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Regulatory and Structural Properties of the High-Affinity Phosphate Acquisition System in Saccharomyces cerevisiae
Stockholm University, Faculty of Science, Department of Biochemistry and Biophysics.
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: urn:nbn:se:su:diva-240ISBN: 91-7265-936-X (print)OAI: oai:DiVA.org:su-240DiVA: diva2:191382
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
List of papers
1. Intracellular localization of an active green fluorescent proteintagged Pho84 phosphate permease in Saccharomyces cerevisiae
Open this publication in new window or tab >>Intracellular localization of an active green fluorescent proteintagged Pho84 phosphate permease in Saccharomyces cerevisiae
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1999 In: FEBS letters, ISSN 0014-5793, Vol. 462, 37-42 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23325 (URN)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
2. Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae
Open this publication in new window or tab >>Properties of the cysteine-less Pho84 phosphate transporter of Saccharomyces cerevisiae
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2001 In: Biochemical and Biophysical Research Communications, ISSN 0006-291X, Vol. 287, no 4, 837- 842 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23326 (URN)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
3. Mutagenic and functional analysis of the C-terminus of Saccharomyces cerevisiae Pho84 phosphate transporter
Open this publication in new window or tab >>Mutagenic and functional analysis of the C-terminus of Saccharomyces cerevisiae Pho84 phosphate transporter
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2002 In: FEBS letters, ISSN 0014-5793, Vol. 526, 31-37 p.Article in journal (Refereed) Published
Identifiers
urn:nbn:se:su:diva-23327 (URN)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16Bibliographically approved
4. The effects of methylphosphonate, a phosphate analog, on the expression and degradation of the highaffinity phosphate transporter Pho84, in Saccharomyces cerevisiae
Open this publication in new window or tab >>The effects of methylphosphonate, a phosphate analog, on the expression and degradation of the highaffinity phosphate transporter Pho84, in Saccharomyces cerevisiae
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2004 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 45, 14444-14453 p.Article in journal (Refereed) Published
Abstract [en]

In Saccharomyces cerevisiae, the Pho84 high-affinity transport system is the major phosphate transporter activated when the cells experience a limitation in external phosphate. In this study, we have compared the phosphate-responsive mechanism of cells expressing PHO84 with a Δpho84 strain by use of a phosphate analogue, methylphosphonate, which was judged to be suitable for assessment of phosphate homeostasis in the cells. Intracellular levels of the analogue, which in several respects mimicks phosphate, were monitored by 31P NMR spectroscopy. Results show that methylphosphonate is a nonhydrolyzable and nonutilizable analogue that cannot be used to replenish phosphate or polyphosphate in yeast cells grown under conditions of phosphate limitation. However, the presence of methylphosphonate under such conditions represses the Pho5 acidic phosphatase activity of PHO84 cells, a finding that implies a direct role of the analogue in the regulation of phosphate-responsive genes and/or proteins. Likewise, accumulation of the Pho84 protein at the plasma membrane of the same cells is inhibited by methylphosphonate, although the derepressive expression of the PHO84 gene is unperturbed. Thus, a post-transcriptional regulation is suggested. Supportive of this suggestion is the fact that addition of methylphosphonate to cells with abundant and active Pho84 at the plasma membrane causes enhanced internalization of the Pho84 protein. Altogether, these observations suggest that the Pho84 transporter is regulated not only at the transcriptional level but also by a direct molecule-sensing mechanism at the protein level.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-23328 (URN)10.1021/bi049327t (DOI)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2010-07-07Bibliographically approved
5. The structure and function of GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae
Open this publication in new window or tab >>The structure and function of GTP binding protein Gtr1 and its role in phosphate transport in Saccharomyces cerevisiae
2005 (English)In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, 511-517 p.Article in journal (Refereed) Published
Abstract [en]

The Pho84 high-affinity phosphate permease is the primary phosphate transporter in the yeast Saccharomyces cerevisiae under phosphate-limiting conditions. The soluble G protein, Gtr1, has previously been suggested to be involved in the derepressible Pho84 phosphate uptake function. This idea was based on a displayed deletion phenotype of Δgtr1 similar to the Δpho84 phenotype. As of yet, the mode of interaction has not been described. The consequences of a deletion of gtr1 on in vivo Pho84 expression, trafficking and activity, and extracellular phosphatase activity were analyzed in strains synthesizing either Pho84−green fluorescent protein or Pho84−myc chimeras. The studies revealed a delayed response in Pho84-mediated phosphate uptake and extracellular phosphatase activity under phosphate-limiting conditions. EPR spectroscopic studies verified that the N-terminal G binding domain (residues 1−185) harbors the nucleotide responsive elements. In contrast, the spectra obtained for the C-terminal part (residues 186−310) displayed no evidence of conformational changes upon GTP addition.

National Category
Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
Identifiers
urn:nbn:se:su:diva-23329 (URN)10.1021/bi048659v (DOI)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2010-08-09Bibliographically approved
6. Structural modeling of dual-affinity purified Pho84 phosphate transporter
Open this publication in new window or tab >>Structural modeling of dual-affinity purified Pho84 phosphate transporter
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.

Keyword
Dual-affinity purification; FLAG epitope; Electron paramagnetic resonance; Phosphate transport; Pho84; Yeast
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:su:diva-23330 (URN)10.1016/j.febslet.2004.11.012 (DOI)
Note
Part of urn:nbn:se:su:diva-240Available from: 2004-09-16 Created: 2004-09-16 Last updated: 2010-08-09Bibliographically approved

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