Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including re-modelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 physically interacts with the plasma-membrane associated phosphatidylinositol 4-kinase Stt4. Here we demonstrate that phenotypes of ypp1 and stt4 conditional mutants are identical, namely osmoremedial temperature sensitivity, hypersensitivity to cell wall destabilizers and defective organization of actin. We go on to show that overexpression of STT4 suppresses the temperature sensitive growth defect of ypp1 mutants. In contrast, overexpression of genes encoding the other two phosphatidylinositol 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependant events at the plasma membrane, as opposed to a general role in overall metabolism of phosphatidylinositol 4-phosphate. Use of a pleckstrin homology domain sensor reveals there are substantially less plasma membrane associated 4-phosphorylated phosphoinositides in ypp1 mutants, in comparison to wild-type cells. Furthermore, in vivo labelling with [3H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality at non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphatidylinositol 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of phosphatidylinositol 4-phosphate in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects these two mutations have on levels of this phosphoinositide.
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|Evidence ID||Analyze ID||Gene||Gene Systematic Name||Phenotype||Experiment Type||Experiment Type Category||Mutant Information||Strain Background||Chemical||Details||Reference|
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