Abstract 2365 Class I phosphatidylinositol transfer proteins control organelle membrane lipid composition beyond supporting phosphoinositides synthesis

Phosphatidylinositol (PI) is one of the structural phospholipids in cellular membranes and serves as the substrate to form the seven phosphorylated inositol lipid species, collectively called phosphoinositides (PPIns). PPIns have been known to play multiple regulatory roles in a variety of cellular functions associated with organelle membranes. Since PI is synthesized in the ER, while PPIns are mostly formed in other organelle membranes, PI must reach these organelles. However, the process(es) by which PI transport is achieved is poorly understood. It has been widely assumed, but not convincingly proven, that PI transfer proteins (PITP) deliver PI from the ER to other cellular membranes. Here we used a recently identified highly potent and selective Class I PITP inhibitor to investigate the contribution of Class I PITPs to the generation of PPIns in various organelle membranes. We found that inhibition of Class I PITPs rapidly decreased PI4P levels in the plasma membrane (PM) and Rab7-positive late endosome/lysosomes, but, surprisingly, did not alter PPIns within the Golgi compartment and early endosomes. Recovery of PI 4,5-bisphosphate (PI(4,5)P2) after strong PLC activation in the PM required both the Class I and Class II PITPs. Furthermore, lipidomic analysis of cells treated with the PITP inhibitor, revealed additional changes in other lipid classes that were specific to selected fatty acyl forms, including reduced levels of phosphatidylserine, di- and triacylglycerols, as well as a prominent increase in unsaturated phosphatidic acid. Lastly, we solved the X-ray structure of PITPNA bound to the inhibitor to gain further insights into its mechanism of inhibitory actions. These studies demonstrate that inhibition of Class I PITPs not only affects PPIn pools, but also has a profound impact on the overall lipid composition of cellular membranes.