| The yeast Saccharomyces cerevisiae contains a family of zinc responsive membrane proteins named IZH1-4 (I&barbelow;mplicated in Z&barbelow;inc H&barbelow;omeostasis). All members of this superfamily, named PAQR (P&barbelow;rogestin, A&barbelow;dipoQ&barbelow;-R&barbelow;eceptor), are putative membrane proteins with at least seven transmembrane domains. Our vast interest in this family stems from recent characterization of five of the eleven human homologues. PAQR1 and PAQR2 are plasma membrane receptors of adiponectin, a hormone that regulates glucose uptake and fatty acid oxidation and may play a key role in the treatment of obesity and Type 2 diabetes. Additionally, PAQR7, PAQR8, and PAQR5, are membrane progestin receptors that mediate rapid signaling responses characteristic of G-protein coupled receptors. The objective of this project is to characterize the role of the PAQR family in lipid mediated cellular signaling. We have evidence to suggest the IZH's are involved in metal and lipid metabolism. Mutants are sensitive to high concentrations of zinc and conditions that affect the cell wall integrity kinase pathway such as caffeine and sodium orthovanadate.;To confirm our genetic evidence at the protein level, we chose to globally examine changes in reversible phosphorylation upon progesterone ligand binding to PAQR7 as a model system. We utilized 2-D gel electrophoresis, in conjunction with a fluorescent phosphoprotein stain and HPLC tandem mass spectrometric analysis, to identify proteins showing a significant change in phosphorylation upon treatment. Two of the proteins, Lsp1p (l&barbelow;ong chain bases s&barbelow;timulate p&barbelow;hosphorylation) and Pil1p (p&barbelow;hosphorylation is i&barbelow;nhibited by l&barbelow;ong chain bases) were of particular interest and warranted further exploration. A TAP (t&barbelow;andem a&barbelow;ffinity p&barbelow;urification)-tagged version of Lsp1p under control of its own promoter was used to purify both Lsp1p and Pil1p, since the proteins interact. Upon mass spectrometric analysis, previously unknown phosphorylation sites on Pil1p and Lsp1p were identified and quantified. Investigation of the PAQR proteins in yeast paves the way toward understanding more complicated and medically relevant systems in mammalian cells. |
