Abstract
The budding yeast Saccharomyces cerevisiae serves as an effective model organism for many cellular pathways including phosphate transport, accumulation, and storage. In S. cerevisiae, phosphate is actively transported across the plasma membrane via several phosphate carriers and is then transported into the acidic vacuole (roughly equivalent to the mammalian lysosome with degradative functions but with additional storage functions, such as calcium) where it is synthesized into volutin, a storage form of polyphosphate, found in many organisms. We have been studying volutin granule formation in wild type cells to determine the physiological requirements for formation and in mutants to determine the pathway by which the volutin biosynthetic proteins are transported to the vacuole. Undertaking an analysis of volutin formation in yeast vacuoles by blocking vacuole function with pharmacological agents, such as ionomycin and CCCP, we see that vacuole pH as well as vacuolar calcium seems critical for volutin formation. Different blocks in vacuolar protein sorting have differential effects on volutin granule accumulation, with volutin granule formation seen in all mutant strains thus far tested, except for vps33, a mutant cell strain lacking all vacuolar structure. Our data are consistent with pleiotrophic effects of vacuolar physiological function blocks leading to a decrease in volutin formation.
Highlights
Phosphate is an essential inorganic molecule required for diverse cellular functions including nucleotide anabolism, bone remodeling and growth, and phosphorylation of a variety of substrates for function or augmentation ofHow to cite this paper: Marshall, P.A., De La Rosa, D.B., Sanchez, L.G. and Starr, M.L. (2014) Analysis of Volutin Granule Formation in Saccharomyces cerevisiae
We were interested in determining the pathway by which volutin granule proteins are trafficked to the vacuole, and so undertook volutin granule staining in a series of vps mutants (Figures 2-6)
Extensive volutin granule formation is seen in all mutant strains, except for Δvps33, a mutant cell strain lacking all vacuolar structure
Summary
Phosphate is an essential inorganic molecule required for diverse cellular functions including nucleotide anabolism, bone remodeling and growth, and phosphorylation of a variety of substrates for function or augmentation of. When the intracellular phosphate level is high, the high affinity transporter Pho84p is targeted for degradation at the vacuole, and Pho4p is phosphorylated and excluded from the nucleus, and cells do not transcribe genes necessary for phosphate storage and accumulation [7]-[9]. When intracellular phosphate is low, Pho84p remains active; Pho4p binds to DNA and transcribes its target genes, and cells upregulate a myriad of genes necessary for phosphate storage and accumulation. We have been studying volutin granule formation in wild type cells, in cell strains lacking the ability to traffic some or all proteins to the yeast vacuole (termed vacuolar protein sorting or vps mutants) [20], and in cells treated with a variety of pharmacological agents. Several strains carrying a vps mutant are still able to make volutin and cells treated with inhibitors of vacuolar function are able to form volutin
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