Project: Deciphering the molecular mechanisms of phosphatidylinositol distribution
About
THE phosphatidylinositol (PI) is a unique lipid because it is the precursor of phosphoinositides, such as PI4P and PI(4,5)P2, These proteins play key roles in the eukaryotic cell. However, the way in which PI is exported from the endoplasmic reticulum (ER), where it is produced, to the Golgi apparatus and the plasma membrane for conversion into phosphoinositides remains poorly understood. Various proteins capable of transferring PI between membranes could perform this task, but this is not well established.
With this project, we want to show that some of them, Sec14, PITPα And PITPβ, coupled with PI4P metabolism and other lipid exchange processes, can transfer PI from the ER to the Golgi apparatus. Next, we will establish how the protein Nir2 can ensure rapid and sustained exchanges of PI and phosphatidic acid between the ER and the plasma membrane during the PI cycle, a metabolic process that regenerates PI(4,5)P2 to maintain human cell signaling functions. Finally, we will define if Sfh3 is a sterol/PI exchanger and what is its function in yeast, perhaps revealing unexpected links between PI and sterol metabolism.
Our team has rebuilt in vitro We will study an ER/Golgi apparatus interface and measure the coupling between the activity of a PI transfer protein, PI4P metabolism, and a sterol/PI4P exchange process. By developing this system and click-chemistry approaches with lipids synthesized by JM Brunel's team (MCT, Marseille), we will demonstrate how PI transfer proteins can function at the interface between the ER and other organelles. With A. Copic (CRBM), we will study these proteins in human and yeast cells, using recent tools to detect PI in organelles. This will provide novel data on the molecular basis of PI distribution in eukaryotic cells.
(HAS) Transfer of dehydroergosterol (DHE), a fluorescent sterol, via Osh4-mediated sterol/PI4P exchanges, driven by a PI4P concentration gradient generated by PI 4-kinase from PI and ATP, and Sac1, which hydrolyzes PI4P to PI. Endoplasmic reticulum-mimicking (ER-like) liposomes containing DOGS-NTA-Ni2+ and covered with Sac1[1-522]His6 were added to an equal amount of PI-enriched, PI4K-coated Golgi-type liposomes. Then, Osh4, alone or with ATP, was injected. A DHE/DNS-PE FRET signal (blue curve) shows that Osh4 actively transfers DHE to the Golgi-type liposomes when PI4P is synthesized and degraded. (B) Golgi-type liposomes coated with PI4K were mixed with ER-type liposomes containing PI. The addition of the PC/PI exchanger Sec14 in the presence of ATP (pink curve) allowed the appearance of PI4P on the surface of the Golgi-type liposomes to be measured using an NBD-PH fluorescent sensor.FAPP, indicating that Sec14 can transport PI from ER-type liposomes to Golgi-type liposomes. (C) ER-type liposomes containing DHE and PI decorated with Sac1[1-522]His6 were added to PI4K-functionalized Golgi-type liposomes. Then, Osh4 and ATP, with or without Sec14, were injected. Active DHE transport was measured only in the presence of ATP, indicating that Sec14 transports PI to PI 4-kinase, thus enabling sterol/PI4P exchange via Osh4.