Control of ROP function by membrane lipids during plant exocytosis and endocytosis

在拟议的项目中，我们想要阐明膜脂如何影响植物中ROP蛋白的生理功能。在我们之前和前期的工作中，我们已经描述了肌醇磷脂对ROP依赖的肌动蛋白结构的调节作用，并发现了Rop与肌醇磷脂和PI4P5-激酶的相互作用。我们进一步发现，磷脂酰肌醇和PI4P5-激酶与动态的质膜微区有关，这种微区依赖于鞘磷脂和甾醇的存在。另一些研究表明，Rop与膜微区有关，而且这种结合也受磷脂PtdSer的影响。综合观察表明，磷脂在控制植物ROP功能中的作用比之前所估计的更复杂。ROPs同时受到磷脂酰肌醇和PtdSer的影响，这提出了一个问题，即这些脂类对ROPs是单独的、协同的、拮抗的还是单独的作用。我们提出了一个两部分的项目来阐明调节性磷脂如何影响ROPs与膜微域的结合，以及这些相互作用的生理效应是什么。这两部分分别从脂侧和蛋白质侧阐明了ROP与膜的相互作用，并将为膜微域在ROP功能中的作用提供补充信息：第一部分是膜脂组成的影响，以描述磷脂酰肌醇和PtdSer如何促进ROP被招募到的质膜微域的形成。第二部分讨论了调节的蛋白质-脂质相互作用的方面，以确定是否需要将Rop与质膜微区中的某些脂质结合来实现Rop的生理效应。拟议的实验将结合我们对未标记膜磷脂的定量生化分析平台的使用，以及我们在细胞生物学和脂生物传感器方面的熟练程度，来分析一系列在膜脂代谢方面存在明确缺陷的现有拟南芥突变体。

In the proposed project we want to elucidate how membrane lipids influence the physiological function of ROP proteins in plants. In our previous and preliminary work we have described regulatory effects of phosphoinositides on ROP-dependent actin structures and found interactions of ROPs with phosphoinositides and with PI4P 5-kinases. We furthermore found that phosphoinositides and PI4P 5-kinases associate with dynamic plasma membrane microdomains, which depend on the presence of sphingolipids and sterols. Others have recently shown that ROPs associate with membrane microdomains and that this association is influenced also by the phospholipid PtdSer. The combined observations indicate a more complex role for phospholipids in the control of ROP function in plants than previously appreciated. ROPs are influenced by both phosphoinositides and PtdSer, raising the question whether these lipids have individual, synergistic or antagonistic, or separate effects on ROPs.We propose a two part project to elucidate how regulatory phospholipids influence the association of ROPs with membrane microdomains, and what the physiological effects of these interactions are. The two parts aim to elucidate ROP-membrane interactions from the lipid side and from the protein side, respectively, and will provide complementary information on the role of membrane microdomains in ROP function:Part 1 addresses effects of membrane lipid composition to delineate how phosphoinositides and PtdSer contribute to the formation of plasma membrane microdomains to which ROPs are recruited. Part 2 addresses aspects of modulated protein-lipid interactions to determine whether association of ROPs with certain lipids in plasma membrane microdomains is required for physiological effects of ROPs. The proposed experiments will combine the use of our platform for the quantitative biochemical analysis of unlabeled membrane phospholipids with our proficiency in cell biology and lipid biosensors to analyze a range of available Arabidopsis mutants with well-defined defects in membrane lipid metabolism.