Regulatory and Structural Properties of the High-Affinity Phosphate Acquisition System in Saccharomyces cerevisiae

Sammanfattning: 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.