期刊
BIOCHEMICAL JOURNAL
卷 415, 期 -, 页码 455-466出版社
PORTLAND PRESS LTD
DOI: 10.1042/BJ20080209
关键词
lipid kinase; phosphoinositide; Saccharomyces cerevisiae; signalling; Stt4; Ypp1
资金
- Biotechnology and Biological Research Council (BBSRC) [BB/E02055O/1]
- KC Wong Foundation
- China Scholarship Council
- BBSRC [BB/E020550/1, BB/E001521/1] Funding Source: UKRI
- MRC [G0601097] Funding Source: UKRI
- Biotechnology and Biological Sciences Research Council [BB/E001521/1, BB/E020550/1] Funding Source: researchfish
- Medical Research Council [G0601097] Funding Source: researchfish
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, Pik 1 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 plasmamembrane-associated 4-phosphorylated phosphoinositides in ypp1 mutants in comparison with wild-type cells. Furthermore, in vivo labelling with [H-3]inositol indicates a dramatic reduction in the level of phosphatidylinositol 4-phosphate in yppl 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 sac1 Delta cells is restored in combination with a ypp1 mutant, consistent with the opposing effects that these two mutations have oil levels of this phosphoinositide.
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