Potassium is an essential intracellular ion, and a sufficient intracellular concentration of it is crucial for many processes; therefore it is fundamental for cells to precisely regulate K+ uptake and efflux through the plasma membrane. The uniporter Trk1 is a key player in K+ acquisition in yeasts. The TRK1 gene is expressed at a low and stable level; thus the activity of the transporter needs to be regulated at a posttranslational level. S. cerevisiae Trk1 changes its activity and affinity for potassium ion quickly and according to both internal and external concentrations of K+, as well as the membrane potential. The molecular basis of these changes has not been elucidated, though phosphorylation is thought to play an important role. In this study, we examined the role of the second, short, and highly conserved intracellular hydrophilic loop of Trk1 (IL2), and identified two phosphorylable residues (Ser882 and Thr900) as very important for 1) the structure of the loop and consequently for the targeting of Trk1 to the plasma membrane, and 2) the upregulation of the transporter's activity reaching maximal affinity under low external K+ conditions. Moreover, we identified three residues (Thr155, Ser414, and Thr900) within the Trk1 protein as strong candidates for interaction with 14-3-3 regulatory proteins, and showed, in an in vitro experiment, that phosphorylated Thr900 of the IL2 indeed binds to both isoforms of yeast 14-3-3 proteins, Bmh1 and Bmh2.

Department of Agricultural Chemistry Edaphology and Microbiology University of Córdoba 140 71 Córdoba Spain

Institute of Physiology of the Czech Academy of Sciences Laboratory of Membrane Transport 14200 Prague 4 Czech Republic

Institute of Physiology of the Czech Academy of Sciences Laboratory of Structural Biology of Signaling Proteins Division BIOCEV 25250 Vestec 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(1):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(4):177–193. doi: 10.1002/yea.3355. PubMed DOI

Sasikumar A., Killiea D., Kennedy B., Brem R. Potassium restriction boosts vacuolar acidity and extends lifespan in yeast. Exp Gerontol. 2019;120:101–106. doi: 10.1016/j.exger.2019.02.001. PubMed DOI PMC

Ramos J., Arino J., Sychrova H. Alkali-metal-cation influx and efflux systems in nonconventional yeast species. FEMS Microbiol Lett. 2011;317(1):1–8. PubMed

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 A. 2021;1863(1):83486. doi: 10.1016/j.bbamem.2020.183486. 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(7):2848–2859. doi: 10.1128/mcb.8.7.2848-2859.1988. PubMed DOI PMC

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(1):23–28. doi: 10.1007/s12223-011-0009-1. PubMed DOI

Borovikova D., Herynkova P., Rapoport A., Sychrova H. Potassium uptake system Trk2 is crucial for yeast cell viability during anhydrobiosis. FEMS Microbiol Lett. 2014;350(1):28–33. doi: 10.1111/1574-6968.12344. PubMed DOI

Dušková M., Cmunt D., Papoušková K., Masaryk J., Sychrová H. Minority potassium-uptake system Trk2 has a crucial role in yeast survival of glucose-induced cell death. Microbiology. 2021;167 doi: 10.1099/mic.0.001065. PubMed DOI

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(3):767–780. doi: 10.1046/j.1365-2958.2003.03335. PubMed DOI

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(5):508–517. doi: 10.1111/j.1567-1364.2010.00630. PubMed DOI

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 A. 2015;1848(5):1183–1195. doi: 10.1016/j.bbamem.2015.02.007. PubMed DOI

Kale D., Spurny P., Shamayeva K., Spurna K., Kahoun D., Ganser D., Zayats V., Ludwig J. The S. cerevisiae cation translocation protein Trk1 is functional without its “long hydrophilic Loop” But LHL regulates cation translocation activity and selectivity. Biochim Biophys A. 2019;1861(8):1476–1488. doi: 10.1016/j.bbamem.2019.06.010. PubMed DOI

Rivetta A., Slayman C., Kuroda T. Quantitative modeling of chloride conductance in yeast TRK potassium transporters. Biophys J. 2005;89(4):2412–2426. doi: 10.1529/biophysj.105.066712. PubMed DOI PMC

Masaryk J., Sychrova H. Yeast Trk1 potassium transporter gradually changes its affinity in response to both external and internal signals. J Fungi. 2022;8(5):432. doi: 10.3390/jof8050432. PubMed DOI PMC

Helbig A., Rosati S., Pijnappel P., van Breukelen B., Timmers M., Mohammed S., Slijper M., Heck A. Perturbation of the yeast N-acetyltransferase NatB induces elevation of protein phosphorylation levels. BMC Genom. 2010;11:685. doi: 10.1186/1471-2164-11-685. PubMed DOI PMC

Li X., Gerber S., Rudner A., Beausoleil S., Haas W., Villén J., Elias J., Gygi S. Large-scale phosphorylation analysis of alpha-factor-arrested Saccharomyces cerevisiae. J Proteome Res. 2007;6(3):1190–1197. doi: 10.1021/pr060559j. PubMed DOI

Albuquerque C., Smolka M., Payne S., Bafna V., Eng J., Zhou H. A multidimensional chromatography technology for In-depth phosphoproteome analysis. Mol Cell Proteom. 2008;7:1389–1396. doi: 10.1074/mcp.M700468-MCP200. PubMed DOI PMC

Swaney D., Beltrao P., Starita L., Guo A., Rush J., Fields S., Krogan L., Vilén J. Global analysis of phosphorylation and ubiquitylation cross-talk in protein degradation. Nat Methods. 2013;10(7):676–682. doi: 10.1038/nmeth.2519. PubMed DOI PMC

Holt L., Tuch B., Vilén J., Johnson A., Gygi S., Morgan D. Global analysis of Cdk1 substrate phosphorylation sites provides insights into evolution. Science. 2009;325(5948):1682–1686. doi: 10.1126/science.1172867. PubMed DOI PMC

Perez-Valle J., Jenkins H., Merchan S., Montiel V., Ramos J., Sharma S., Serrano R., Yenush L. Key role for intracellular K+ and protein kinases Sat4/Hal4 and Hal5 in the plasma membrane stabilization of yeast nutrient transporters. Mol Cell Biol. 2007;27(16):5725–5736. doi: 10.1128/MCB.01375-06. PubMed DOI PMC

Portillo F., Mulet J., Serrano R. A role for the non-phosphorylated form of yeast Snf1: tolerance to toxic cations and activation of potassium transport. FEBS Lett. 2005;579(2):512–516. doi: 10.1016/j.febslet.2004.12.019. PubMed DOI

Casado C., Yenush L., Melero C., Ruiz C., Serrano R., Perez-Valle J., Arino J., Ramos J. Regulation of Trk-dependent potassium transport by the calcineurin pathway involves the Hal5 kinase. FEBS Lett. 2010;584(11):2415–2420. doi: 10.1016/j.febslet.2010.04.042. PubMed DOI

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(19):8683–8692. doi: 10.1128/MCB.25.19.8683-8692.2005. PubMed DOI PMC

Forment J., Mulet J., Vicente O., Serrano R. The yeast SR protein kinase Sky1p modulates salt tolerance, membrane potential and the Trk1,2 potassium transporter. Biochim Biophys A. 2002;1565(1):36–40. doi: 10.1016/s0005-2736(02)00503-5. PubMed DOI

Bridges D., Moorhead G. 14-3-3 proteins: a number of functions for a numbered protein. Sci STKE. 2004;296:10. doi: 10.1126/stke.2962005re10. PubMed DOI

Shi L., Ren A., Zhu J., Yu H., Jiang A., Zheng H., Zhao M. 14-3-3 Proteins: a window for a deeper understanding of fungal metabolism and development. World J Microbiol Biotechnol. 2019;35(2):24. doi: 10.1007/s11274-019-2597. PubMed DOI

Smidova A., Alblova M., Kalabova D., Psenakova K., Rosulek M., Herman P., Obsil T., Obsilova V. 14-3-3 Protein masks the nuclear localization sequence of caspase-2. FEBS J. 2018;285(22):4196–4213. doi: 10.1111/febs.14670. PubMed DOI

Kalabova D., Filandr F., Alblova M., Petrvalska O., Horvath M., Man P., Obsil T., Obsilova V. 14-3-3 Protein binding blocks the dimerization interface of caspase-2. FEBS J. 2020;287(16):3494–3510. doi: 10.1111/febs.15215. PubMed DOI

van Heusden G. 14-3-3 Proteins: insights from genome-wide studies in yeast. Genomics. 2009;94(5):287–293. doi: 10.1016/j.ygeno.2009.07.004. PubMed DOI

Bruckmann A., Steensma H., de Mattos M., van Heusden G. Regulation of transcription by Saccharomyces cerevisiae 14-3-3 Proteins. Biochem J. 2004;382(3):867–875. doi: 10.1042/BJ20031885. PubMed DOI PMC

Kakiuchi K., Yamauchi Y., Taoka M., Iwago M., Fujita T., Ito T., Song S., Isobe T., Ichimura T. Proteomic analysis of in vivo 14-3-3 interactions in the yeast Saccharomyces cerevisiae. Biochemistry. 2007;46(26):7781–7792. doi: 10.1021/bi700501t. PubMed DOI

van Heusden G., Steensma H. Yeast 14-3-3 proteins. Yeast. 2006;23(3):159–171. doi: 10.1002/yea.1338. 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(3):734. doi: 10.3390/ijms20030734. PubMed DOI PMC

Smidova A., Stankova K., Petrvalska O., Lazar J., Sychrova H., Obsil T., Zimmermannova O., Obsilova V. The Activity of Saccharomyces cerevisiae Na+, K+/H+ Antiporter Nha1 Is negatively regulated by 14-3-3 protein binding at serine 481. Biochim Biophys A. 2019;1866(12) doi: 10.1016/j.bbamcr.2019.118534. PubMed DOI

Duby G., Poreba W., Piotrowiak D., Bobik K., Derua R., Waelkens E., Boutry M. Activation of plant plasma membrane H+-ATPase by 14-3-3 proteins is negatively controlled by two phosphorylation sites within the H+-ATPase C-terminal region. J Biol Chem. 2009;284(7):4213–4221. doi: 10.1074/jbc.M807311200. PubMed DOI

Sottocornola B., Gazzarrini S., Olivari C., Romani G., Valbuzzi P., Thiel G., Moroni A. 14-3-3 proteins regulate the potassium channel KAT1 by dual modes. Plant Biol. 2008;10(2):231–236. doi: 10.1111/j.1438-8677.2007.00028. PubMed DOI

Guldener U., Fielder T., Beinhauer J., Hegemann J. A new efficient gene disruption cassette for repeated use in budding yeast. Nucleic Acid Res. 1996;24(13):2519–2524. doi: 10.1093/nar/24.13.2519. PubMed DOI PMC

Hanscho M., Ruckerbauer D.E., Chauhan N., Hofbauer H.F., Krahulec S., Nidetzky B., Kohlwein S.D., Zanghellini J., Natter K. Nutritional requirements of the BY series of Saccharomyces cerevisiae strains for optimum growth. FEMS Yeast Res. 2012;12(7):796–808. doi: 10.1111/j.1567-1364.2012.00830. 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 A. 2019;1866(9):1376–1388. doi: 10.1016/j.bbamcr.2019.05.005. PubMed DOI

Crooks G., Hon G., Chandonia J., Brenner S. WebLogo: a sequence logo generator. Genome Res. 2004;14(6):1188–1190. doi: 10.1101/gr.849004. PubMed DOI PMC

Blom N., Gammeltoft S., Brunak S. Sequence and structure-based prediction of eukaryotic protein phosphorylation sites. J Mol Biol. 1999;294(5):1351–1362. doi: 10.1006/jmbi.1999.3310. PubMed DOI

Madeira F., Tinti M., Murugesan G., Berrett E., Stafford M., Toth R., Cole C., MacKintosh C., Barton G. 14-3-3-Pred: improved methods to predict 14-3-3-binding phosphopeptides. Bioinformatics. 2015;31(14):2276–2283. doi: 10.1093/bioinformatics/btv133. (oi) PubMed DOI PMC

Jumper J., Evans R., Pritzel A., Green T., Figurnov M., Ronneberger O., Tunyasuvunakool K., Bates R., Zidek A., Potapenko A., Bridgland A., Meyer C., Kohl S., Ballard A., Cowie A., Romera-Paredes B., Nikolov S., Jain R., Adler J., Back T., Petersen S., Reiman D., Clancy E., Zielinski M., Steinegger M., Pacholska M., Berghammer T., Bodestein S., Silver D., Vinalys O., Senior A., Kavukcuoglu K., Kohli P., Hassabis D. Highly accurate protein structure prediction with AlphaFold. Nature. 2021;596(7873):583–589. doi: 10.1038/s41586-021-03819-2. PubMed DOI PMC

Varadi M., Anyango S., Deshpande M., Nair S., Natassia C., Yordanova G., Yuan D., Stroe O., Wood G., Laydon A., Zidek A., Green T., Tunyasuvunakool K., Petersen S., Jumper J., Clancy E., Green R., Vora A., Lutfi M., Figurnov M., Cowie A., Hobbs N., Kohli P., Kleywegt G., Birney E., Hassabis D., Velnkar S. AlphaFold protein structure database: massively expanding the structural coverage of protein-sequence space with high-accuracy models. Nucleic Acid Res. 2021;50(1):439–444. doi: 10.1093/nar/gkab1061. 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(1):14–25. doi: 10.1002/pro.3235. PubMed DOI PMC

Veisova D., Rezabkova L., Stepanek M., Novotna P., Herman P., Vecer J., Obsil T., Obsilova V. The C-terminal segment of yeast BMH Proteins exhibits different structure compared to other 14-3-3 protein isoforms. Biochemistry. 2010;49(18):3853–3861. doi: 10.1021/bi100273k. PubMed DOI

Pohl P., Joshi R., Petrvalska O., Obsil T., Obsilova V. 14-3-3-protein regulates nedd4-2 by modulating interactions between HECT and WW domains. Commun. Biol. 2021;4:899. doi: 10.1038/s42003-021-02419-0. PubMed DOI PMC

Miranda M., Bashi E., Vylkova S., Edgerton M., Slayman C., Rivetta A. Conservation and dispersion of sequence and function in fungal TRK potassium transporters: Focus on Candida albicans. FEMS Yeast Res. 2009;9(2):278–292. doi: 10.1111/j.1567-1364.2008.00471. PubMed DOI

Eraso P., Mazon M., Portillo F. Yeast protein kinase Ptk2 localizes at the plasma membrane and phosphorylates in vitro the C-terminal peptide of the H+-ATPase. Biochim Biophys A. 2006;1758(2):164–170. doi: 10.1016/j.bbamem.2006.01.010. PubMed DOI

de Nadal E., Posas F. The HOG pathway and the regulation of osmoadaptive responses in yeast. FEMS Yeast Res. 2022;22(1):foac013. doi: 10.1093/femsyr/foac013. PubMed DOI PMC

Taylor I., Wang Y., Seitz K., Baer J., Bennewitz S., Mooney B., Walker J. Analysis of phosphorylation of the receptor-like protein kinase HAESA during arabidopsis floral abscission. PLoS One. 2016;11(1) doi: 10.1371/journal.pone.0147203. PubMed DOI PMC

White D., Unwin R., Bidels E., Pierce A., Teng H., Muter J., Greystoke B., Somerville T., Grifiths J., Lovell S., Somervaille T., Delwel R., Whetton A., Meyer S. Phosphorylation of the leukemic oncoprotein EVI1 on serine 196 modulates DNA Binding, transcriptional repression and transforming ability. PloS One. 2013;8(6) doi: 10.1371/journal.pone.0066510. PubMed DOI PMC

Vlastaridis P., Kyriakidou P., Chaliotis A., Van de Peer Y., Oliver S., Amoutzias G. Estimating the total number of phosphoproteins and phosphorylation sites in eukaryotic proteomes. Gigascience. 2017;6(2):1–11. doi: 10.1093/gigascience/giw015. PubMed DOI PMC

Davidson A. Mechanism of coupling of transport to hydrolysis in bacterial ATP-binding cassette transporters. J Bacteriol. 2002;184(5):1225–1233. doi: 10.1128/JB.184.5.1225-1233.2002. PubMed DOI PMC

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

Chen Z., Cole P. Synthetic approaches to protein phosphorylation. Curr Opin Chem Biol. 2015;28:115–122. doi: 10.1016/j.cbpa.2015.07.001. PubMed DOI PMC

Rezaei-Ghaleh N., Amininasab M., Kumar S., Walter J., Zweckstetter M. Phosphorylation modifies the molecular stability of β-amyloid deposits. Nat Commun. 2016;7:11359. doi: 10.1038/ncomms11359. 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(5):920–929. doi: 10.1093/emboj/21.5.920. 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 Comm. 2021;12(1) doi: 10.1038/s41467-021-26782-y. PubMed DOI PMC

Camoni L., Visconti S., Aducci P., Marra M. From plant physiology to pharmacology: fusicoccin leaves the leaves. Planta. 2019;249(1):49–57. doi: 10.1007/s00425-018-3051-2. PubMed DOI

The yeast 14-3-3 proteins Bmh1 and Bmh2 regulate key signaling pathways