Non-vesicular phosphatidylinositol transfer plays critical roles in defining organelle lipid composition.

Phosphatidylinositol (PI) is the precursor lipid for the minor phosphoinositides (PPIns), which are critical for multiple functions in all eukaryotic cells. It is poorly understood how phosphatidylinositol, which is synthesized in the ER, reaches those membranes where PPIns are formed. Here, we used VT01454, a recently identified inhibitor of class I PI transfer proteins (PITPs), to unravel their roles in lipid metabolism, and solved the structure of inhibitor-bound PITPNA to gain insight into the mode of inhibition. We found that class I PITPs not only distribute PI for PPIns production in various organelles such as the plasma membrane (PM) and late endosomes/lysosomes, but that their inhibition also significantly reduced the levels of phosphatidylserine, di- and triacylglycerols, and other lipids, and caused prominent increases in phosphatidic acid. While VT01454 did not inhibit Golgi PI4P formation nor reduce resting PM PI(4,5)P2 levels, the recovery of the PM pool of PI(4,5)P2 after receptor-mediated hydrolysis required both class I and class II PITPs. Overall, these studies show that class I PITPs differentially regulate phosphoinositide pools and affect the overall cellular lipid landscape. Synopsis: Class I phosphatidylinositol (PI) transfer proteins (PITPs) have been proposed to supply PI to the lipid kinases that produce mono-phosphorylated phosphoinositides, PI4P and PI3P. A novel inhibitor of class I PITPs illuminates how these proteins contribute to the maintenance of various phosphoinositide pools in intact cells. PITPs provide PI to the plasma membrane and late endosomes for generation of PI4P in those organelle membranes. Acute PITP inhibition does not affect PI4P levels in the Golgi and only has a small impact on PI3P levels in early endosomes. PI(4,5)P2 maintenance in the plasma membrane upon phospholipase C activation requires both class I and class II PITPs. The crystal structure of inhibitor-bound PITPNA reveals an "open" structure identical to the reported cargo-free structures. PTIP Inhibition affects the levels of several lipid classes. Inhibition of specific lipid transfer proteins reveals how pools of key signalling lipids are maintained in the plasma membrane and late endosome. [ABSTRACT FROM AUTHOR]