DECIPHERING NEW LIPID EXCHANGE MECHANISMS THAT CONTROL LIPID DISTRIBUTION IN THE CELL – ExCHANGE

ExCHANGE is a 4-years multidisciplinary research project that aims to study at the cellular, molecular and atomic level, novel lipid exchange mechanisms mediated by ORP/Osh proteins and driven by PI4P metabolism. This project will benefit from the collaboration of three complementary teams: a team expert in membrane biochemistry and cell biology, led by G.Drin (IPMC, Sophia Antipolis), a team specialist in X-ray crystallography and drug-design led by W.Bourguet (CBS, Montpellier) and a group expert in yeast biology and genetics, lead by A.Copic (IJM, Paris).

The building-blocks of cell membranes are lipids, a group of more than 1000 subspecies. These lipids are precisely allocated within the cell - each organelle has its own lipid composition, and consequently physical features and a molecular identity that are critical for the localization and function of many proteins. Decrypting the mechanisms underlying lipid homeostasis is essential for understanding multiple cellular and physiological functions and the molecular bases of many diseases.

We study lipid transport, one of the key processes that maintain intracellular lipid distribution. The endoplasmic reticulum (ER) is the main cellular lipid factory. These lipids, once synthesized, must be conveyed to other compartments (Golgi apparatus, plasma membrane (PM)). Specialized proteins help lipids, highly hydrophobic, to cross the aqueous medium separating organelle membranes. With the discovery that ORP/Osh proteins, an evolutionary conserved family in eukaryotes, exchange phosphatidylinositol-4-phosphate PI4P with sterol or phosphatidylserine (PS), we propose new models to explain how key lipids are transported vectorially between organelles.

PI4P is a lipid that is prominent in the trans-Golgi and the PM but is absent from the ER. We showed that this PI4P unbalance is used by ORP/Osh proteins to transport sterol or phosphatidylserine from the ER to the trans-Golgi/PM. This explains how cells maintain high sterol and PS levels in the PM, thereby ensuring its impermeability and its signaling competence. We also better understand, as PI4P synthesis depends on ATP, how cells provide energy to trace and maintain lipid transport routes. We aim to address novel key questions arising from these findings.

1) PS is precisely distributed within the cell between and across membranes and this is crucial for cell functions. Osh6p and Osh7p create a PS gradient at ER/PM interface by PS/PI4P exchange but we have almost no evidence on how this exchange process is coupled to PI4P metabolism and about the role of other lipids. Likewise, we do not know whether PS/PI4P exchanges only occur at sites where the ER and PM are close, how Osh6/7p are localized to these sites, and whether they contribute to PS asymmetry. To address these issues, we will fully characterize Osh6/Osh7p activity in yeast and in vitro, with advanced tools to measure PS/PI4P exchanges.

2) Sequence & structural analyses suggest that some ORP/Osh proteins bind PI4P and a second lipid ligand that is neither sterol nor PS. We posit that these ORP/Osh proteins transport, via a PI4P-driven exchange mechanism, various lipids whose nature remains to be defined. We plan to identify the second ligand of some ORP/Osh proteins, and describe how these ligands are recognized using X-ray crystallography, then explore the role of these proteins in lipid homeostasis.

3) OSBP has a key role in the replication of various viruses in human cells. By sterol/PI4P exchange, it delivers sterol en masse into organelles where viral proteins elicit PI4P overproduction to support the genesis of replication platforms. Molecules termed ORPhilins target OSBP and display a strong antiviral action. An urgent task is to solve the X-ray structure of the sterol-binding domain of OSBP bound to these molecules to describe at the atomic level how they inhibit OSBP. Such analyses would serve to create novel pharmacological entities by drug design.