Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes

Several members of the evolutionarily conserved oxysterol-binding protein (OSBP)-related proteins(ORP)/OSBP homologs (Osh) family have recently been found ...

Université Côte d’Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire

A cellular membrane is highly deformable: during the last decade, numerous studies have dissected at the molecular scale how during vesicular transport various proteins could deform a membrane or recognize membrane deformation. We will discuss how the activity of ArfGAP1 and GMAP-210, two proteins involved in vesicular transport, is regulated by membrane curvature thanks to an ALPS motif. Since a membrane is not solely defined by its shape but also by its lipids composition, we will show how others proteins are adapted to other lipid compositions to recognize membrane shape.

[Regulation of vesicular transport by membrane curvature].

Numerous data have been collected on lipid-binding amphipathic helices involved in membrane-remodeling machineries and vesicular transport. Here we describe how, with regard to lipid composition, the physicochemical features of some amphipathic helices explain their ability to recognize membrane curvature or to participate in membrane remodeling. We propose that sensing highly-curved membranes requires that the polar and hydrophobic faces of the helix do not cooperate in lipid binding. A more detailed description of the interaction between amphipathic helices and lipids is however needed; notably to explain how new helices contribute to detection of modest changes in curvature or even negative curvature.

Amphipathic helices and membrane curvature.

Lipids are unevenly distributed within and between cell membranes, thus defining organelle identity. Such distribution relies on local metabolic branches and mechanisms that move lipids. These processes are regulated by feedback mechanisms that decipher topographical information in organelle membranes and then regulate lipid levels or flows. In the endoplasmic reticulum, the major lipid source, transcriptional regulators and enzymes sense changes in membrane features to modulate lipid production. At the Golgi apparatus, lipid-synthesizing, lipid-flippase, and lipid-transport proteins (LTPs) collaborate to control lipid balance and distribution within the membrane to guarantee remodeling processes crucial for vesicular trafficking. Open questions exist regarding LTPs, which are thought to be lipid sensors that regulate lipid synthesis or carriers that transfer lipids between organelles across long distances or in contact sites. A novel model is that LTPs, by exchanging two different lipids, exploit one lipid gradient between two distinct membranes to build a second lipid gradient. 

Topological regulation of lipid balance in cells.

In eukaryotic cells, phosphatidylserine (PS) is synthesized in the endoplasmic reticulum (ER) but is highly enriched in the plasma membrane (PM), where it contributes negative charge and to specific recruitment of signaling proteins. This distribution relies on transport mechanisms whose nature remains elusive. Here, we found that the PS transporter Osh6p extracted phosphatidylinositol 4-phosphate (PI4P) and exchanged PS for PI4P between two membranes. We solved the crystal structure of Osh6p:PI4P complex and demonstrated that the transport of PS by Osh6p depends on PI4P recognition in vivo. Finally, we showed that the PI4P-phosphatase Sac1p, by maintaining a PI4P gradient at the ER/PM interface, drove PS transport. Thus, PS transport by oxysterol-binding protein-related protein (ORP)/oxysterol-binding homology (Osh) proteins is fueled by PI4P metabolism through PS/PI4P exchange cycles. 

INTRACELLULAR TRANSPORT. Phosphatidylserine transport by ORP/Osh proteins is driven by phosphatidylinositol 4-phosphate.

Lipids are precisely distributed in cell membranes, along with associated proteins defining organelle identity. Because the major cellular lipid factory is the endoplasmic reticulum (ER), a key issue is to understand how various lipids are subsequently delivered to other compartments by vesicular and non-vesicular transport pathways. Efforts are currently made to decipher how lipid transfer proteins (LTPs) work either across long distances or confined to membrane contact sites (MCSs) where two organelles are at close proximity. Recent findings reveal that proteins of the oxysterol-binding protein related-proteins (ORP)/oxysterol-binding homology (Osh) family are not all just sterol transporters/sensors: some can bind either phosphatidylinositol 4-phosphate (PtdIns(4)P) and sterol or PtdIns(4)P and phosphatidylserine (PS), exchange these lipids between membranes, and thereby use phosphoinositide metabolism to create cellular lipid gradients. Lipid exchange is likely a widespread mechanism also utilized by other LTPs to efficiently trade lipids between organelle membranes. Finally, the discovery of more proteins bearing a lipid-binding module (SMP or START-like domain) raises new questions on how lipids are conveyed in cells and how the activities of different LTPs are coordinated.

New molecular mechanisms of inter-organelle lipid transport.

Lipids are unevenly distributed within eukaryotic cells, allowing various processes to work in an optimized membrane environment. Rather than following vesicular trafficking pathways, certain lipids are transported by lipid transfer proteins (LTPs), some at sites of close apposition of two organelles called membrane contact sites (MCSs). An important question is whether or not LTPs are able to convey lipids to create and maintain the lipid gradients observed between organelles. Recent data shows that LTPs from the ORP/Osh family transport sterols and phosphatidylserine (PS) to the Golgi and plasma membrane, respectively, through counter-exchange for the phosphoinositide phosphatidylinositol 4-phosphate (PI4P). Coupling PI4P-driven exchange to PI4P metabolism allows for an accumulation of these lipids in specific organelles and for a regulation of ORP/Osh proteins in MCSs. Additionally, data indicate that PI4P/sterol exchanges are hijacked by various virus strains to generate replication organelles. Compounds called ORPhilins block PI4P/sterol exchange and have thereby powerful antiviral activities, indicating in turn that some ORP/Osh proteins might be relevant pharmaceutical targets.

Running up that hill: How to create cellular lipid gradients by lipid counter-flows.

Discoveries spanning several decades have pointed to vital membrane lipid trafficking pathways involving both vesicular and non-vesicular carriers. But the relative contributions for distinct membrane delivery pathways in cell growth and organelle biogenesis continue to be a puzzle. This is because lipids flow from many sources and across many paths via transport vesicles, non-vesicular transfer proteins, and dynamic interactions between organelles at membrane contact sites. This forum presents our latest understanding, appreciation, and queries regarding the lipid transport mechanisms necessary to drive membrane expansion during organelle biogenesis and cell growth.

Membrane dynamics and organelle biogenesis-lipid pipelines and vesicular carriers.

Recent findings unveiled that Oxysterol-binding protein-related proteins (ORP)/Oxysterol-binding homology (Osh) proteins, which constitute a major family of lipid transfer proteins (LTPs), conserved among eukaryotes, are not all mere sterol transporters or sensors. Indeed, some of them are able to exchange sterol for phosphatidylinositol-4-phosphate (PI4P) or phosphatidylserine (PS) for PI4P between membranes and thereby to use PI4P metabolism to generate sterol or PS gradients in the cell, respectively. Here, we describe a full strategy to measure in vitro a sterol/PI4P exchange process between artificial membranes using Förster resonance energy transfer (FRET)-based assays and a standard spectrofluorometer. Such an approach can serve to better characterize the activity of known sterol/PI4P exchangers, but also to reveal whether ill-defined ORP/Osh proteins or LTPs belonging to other families have such an exchange activity. Besides, this protocol is amenable to test whether molecules can act as Orphilins, which have been found to inhibit the sterol/PI4P exchange activity of certain ORPs. Last, our strategy to measure in real-time PI4P transport using a known lipid-binding domain can serve as a basis for the design of novel in vitro protocols aiming to detect other lipid species.

In Vitro Strategy to Measure Sterol/Phosphatidylinositol-4-Phosphate Exchange Between Membranes.

A central assumption is that lipid transfer proteins (LTPs) bind transiently to organelle membranes to distribute lipids in the eukaryotic cell. Osh6p and Osh7p are yeast LTPs that transfer phosphatidylserine (PS) from the endoplasmic reticulum (ER) to the plasma membrane (PM) via PS/phosphatidylinositol-4-phosphate (PI4P) exchange cycles. It is unknown how, at each cycle, they escape from the electrostatic attraction of the PM, highly anionic, to return to the ER. Using cellular and in vitro approaches, we show that Osh6p reduces its avidity for anionic membranes once it captures PS or PI4P, due to a molecular lid closing its lipid-binding pocket. Thus, Osh6p maintains its transport activity between ER- and PM-like membranes. Further investigations reveal that the lid governs the membrane docking and activity of Osh6p because it is anionic. Our study unveils how an LTP self-limits its residency time on membranes, via an electrostatic switching mechanism, to transfer lipids efficiently.

Guillaume Drin

Université Côte d’Azur, Centre National de la Recherche Scientifique, Institut de Pharmacologie Moléculaire et Cellulaire

Fluorescence-Based Measurements of Phosphatidylserine/Phosphatidylinositol 4-Phosphate Exchange Between Membranes