Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane.
Ypp1/YGR198w plays an essential role in phosphoinositide signalling at the plasma membrane.
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DOI：
10.1042/bj20080209
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发表时间：
2008-11
期刊：
影响因子：
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通讯作者：
C. Zhai;Kuoyu Li;V. Markaki;J. Phelan;K. Bowers;F. Cooke;B. Panaretou
中科院分区：
文献类型：
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作者：
C. Zhai;Kuoyu Li;V. Markaki;J. Phelan;K. Bowers;F. Cooke;B. Panaretou
肌醇磷脂通过真核细胞质膜传递的信号在许多过程中起着重要作用，包括肌动蛋白细胞骨架的重塑、囊泡运输和细胞表面的信号传递。在酿酒酵母中进行的蛋白质组筛选显示，Ypp1与质膜相关的磷脂酰肌醇4-激酶Stt4物理上相互作用。在本研究中，我们证明了ypp1和stt4条件突变体的表型是相同的，即渗透中层温度敏感性、对细胞壁不稳定剂的超敏以及肌动蛋白的组织缺陷。我们进一步证明，STT4的过表达抑制了ypp1突变体对温度敏感的生长缺陷。相反，编码另外两个磷酸肌醇4-激酶的基因在酵母中的过度表达，Pik1和Lsb6，并不抑制这种表型。这意味着Ypp1在质膜上的Stt4依赖事件中发挥作用，而不是在磷脂酰肌醇4-磷酸的整体代谢中发挥一般作用。Pleckstrin同源结构域传感器的使用表明，与野生型细胞相比，ypp1突变体中质膜相关的4-磷酸化磷酸肌醇的数量大大减少。此外，用[(3)H]肌醇在体内标记表明，在ypp1突变体中，磷脂酰肌醇4-磷酸水平显著降低。这是在不允许的条件下导致ypp1突变体致死的主要原因，因为限制SAC1磷酸肌醇4-磷酸磷酸酶的活性会导致活性的恢复。此外，与Sac1Delta细胞中PtdIns4P水平升高相关的内吞缺陷与ypp1突变体一起被修复，这与这两个突变对这种磷脂酰肌醇水平的相反影响是一致的。
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.