.1567-1364.2010.00630.x. PubMed DOI

Zimmermannova O., Felcmanova K., Rosas-Santiago P., Papouskova K., Pantoja O., Sychrova H. Erv14 cargo receptor participates in regulation of plasma-membrane potential, intracellular pH and potassium homeostasis via its interaction with K+-specific transporters Trk1 and Tok1. Biochim. Biophys. Acta. 2019;1866:1376–1388. doi: 10.1016/j.bbamcr.2019.05.005. PubMed DOI

Petrezselyova S., Ramos J., Sychrova H. Trk2 transporter is a relevant player in K+ supply and plasma-membrane potential control in Saccharomyces cerevisiae. Folia Microbiol. 2011;56:23–28. doi: 10.1007/s12223-011-0009-1. PubMed DOI

Kodedova M., Sychrova H. Changes in the sterol composition of the plasma membrane affect membrane potential, salt tolerance and the activity of multidrug resistance pumps in Saccharomyces cerevisiae. PLoS ONE. 2015;10:e0139306. doi: 10.1371/journal.pone.0139306. PubMed DOI PMC

Goddard T., Huang C., Meng E., Pettersen E., Couch G., Morris J., Ferrin T. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 2018;27:14–25. doi: 10.1002/pro.3235. PubMed DOI PMC

Herrera R., Alvarez M., Gelis S., Kodedova M., Sychrova H., Kschischo M., Ramos J. Role of Saccharomyces cerevisiae Trk1 in stabilization of intracellular potassium content upon changes in external potassium levels. Biochim. Biophys. Acta. 2014;1838:127–133. doi: 10.1016/j.bbamem.2013.08.022. PubMed DOI

Orij R., Bruhl S., Smits G. Intracellular pH is a tightly controlled signal in yeast. Biochim. Biophys. Acta. 2011;1810:933–944. doi: 10.1016/j.bbagen.2011.03.011. PubMed DOI

Zahumensky J., Janickova I., Drietomska A., Svenkrtova A., Hlavacek O., Hendrych T., Plasek J., Sigler K., Gaskova D. Yeast Tok1p channel is a major contributor to membrane potential maintenance under chemical stress. Biochim. Biophys. Acta. 2017;1859:1974–1985. doi: 10.1016/j.bbamem.2017.06.019. PubMed DOI

Hanelt I., Tholema N., Kroning N., Vor der Bruggen M., Wunnicke D., Bakker E. KtrB, a member of the superfamily of K+ transporters. Eur. J. Cell. Biol. 2011;90:696–704. doi: 10.1016/j.ejcb.2011.04.010. PubMed DOI

Parker J., Newstead S. Molecular basis of nitrate uptake by the plant nitrate transporter NRT1.1. Nature. 2014;507:68–72. doi: 10.1038/nature13116. PubMed DOI PMC

Liu K., Tsay Y. Switching between the two action modes of the dual-affnity nitrate transporter Chl1 by phosphorylation. EMBO J. 2003;22:1005–1013. doi: 10.1093/emboj/cdg118. PubMed DOI PMC

Tsay Y. How to switch affinity. Nature. 2014;507:44–45. doi: 10.1038/nature13063. PubMed DOI

Sun J., Zheng N. Molecular mechanism underlying the plant Nrt1.1 dual-affinity nitrate transporter. Front. Physiol. 2015;6:386. doi: 10.3389/fphys.2015.00386. PubMed DOI PMC

Reifenberger E., Boles E., Ciriacy M. Kinetic characterization of individual hexose transporters of Saccharomyces cerevisiae and their relation to the triggering mechanisms of glucose repression. Eur. J. Biochem. 1997;245:324–333. doi: 10.1111/j.1432-1033.1997.00324.x. PubMed DOI

Fu H., Luan S. AtKUP1: A dual-affinity K+ transporter from Arabidopsis. Plant Cell. 1998;10:63–73. doi: 10.1105/tpc.10.1.63. PubMed DOI PMC

Ruiz-Castilla F., Bieber J., Caro G., Michan C., Sychrova H., Ramos J. Regulation and activity of CaTrk1, CaAcu1 and CaHak1, the three plasma membrane potassium transporters in Candida albicans. Biochim. Biophys. Acta. 2021;1863:183486. doi: 10.1016/j.bbamem.2020.183486. PubMed DOI

Capera J., Serrano-Novillo C., Navarro-Pérez M., Cassinelli S., Felipe A. The potassium channel odyssey: Mechanisms of traffic and membrane arrangement. Int. J. Mol. Sci. 2019;20:734. doi: 10.3390/ijms20030734. PubMed DOI PMC

Ashraf K., Josts I., Moshbahi K., Kelly S., Byron O., Smith B., Walker D. The potassium binding protein Kbp is a cytoplasmic potassium sensor. Structure. 2016;24:741–749. doi: 10.1016/j.str.2016.03.017. PubMed DOI

Herrera R., Alvarez M., Gelis S., Ramos J. Subcellular potassium and sodium distribution in Saccharomyces cerevisiae wild-type and vacuolar mutants. Biochem. J. 2013;454:525–532. doi: 10.1042/BJ20130143. PubMed DOI

Levin E., Zhiu M. Recent progress on the structure and function of the TrkH/KtrB ion channel. Curr. Opin. Struct. Biol. 2014;27:95–101. doi: 10.1016/j.sbi.2014.06.004. PubMed DOI PMC

Lam F., Ghaderi A., Fink G., Stephanopoulos G. Engineering alcohol tolerance in yeast. Science. 2014;346:71–75. doi: 10.1126/science.1257859. PubMed DOI PMC

Henriques S., Mira N., Sa-Correia I. Genome-wide search for candidate genes for yeast robustness improvement against formic acid reveals novel susceptibility (Trk1 and positive regulators) and resistance (Haa1-regulon) determinants. Biotechnol. Biofuels. 2017;10:96. doi: 10.1186/s13068-017-0781-5. PubMed DOI PMC

Xu X., Williams T., Divne C., Pretorius I., Paulsen I. Evolutionary engineering in Saccharomyces cerevisiae a TRK1-dependent potassium influx mechanism for propionic acid tolerance. Biotechnol. Biofuels. 2019;12:97. doi: 10.1186/s13068-019-1427-6. PubMed DOI PMC

Reisser C., Dick C., Kruglyak L., Botstein D., Schacherer J., Hess D. Genetic basis of ammonium toxicity resistance in a sake strain of yeast: A mendelian case. G3 Genes Genomes Genet. 2013;3:733–740. doi: 10.1534/g3.113.005884. PubMed DOI PMC

Llopis-Torregrosa V., Vaz C., Monteoliva R., Ryman K., Engstrom Y., Gacser A., Gil C., Lungdahl P., Sychrova H. Trk1-mediated potassium uptake contributes to cell-surface properties and virulence of Candida glabrata. Sci. Rep. 2019;9:7529. doi: 10.1038/s41598-019-43912-1. PubMed DOI PMC

Petrezselyova S., Zahradka J., Sychrova H. Saccharomyces cerevisiae BY4741 and W303-1A laboratory strains differ in salt tolerance. Fungal Biol. 2010;114:144–150. doi: 10.1016/j.funbio.2009.11.002. PubMed DOI

Sikorski R., Hieter P. A system of shuttle vectors and yeast host strains designed for efficient anipulation of DNA in Saccharomyces cerevisiae. Genetics. 1989;122:19–27. doi: 10.1093/genetics/122.1.19. PubMed DOI PMC

Hill J., Myers A., Koerner T., Tzagoloff A. Yeast/E. coli shuttle vectors with multiple unique restriction sites. Yeast. 1986;2:163–167. doi: 10.1002/yea.320020304. PubMed DOI

The superior growth of Kluyveromyces marxianus at very low potassium concentrations is enabled by the high-affinity potassium transporter Hak1

The Hrk1 kinase is a determinant of acetic acid tolerance in yeast by modulating H+ and K+ homeostasis

The second intracellular loop of the yeast Trk1 potassium transporter is involved in regulation of activity, and interaction with 14-3-3 proteins

Dimerisation of the Yeast K+ Translocation Protein Trk1 Depends on the K+ Concentration