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

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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.