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  • 1.
    Sengottaiyan, Palanivelu
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Petrlova, Jitka
    Lund university.
    Lagerstedt, Jens
    Lund university.
    Ruiz-Pavon, Lorena
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Budamagunta, Madhu
    University of California, USA.
    Voss, John
    University of California, USA.
    Persson, Bengt L.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Characterization of the biochemical and biophysical properties of the Saccharomyces cerevisiae phosphate transporter Pho892013In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 436, no 3, p. 551-556Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, Pho89 mediates a cation-dependent transport of Pi across the plasma membrane. This integral membrane protein belongs to the Inorganic Phosphate Transporter (PiT) family, a group that includes the mammalian Na+/Pi cotransporters Pit1 and Pit2. Here we report that the Pichia pastoris expressed recombinant Pho89 was purified in the presence of Foscholine-12 and functionally reconstituted into proteoliposomes with a similar substrate specificity as observed in an intact cell system. The alpha-helical content of the Pho89 protein was estimated to 44%. EPR analysis showed that purified Pho89 protein undergoes conformational change upon addition of substrate. 

  • 2.
    Sengottaiyan, Palanivelu
    et al.
    Linnaeus University, Faculty of Health and Life Sciences, Department of Chemistry and Biomedical Sciences.
    Ruiz-Pavon, Lorena
    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.
    Functional expression, purification and reconstitution of the recombinant phosphate transporter Pho89 of Saccharomyces cerevisiae2013In: The FEBS Journal, ISSN 1742-464X, E-ISSN 1742-4658, Vol. 280, no 3, p. 965-975Article in journal (Refereed)
    Abstract [en]

    The Saccharomyces cerevisiae high-affinity phosphate transporter Pho89 is a member of the inorganic phosphate (Pi) transporter (PiT) family, and shares significant homology with the type III Na+/Pi symporters, hPit1 and hPit2. Currently, detailed biochemical and biophysical analyses of Pho89 to better understand its transport mechanisms are limited, owing to the lack of purified Pho89 in an active form. In the present study, we expressed functional Pho89 in the cell membrane of Pichia pastoris, solubilized it in Triton X-100 and foscholine-12, and purified it by immobilized nickel affinity chromatography combined with size exclusion chromatography. The protein eluted as an oligomer on the gel filtration column, and SDS/PAGE followed by western blotting analysis revealed that the protein appeared as bands of approximately 63, 140 and 520 kDa, corresponding to the monomeric, dimeric and oligomeric masses of the protein, respec- tively. Proteoliposomes containing purified and reconstituted Pho89 showed Na+-dependent Pi transport activity driven by an artificially imposed electrochemical Na+ gradient. This implies that Pho89 operates as a symporter. Moreover, its activity is sensitive to the Na+ ionophore monensin. To our knowledge, this study represents the first report on the functional reconstitution of a Pi-coupled PiT family member. 

  • 3.
    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.

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