Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane.
Phosphoinositide signalling through the eukaryotic plasma membrane makes essential contributions to many processes, including remodelling of the actin cytoskeleton, vesicle trafficking and signalling from the cell surface. A proteome-wide screen performed in Saccharomyces cerevisiae revealed that Ypp1 interacts physically with the plasma-membrane-associated phosphoinositide 4-kinase, Stt4. In the present study, 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 phosphoinositide 4-kinases in yeast, Pik1 and Lsb6, do not suppress this phenotype. This implies a role for Ypp1 in Stt4-dependent 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 that there are substantially fewer plasma-membrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [(3)H]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in ypp1 mutants. This is the principal cause of lethality under non-permissive conditions in ypp1 mutants, as limiting the activity of the Sac1 phosphoinositide 4-phosphate phosphatase leads to restoration of viability. Additionally, the endocytic defect associated with elevated levels of PtdIns4P in sac1Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have on levels of this phosphoinositide.
Zhai
Chao
C
Pharmaceutical Science Division, King's College London, Franklin-Wilkins Building, 150 Stamford Street, London SE1 9NH, UK.
Li
Kuoyu
K
Markaki
Valentini
V
Phelan
John P
JP
Bowers
Katherine
K
Cooke
Frank T
FT
Panaretou
Barry
B
D019870:1-Phosphatidylinositol 4-Kinase / Q000235:genetics / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000235:genetics / Q000378:metabolism*; D000483:Alleles; D002462:Cell Membrane / Q000378:metabolism*; D004705:Endocytosis; D020935:MAP Kinase Signaling System; D009154:Mutation; D010641:Phenotype; D018129:Phosphatidylinositol Phosphates / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D015398:Signal Transduction
D033942:Adaptor Proteins, Vesicular Transport; D018129:Phosphatidylinositol Phosphates; D029701:Saccharomyces cerevisiae Proteins; C522639:YPP1 protein, S cerevisiae; C037178:phosphatidylinositol 4-phosphate; D019870:1-Phosphatidylinositol 4-Kinase; C112465:STT4 protein, S cerevisiae