Summary It is well understood how lipids are synthesized and metabolized in cells and that many lipids exhibit signalling functions to regulate cellular processes in a spatially and temporally defined way. The latter requires the build-up and turnover of lipid species in membranes either in a site-specific fashion or, alternatively, a directed form of lipid transport. This work aims to investigate the intracellular transfer of lipids from one membrane to another by several proteins that we discovered to be involved in lipid transport. In the previous funding period, we synthesized multifunctional lipid derivatives of five phosphoinositides and four common glycerophospholipids. These feature a photo-activatable protecting group (”cage”) to release the lipid derivative by light and a photo-crosslinking diazirine to covalently attach the lipid derivative to binding proteins. An alkyne group for click chemistry is useful for isolating lipid-protein conjugates or for determining the lipid location in cells by fluorescent tagging and microscopy. In published work, we identified specific lipid binding proteins for phosphatidylinositol 3,4,5-trisphosphate (PIP3), phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2], and phosphatidylinositol (PI). Because the “caged” derivatives accumulated in endomembranes, we observed changes in their subsequent cellular distribution after uncaging with light. All three phosphoinositides transferred to the plasma membrane (PM) within 30 to 120 sec. Such transport is known for PI but has never been described for PIP3 or PI(3,4)P2. We then identified putative lipid transport proteins via proteomic analysis and used siRNAs to block lipid transport. We found two hits that were required for transporting PIP3 and PI(3,4)P2: cytosolic MPP6 and transmembrane ATP11A. Knockdown of both reduced internalization of EGF receptor, indicating effects on PIP3 signalling. In this work, we will characterize the lipid transport by these two proteins with respect to lipid specificity (Aim 1.1). For this aim, we will improve our current method of precisely quantifying lipid transport in cells (Aim 1.2). We will validate our findings in-vitro by using recombinant proteins including those with point mutations of key residues to study protein-lipid interactions with biophysical and biochemical methods (Aim 1.3). We will increase rigor by analysing the cellular lipid composition by mass spectrometry after uncaging lipid derivatives (Aim 1.4). In Aim 2, we will demonstrate the need of MPP6 mobility for lipid transport. We will develop a light-switchable MPP6 using the LOV2 technique that will replace endogenous MPP6 (by gene editing) and will be located at the plasma membrane (PM) until we illuminate the cells with 488 nm light. We hypothesize that lipid transport will only be possible if MPP6 is liberated from the PM. We will also test for lipid retro-transport by MPP6 from the PM to endomembranes. For this, we will synthesize lipid derivatives tha...