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

在这个项目中，我们希望阐明膜脂如何影响ROP蛋白在植物中的生理功能。在我们以前的和初步的工作中，我们已经描述了磷酸肌醇对ROP依赖的肌动蛋白结构的调节作用，并发现ROP与磷酸肌醇和PI 4P 5-激酶的相互作用。我们还发现，磷酸肌醇和PI 4P 5-激酶与动态质膜微区，这取决于鞘脂和甾醇的存在。其他人最近表明，ROP与膜微区相关，这种关联也受到磷脂PtdSer的影响。综合观察表明磷脂在控制植物ROP功能中的作用比以前认识到的更复杂。ROPs的影响由磷脂酰肌醇和PtdSer，提出的问题是否这些脂质有单独的，协同或拮抗，或单独的影响ROPs。我们提出了一个两部分的项目来阐明调节磷脂如何影响与膜微区的ROPs协会，以及这些相互作用的生理效应是什么。这两个部分的目的是阐明ROP-膜相互作用从脂质方面和从蛋白质方面，分别，并将提供补充信息的作用，膜微结构域在ROP功能：第1部分解决膜脂质组合物的影响，描绘如何磷酸肌醇和PtdSer有助于质膜微结构域的形成，其中ROP被招募。第2部分涉及调制蛋白质-脂质相互作用的方面，以确定ROP与质膜微区中的某些脂质的关联是否是ROP的生理效应所需的。拟议的实验将结合联合收割机使用我们的平台，我们在细胞生物学和脂质生物传感器的熟练程度，分析了一系列可用的拟南芥突变体与明确定义的膜脂代谢缺陷的未标记的膜磷脂的定量生化分析。

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.