Guillaume Drin

A general amphipathic alpha-helical motif for sensing membrane curvature.

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic α-helix: its polar face is weakly charged, containing mainly serine and threonine residues. We show that this feature explains the specificity of ArfGAP1 for curved versus flat lipid membranes. We built an algorithm to identify other potential amphipathic α-helices rich in serine and threonine residues in protein databases. Among the identified sequences, we show that three act as membrane curvature sensors. In the golgin GMAP-210, the sensor may serve to trap small vesicles at the end of a long coiled coil. In Osh4p/Kes1p, which transports sterol between membranes, the sensor controls access to the sterol-binding pocket. In the nucleoporin Nup133, the sensor corresponds to an exposed loop of a β-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.

HELIQUEST: a web server to screen sequences with specific α-helical properties

SUMMARYnHELIQUEST calculates the physicochemical properties and amino acid composition of an alpha-helix and screens databank to identify protein segments possessing similar features. This server is also dedicated to mutating helices manually or automatically by genetic algorithm to design analogues of defined features.nnnAVAILABILITYnhttp://heliquest.ipmc.cnrs.fr.

Amphipathic helices and 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.

A Four-Step Cycle Driven by PI(4)P Hydrolysis Directs Sterol/PI(4)P Exchange by the ER-Golgi Tether OSBP

Several proteins at endoplasmic reticulum (ER)-Golgi membrane contact sites contain a PH domain that interacts with the Golgi phosphoinositide PI(4)P, a FFAT motif that interacts with the ER protein VAP-A, and a lipid transfer domain. This architecture suggests the ability to both tether organelles and transport lipids between them. We show that in oxysterol binding protein (OSBP) these two activities are coupled by a four-step cycle. Membrane tethering by the PH domain and the FFAT motif enables sterol transfer by the lipid transfer domain (ORD), followed by back transfer of PI(4)P by the ORD. Finally, PI(4)P is hydrolyzed in cis by the ER protein Sac1. The energy provided by PI(4)P hydrolysis drives sterol transfer and allows negative feedback when PI(4)P becomes limiting. Other lipid transfer proteins are tethered by the same mechanism. Thus, OSBP-mediated back transfer of PI(4)P might coordinate the transfer of other lipid species at the ER-Golgi interface.

ArfGAP1 responds to membrane curvature through the folding of a lipid packing sensor motif

ArfGAP1 promotes GTP hydrolysis in Arf1, a small G protein that interacts with lipid membranes and drives the assembly of the COPI coat in a GTP‐dependent manner. The activity of ArfGAP1 increases with membrane curvature, suggesting a negative feedback loop in which COPI‐induced membrane deformation determines the timing and location of GTP hydrolysis within a coated bud. Here we show that a central sequence of about 40 amino acids in ArfGAP1 acts as a lipid‐packing sensor. This ALPS motif (ArfGAP1 Lipid Packing Sensor) is also found in the yeast homologue Gcs1p and is necessary for coupling ArfGAP1 activity with membrane curvature. The ALPS motif binds avidly to small liposomes and shows the same hypersensitivity on liposome radius as full‐length ArfGAP1. Site‐directed mutagenesis, limited proteolysis and circular dichroism experiments suggest that the ALPS motif, which is unstructured in solution, inserts bulky hydrophobic residues between loosely packed lipids and forms an amphipathic helix on highly curved membranes. This helix differs from classical amphipathic helices by the abundance of serine and threonine residues on its polar face.

Osh4p exchanges sterols for phosphatidylinositol 4-phosphate between lipid bilayers

The yeast Kes1p/Osh4p protein functions as a sterol/PI(4)P exchanger between lipid membranes, which suggests the possibility of creating a sterol gradient via phosphoinositide metabolism.

Asymmetric Tethering of Flat and Curved Lipid Membranes by a Golgin

Golgins, long stringlike proteins, tether cisternae and transport vesicles at the Golgi apparatus. We examined the attachment of golgin GMAP-210 to lipid membranes. GMAP-210 connected highly curved liposomes to flatter ones. This asymmetric tethering relied on motifs that sensed membrane curvature both in the N terminus of GMAP-210 and in ArfGAP1, which controlled the interaction of the C terminus of GMAP-210 with the small guanine nucleotide–binding protein Arf1. Because membrane curvature constantly changes during vesicular trafficking, this mode of tethering suggests a way to maintain the Golgi architecture without compromising membrane flow.

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

Membrane contact sites promote lipid exchange Most membrane lipids are manufactured in the endoplasmic reticulum (ER). Different organelles and the plasma membrane (PM) have distinct phospholipid compositions. Chung et al., working in mammalian cells, and Moser von Filseck et al., working in yeast, both describe how a family of proteins is important in maintaining the balance of lipids within the cell. These special proteins accumulate at and tether contact sites between the ER and the PM and promote the exchange of specific phospholipids, which helps to maintain the PMs distinct identity. Science, this issue pp. 428 and 432 A phosphatidylinositol 4-phosphate gradient drives phosphatidylserine transport from the endoplasmic reticulum to the plasma membrane. 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.

Itraconazole Inhibits Enterovirus Replication by Targeting the Oxysterol-Binding Protein

n Summaryn n Itraconazole (ITZ) is a well-known antifungal agent that also has anticancer activity. In this study, wexa0identify ITZ as a broad-spectrum inhibitor of enteroviruses (e.g., poliovirus, coxsackievirus, enterovirus-71, rhinovirus). We demonstrate that ITZ inhibits viral RNA replication by targeting oxysterol-binding protein (OSBP) and OSBP-related protein 4 (ORP4). Consistently, OSW-1, a specific OSBP/ORP4 antagonist, also inhibits enterovirus replication. Knockdown of OSBP inhibits virus replication, whereas overexpression of OSBP or ORP4 counteracts the antiviral effects of ITZ and OSW-1. ITZ binds OSBP and inhibits its function, i.e., shuttling of cholesterol and phosphatidylinositol-4-phosphate between membranes, thereby likely perturbing the virus-induced membrane alterations essential for viral replication organelle formation. ITZ also inhibits hepatitis C virus replication, which also relies on OSBP. Together, these data implicate OSBP/ORP4 as molecular targets of ITZ and point to an essential role of OSBP/ORP4-mediated lipid exchange in virus replication that can be targeted by antiviral drugs.n n

Amphipathic Lipid Packing Sensor Motifs: Probing Bilayer Defects with Hydrophobic Residues

Sensing membrane curvature allows fine-tuning of complex reactions that occur at the surface of membrane-bound organelles. One of the most sensitive membrane curvature sensors, the Amphipathic Lipid Packing Sensor (ALPS) motif, does not seem to recognize the curved surface geometry of membranes per se; rather, it recognizes defects in lipid packing that arise from membrane bending. In a companion paper, we show that these defects can be mimicked by introducing conical lipids in a flat lipid bilayer, in agreement with experimental observations. Here, we use molecular-dynamics (MD) simulations to characterize ALPS binding to such lipid bilayers. The ALPS motif recognizes lipid-packing defects by a conserved mechanism: peptide partitioning is driven by the insertion of hydrophobic residues into large packing defects that are preformed in the bilayer. This insertion induces only minor modifications in the statistical distribution of the free packing defects. ALPS insertion is severely hampered when monounsaturated lipids are replaced by saturated lipids, leading to a decrease in packing defects. We propose that the hypersensitivity of ALPS motifs to lipid packing defects results from the repetitive use of hydrophobic insertions along the monotonous ALPS sequence.