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  • 1.
    Andersson, Michael R.
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Mutational analysis of conserved glutamic acids of Pho89, a Saccharomyces cerevisiae high-affinity inorganic phosphate:Na+ symporter2012In: Biologia (Bratislava), ISSN 0006-3088, E-ISSN 1336-9563, Vol. 67, no 6, p. 1056-1061Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, the high-affinity phosphate transport system comprises the Pho84 and Pho89 permeases. The Pho89 permease catalyzes import of inorganic phosphate in a symport manner by utilizing Na+ ions as co-solute. We have addressed the functional importance of two glutamic acid residues at positions 55 and 491. Both residues are highly conserved amongst members of the inorganic phosphate transporter (PiT) family, which might be an indication of functional importance. Moreover, both residues have been shown to be of critical importance in the hPit2 transporter. We have created site-directed mutations of both E55 and E491 to lysine and glutamine. We observed that in all four cases there is a dramatic impact on the transport activity, and thus it seems that they indeed are of functional importance. Following these observations, we addressed the membrane topology of this protein by using several prediction programs. TOPCONS predicts a 7-5 transmembrane segment organization, which is the most concise topology as compared to the hPiT2 transporter. By understanding the functionality of these residues, we are able to correlate the Pho89 topology to that of the hPiT2, and can now further analyze residues which might play a role in the transport activity.

  • 2.
    Basheer, Shabana
    et al.
    Central Food Technology Research Institute.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Hedström, Martin
    Lund university.
    Thakur, Munna Singh
    Central Food Technology Research Institute.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Mattiasson, Bo
    Lund university.
    A membrane protein based biosensor: Use of a phosphate - H+ symporter membrane protein (Pho84) in the sensing of phosphate ions.2011In: Biosensors & bioelectronics, ISSN 0956-5663, E-ISSN 1873-4235, Vol. 27, no 1, p. 58-63Article in journal (Refereed)
    Abstract [en]

    A label free biosensor for direct detection of inorganic phosphate based on potential-step capacitance measurements has been developed. The high-affinity Pho84 plasma membrane phosphate/proton symporter of Saccharomyces cerevisiae was used as a sensing element. Heterologously expressed and purified Pho84 protein was immobilized on a self-assembled monolayer (SAM) on a capacitance electrode. Changes in capacitance were recorded upon exposure to phosphate compared to the control substance, phosphate analogue methylphosphonate. Hence, even without the explicit use of lipid membranes, the Pho84 membrane protein could retain its capacity of selective substrate binding, with a phosphate detection limit in the range of the apparent in vivo K(m). A linear increase in capacitance was monitored in the phosphate concentration range of 5-25 mu M. The analytical response of the capacitive biosensor is in agreement with that the transporter undergoes significant conformational changes upon exposure to inorganic phosphate, while exposure to the analogue only causes minor responses.

  • 3.
    Lundh, Fredrik
    et al.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mouillon, Jean-Marie
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Samyn, Dieter R.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Stadler, Kent
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Popova, Yulia
    Lagerstedt, Jens
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Thevelein, Johan
    Persson, Bengt L.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Molecular mechanisms controlling phosphate-induced downregulation of the yeast Pho84 phosphate transporter2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 21, p. 4497-4505Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, phosphate uptake is mainly dependent on the proton-coupled Pho84 permease under phosphate-limited growth conditions. Phosphate addition causes Pho84-mediated activation of the protein kinase A (PKA) pathway as well as rapid internalization and vacuolar breakdown of Pho84. We show that Pho84 undergoes phosphate-induced phosphorylation and subsequent ubiquitination on amino acids located in the large middle intracellular loop prior to endocytosis. The attachment of ubiquitin is dependent on the ubiquitin conjugating enzymes Ubc2 and Ubc4. In addition, we show that the Pho84 endocytotic process is delayed in strains with reduced PKA activity. Our results suggest that Pho84-mediated activation of the PKA pathway is responsible for its own downregulation by phosphorylation, ubiquination, internalization, and vacuolar breakdown.

  • 4.
    Ruiz-Pavon, Lorena
    et al.
    Division of Molecular Genetics, Department of Physics, Chemistry and Biology, Linköping University and School of Natural Sciences, Linnaeus university.
    Karlsson, Patrik M.
    Division of Molecular Genetics, Department of Physics, Chemistry and Biology, Linköping University .
    Carlsson, Jonas
    Division of Bioinformatics, Department of Physics, Chemistry and Biology, Linköping University .
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Persson, Bengt L.
    Division of Bioinformatics, Department of Physics, Chemistry and Biology, Linköping University .
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Spetea, Cornelia
    Division of Molecular Genetics, Department of Physics, Chemistry and Biology, Linköping University .
    Functionally important amino acids in the Arabidopsis thylakoid phosphate transporter: Homology modeling and site-directed mutagenesis2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 30, p. 6430-6439Article in journal (Refereed)
    Abstract [en]

    The anion transporter 1 (ANTR1) from Arabidopsis thaliana, homologous to the mammalian members of the solute carrier 17 (SLC17) family, is located in the chloroplast thylakoid membrane. When expressed heterologously in Escherichia coli, ANTR1 mediates a Na+-dependent active transport of inorganic phosphate (Pi). The aim of this study was to identify amino acid residues involved in Pi binding and translocation by ANTR1 and in the Na+ dependence of its activity. A three-dimensional structural model of ANTR1 was constructed using the crystal structure of glycerol 3-phosphate/phosphate antiporter from E. coli as a template. Based on this model and multiple sequence alignments, five highly conserved residues in plant ANTRs and mammalian SLC17 homologues have been selected for site-directed mutagenesis, namely, Arg-120, Ser-124, and Arg-201 inside the putative translocation pathway and Arg-228 and Asp-382 exposed at the cytoplasmic surface of the protein. The activities of the wild-type and mutant proteins have been analyzed using expression in E. coli and radioactive Pi transport assays and compared with bacterial cells carrying an empty plasmid. The results from Pi- and Na+-dependent kinetics indicate the following: (i) Arg-120 and Arg-201 may be important for binding and translocation of the substrate; (ii) Ser-124 may function as a transient binding site for Na+ ions in close proximity to the periplasmic side; (iii) Arg-228 and Asp-382 may participate in interactions associated with protein conformational changes required for full transport activity. Functional characterization of ANTR1 should provide useful insights into the function of other plant and mammalian SLC17 homologous transporters.

  • 5.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Structure/function relationships of inorganic phosphate transporters2011Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    Of the many nutrients that make the metabolic clock tick, inorganic phosphate fulfills an essential role in all yeast (and other organisms), being necessary for both structural and metabolic purposes. In order to transport phosphate into the cell interior, Saccharomyces cerevisiae makes use of two systems, comprising high- or low-affinity transporters, which are responsible for the cellular accumulation of inorganic phosphate. Depending on the extracellular concentration of inorganic phosphate, one of the two systems will be responsible for scavenging phosphate from the surroundings.The present thesis focuses on the high-affinity transport system in S. cerevisiae, i.e. Pho84, which is induced under low phosphate conditions. The expression and degradation of Pho84 is dependent on the availability of phosphate. When confronted with ample amounts of external phosphate, the Pho84 undergoes phosphorylation, prior to ubiquitylation. These events will eventually lead to the removal of Pho84 from the plasma membrane, followed by vacuolar degradation. The Pho84, together with the ANTR1 high-affinity inorganic phosphate transporter of Arabidopsis thaliana, are integral membrane proteins belonging to the major facilitator superfamily. Both are predicted to have 12 transmembrane helices, which have been confirmed by in silico modeling of both proteins, using the glycerol-3-phosphate transporter as template. The obtained models served as a rational start point for the study of the molecular transport mechanisms by means of site-directed mutagenesis and consequently functional and biochemical characterization.The other high-affinity transporter, Pho89, has been studied less because of its lower activity (as compared to the Pho84) and its preferred alkaline operational conditions. In order to study Pho89 in more detail, a quadruple deletions strain (Pho84Δ Pho87Δ Pho90Δ Pho91Δ) was used.In conclusion, this thesis has contributed to broaden the knowledge of structural and functional aspects of inorganic phosphate transporters.

  • 6.
    Samyn, Dieter R.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Andersson, M.
    Ruiz-Pavon, Lorena
    Popova, Y.
    Persson, Bengt L.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Thevelein, J.
    The high-affinity inorganic phosphate transport system of Saccharomyces cerevisiae: a tale of two proteins2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, p. 152-152Article in journal (Other academic)
  • 7.
    Samyn, Dieter R.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Persson, Bengt L.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Inorganic phosphate and sulfate transport in S. cerevisiae2016In: Yeast Membrane Transport / [ed] José Ramos, Hana Sychrova, Maik Kschischo, Springer, 2016, p. 253-269Chapter in book (Refereed)
    Abstract [en]

    Inorganic ions such as phosphate and sulfate are essential macronutrients required for a broad spectrum of cellular functions and their regulation. In a constantly fluctuating environment microorganisms have for their survival developed specific nutrient sensing and transport systems ensuring that the cellular nutrient needs are met. This chapter focuses on the S. cerevisiae plasma membrane localized transporters, of which some are strongly induced under conditions of nutrient scarcity and facilitate the active uptake of inorganic phosphate and sulfate. Recent advances in studying the properties of the high-affinity phosphate and sulfate transporters by means of site-directed mutagenesis have provided further insight into the molecular mechanisms contributing to substrate selectivity and transporter functionality of this important class of membrane transporters.

  • 8.
    Samyn, Dieter R.
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Ruiz-Pavon, Lorena
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Andersson, Michael R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Popova, Yulia
    Katholieke Universiteit Leuven.
    Thevelein, Johan
    Katholieke Universiteit Leuven.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Mutational analysis of putative phosphate- and proton-binding sites in the Saccharomyces cerevisiae Pho84 phosphate:H+ transceptor and its effect on signalling to the PKA and PHO pathways2012In: Biochemical Journal, ISSN 0264-6021, E-ISSN 1470-8728, Vol. 445, p. 413-422Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, the Pho84 phosphate transporter acts as the main provider of phosphate to the cell using a proton symport mechanism, but also mediates rapid activation of the PKA (protein kinase A) pathway. These two features led to recognition of Pho84 as a transceptor. Although the physiological role of Pho84 has been studied in depth, the mechanisms underlying the transport and sensor functions are unclear. To obtain more insight into the structure–function relationships of Pho84, we have rationally designed and analysed site-directed mutants. Using a three-dimensional model of Pho84 created on the basis of the GlpT permease, complemented with multiple sequence alignments, we selected Arg168 and Lys492, and Asp178, Asp358 and Glu473 as residues potentially involved in phosphate or proton binding respectively, during transport. We found that Asp358 (helix 7) and Lys492 (helix 11) are critical for the transport function, and might be part of the putative substrate-binding pocket of Pho84. Moreover, we show that alleles mutated in the putative proton-binding site Asp358 are still capable of strongly activating PKA pathway targets, despite their severely reduced transport activity. This indicates that signalling does not require transport and suggests that mutagenesis of amino acid residues involved in binding of the co-transported ion may constitute a promising general approach to separate the transport and signalling functions in transceptors.

  • 9.
    Samyn, Dieter R.
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences. Cocordia University, Canada.
    Van der Veken, Jeroen
    Inst Agr & Fisheries Res ILVO, Belgium.
    Van Zeebroeck, Griet
    VIB, Belgium;Katholieke Univ Leuven, Belgium.
    Persson, Bengt L.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Karlsson, Björn C. G.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Key Residues and Phosphate Release Routes in the Saccharomyces cerevisae Pho84 Transceptor - The Role of Tyr179 in Functional Regulation2016In: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 291, no 51, p. 26388-26398Article in journal (Refereed)
    Abstract [en]

    Pho84, a major facilitator superfamily (MFS) protein, is the main high-affinity Pi transceptor in Saccharomyces cerevisiae. Although transport mechanisms have been suggested for other MFS members, the key residues and molecular events driving transport by Pi: H+ symporters are unclear. The current Pho84 transport model is based on the inward-facing occluded crystal structure of the Pho84 homologue PiPT in the fungus Piriformospora indica. However, this model is limited by the lack of experimental data on the regulatory residues for each stage of the transport cycle. In this study, an open, inward-facing conformation of Pho84 was used to study the release of Pi. A comparison of this conformation with the model for Pi release in PiPT revealed that Tyr(179) in Pho84 (Tyr150 in PiPT) is not part of the Pi binding site. This difference may be due to a lack of detailed information on the Pi release step in PiPT. Molecular dynamics simulations of Pho84 in which a residue adjacent to Tyr(179), Asp(178), is protonated revealed a conformational change in Pho84 from an open, inward-facing state to an occluded state. Tyr(179) then became part of the binding site as was observed in the PiPT crystal structure. The importance of Tyr(179) in regulating Pi release was supported by site-directed mutagenesis and transport assays. Using trehalase activity measurements, we demonstrated that the release of Pi is a critical step for transceptor signaling. Our results add to previous studies on PiPT, creating a more complete picture of the proton-coupled Pi transport cycle of a transceptor.

  • 10.
    Schothorst, Joep
    et al.
    Katholieke Universiteit Leuven, Belgium.
    Nag Kankipati, Harish
    Katholieke Universiteit Leuven, Belgium.
    Conrad, Michaela
    Katholieke Universiteit Leuven, Belgium.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Van Zeebroeck, Griet
    Katholieke Universiteit Leuven, Belgium.
    Popova, Yulia
    Katholieke Universiteit Leuven, Belgium.
    Rubio-Texeira, Marta
    Katholieke Universiteit Leuven, Belgium.
    Persson, Bengt L.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Thevelein, Johan
    Katholieke Universiteit Leuven, Belgium.
    Yeast nutrient transceptors provide novel insight in the functionality of membrane transporters.2013In: Current Genetics, ISSN 0172-8083, E-ISSN 1432-0983, Vol. 59, no 4, p. 197-206Article in journal (Refereed)
    Abstract [en]

    In the yeast Saccharomyces cerevisiae several nutrient transporters have been identified that possess an additional function as nutrient receptor. These transporters are induced when yeast cells are starved for their substrate, which triggers entry into stationary phase and acquirement of a low protein kinase A (PKA) phenotype. Re-addition of the lacking nutrient triggers exit from stationary phase and sudden activation of the PKA pathway, the latter being mediated by the nutrient transceptors. At the same time, the transceptors are ubiquitinated, endocytosed and sorted to the vacuole for breakdown. Investigation of the signaling function of the transceptors has provided a new read-out and new tools for gaining insight into the functionality of transporters. Identification of amino acid residues that bind co-transported ions in symporters has been challenging because the inactivation of transport by site-directed mutagenesis is not conclusive with respect to the cause of the inactivation. The discovery of nontransported agonists of the signaling function in transceptors has shown that transport is not required for signaling. Inactivation of transport with maintenance of signaling in transceptors supports that a true proton-binding residue was mutagenised. Determining the relationship between transport and induction of endocytosis has also been challenging, since inactivation of transport by mutagenesis easily causes loss of all affinity for the substrate. The use of analogues with different combinations of transport and signaling capacities has revealed that transport, ubiquitination and endocytosis can be uncoupled in several unexpected ways. The results obtained are consistent with transporters undergoing multiple substrate-induced conformational changes, which allow interaction with different accessory proteins to trigger specific downstream events.

  • 11.
    Sengottaiyan, Palanivelu
    et al.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Spetea, Cornelia
    University of Gothenburg.
    Lagerstedt, Jens O.
    Lund University.
    Samyn, Dieter R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Andersson, Michael R.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Ruiz-Pavon, Lorena
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences.
    Persson, Bengt L.
    Linnaeus University, Faculty of Science and Engineering, School of Natural Sciences. Katholieke Universiteit Leuven, Belgium ; Flanders Institute of Biotechnology, Belgium.
    The intrinsic GTPase activity of the Gtr1 protein from Saccharomyces cerevisiae2012In: BMC Biochemistry, ISSN 1471-2091, E-ISSN 1471-2091, Vol. 13, article id 11Article in journal (Refereed)
    Abstract [en]

    Background

    The Gtr1 protein of Saccharomyces cerevisiae is a member of the RagA subfamily of the Ras-like small GTPase superfamily. Gtr1 has been implicated in various cellular processes. Particularly, the Switch regions in the GTPase domain of Gtr1 are essential for TORC1 activation and amino acid signaling [R. Gong, L. Li, Y. Liu, P. Wang, H. Yang, L. Wang, J. Cheng, K.L. Guan, Y. Xu, Genes Dev. 25 (2011) 1668–1673]. Therefore, knowledge about the biochemical activity of Gtr1 is required to understand its mode of action and regulation.

    Results

    By employing tryptophan fluorescence analysis and radioactive GTPase assays, we demonstrate that Gtr1 can adopt two distinct GDP- and GTP-bound conformations, and that it hydrolyses GTP much slower than Ras proteins. Using cysteine mutagenesis of Arginine-37 and Valine-67, residues at the Switch I and II regions, respectively, we show altered GTPase activity and associated conformational changes as compared to the wild type protein and the cysteine-less mutant.

    Conclusions

    The extremely low intrinsic GTPase activity of Gtr1 implies requirement for interaction with activating proteins to support its physiological function. These findings as well as the altered properties obtained by mutagenesis in the Switch regions provide insights into the function of Gtr1 and its homologues in yeast and mammals.

  • 12. Zvyagilskaya, Renata
    et al.
    Lundh, Fredrik
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Samyn, Dieter R.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Granberg, Johanna
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Mouillon, Jean-Marie
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Popova, Yulia
    Thevelein, Johan
    Persson, Bengt L.
    University of Kalmar, School of Pure and Applied Natural Sciences.
    Characterization of the Pho89 phosphate transporter by functional hyperexpression in Saccharomyces cerevisiae.2008In: FEMS yeast research (Print), ISSN 1567-1356, E-ISSN 1567-1364, Vol. 8, no 5, p. 685-696Article in journal (Refereed)
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