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Structural modeling of dual-affinity purified Pho84 phosphate transporter
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
2004 (English)In: FEBS Letters, ISSN 0014-5793, E-ISSN 1873-3468, Vol. 578, no 3, 262-268 p.Article in journal (Refereed) Published
Abstract [en]

The phosphate transporter Pho84 of Saccharomyces cerevisiae is predicted to contain 12 transmembrane (TM) regions, divided into two partially duplicated parts of 6 TM segments. The three-dimensional (3D) organization of the Pho84 protein has not yet been determined. However, the 3D crystal structure of the Escherichia coli MFS glycerol-3-phosphate/phosphate antiporter, GlpT, and lactose transporter, LacY, has recently been determined. On the basis of extensive prediction and fold recognition analyses (at the MetaServer), GlpT was proposed as the best structural template on which the arrangement of TM segments of the Pho84 transporter was fit, using the comparative structural modeling program MODELLER. To initiate an evaluation of the appropriateness of the Pho84 model, we have performed two direct tests by targeting spin labels to putative TM segments 8 and 12. Electron paramagnetic resonance spectroscopy was then applied on purified and spin labeled Pho84. The line shape from labels located at both positions is consistent with the structural environment predicted by the template-generated model, thus supporting the model.

Place, publisher, year, edition, pages
2004. Vol. 578, no 3, 262-268 p.
Keyword [en]
Dual-affinity purification; FLAG epitope; Electron paramagnetic resonance; Phosphate transport; Pho84; Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:su:diva-23330DOI: 10.1016/j.febslet.2004.11.012OAI: oai:DiVA.org:su-23330DiVA: diva2:191381
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2017-12-13Bibliographically approved
In thesis
1. Regulatory and Structural Properties of the High-Affinity Phosphate Acquisition System in Saccharomyces cerevisiae
Open this publication in new window or tab >>Regulatory and Structural Properties of the High-Affinity Phosphate Acquisition System in Saccharomyces cerevisiae
2004 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Inorganic phosphate is an essential nutrient required for the synthesis of many cellular components (e.g., nucleic acids, proteins, lipids and sugars), as well as for meeting metabolic needs (e.g., energy production and translocation). In the case of the unicellular yeast Saccharomyces cerevisiae, the presence of both high- and low-affinity phosphate transporters in the plasma membrane provides for adaptation to environmental variations. Of these systems, the high-affinity Pho84 transport system is the major phosphate transporter activated when the cells have limited access to external phosphate.

This integral membrane protein belongs to the major facilitator superfamily (MFS) and possesses 12 predicted transmembrane domains. Activation of this and other proteins (e.g., extracellular phosphatases) involved in maintaining cellular phosphate homeostasis under conditions of limited availability of external phosphate is controlled primarily by transcriptional regulation. However, the presence of proteins indirectly or directly involved in phosphate transport by Pho84, including Gtr1, has been reported. The Gtr1 protein binds guanine nucleotides and probably functions as a molecular switch.

The present thesis describes the regulated intracellular trafficking and degradation of Pho84 in response to phosphate, as well as to its non-hydrolysable and non-utilizable analog methylphosphonate. The involvement of the Gtr1 protein in high-affinity phosphate uptake has also been examined. Moreover, in vitro and in silico analyses of structural and functional aspects of both the Pho84 and Gtr1 proteins are presented and discussed.

Place, publisher, year, edition, pages
Stockholm: Institutionen för biokemi och biofysik, 2004. 63 p.
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-240 (URN)91-7265-936-X (ISBN)
Public defence
2004-10-07, William-Olssonsalen, Geovetenskapens hus, Svante Arrhenius väg 8 A, Stockholm, 10:00
Opponent
Supervisors
Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved

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