Romain Gautier

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

SUMMARY HELIQUEST 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. AVAILABILITY http://heliquest.ipmc.cnrs.fr.

Polyunsaturated phospholipids facilitate membrane deformation and fission by endocytic proteins

Bending the benefits of polyunsaturates We have often heard that it is beneficial to eat polyunsaturated fatty acids. We also know that some organelles such as synaptic vesicles are extremely rich in polyunsaturated lipids. However, what polyunsaturated lipids do in our body is unclear. Using cell biology, biochemical reconstitutions, and molecular dynamics, Pinot et al. show that polyunsaturated phospholipids can change the response of membranes to proteins involved in membrane curvature sensing, membrane shaping, and membrane fission. Polyunsaturated phospholipids make the plasma membrane more amenable to deformation; facilitate endocytosis; and, in reconstitution experiments, increased membrane fission by the dynamin-endophilin complex. Science, this issue p. 693 Certain membrane lipids adapt their conformation to membrane curvature, facilitating membrane deformation and fission. Phospholipids (PLs) with polyunsaturated acyl chains are extremely abundant in a few specialized cellular organelles such as synaptic vesicles and photoreceptor discs, but their effect on membrane properties is poorly understood. Here, we found that polyunsaturated PLs increased the ability of dynamin and endophilin to deform and vesiculate synthetic membranes. When cells incorporated polyunsaturated fatty acids into PLs, the plasma membrane became more amenable to deformation by a pulling force and the rate of endocytosis was accelerated, in particular, under conditions in which cholesterol was limiting. Molecular dynamics simulations and biochemical measurements indicated that polyunsaturated PLs adapted their conformation to membrane curvature. Thus, by reducing the energetic cost of membrane bending and fission, polyunsaturated PLs may help to support rapid endocytosis.

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.

A sub-nanometre view of how membrane curvature and composition modulate lipid packing and protein recruitment

Two parameters of biological membranes, curvature and lipid composition, direct the recruitment of many peripheral proteins to cellular organelles. Although these traits are often studied independently, it is their combination that generates the unique interfacial properties of cellular membranes. Here, we use a combination of in vivo, in vitro and in silico approaches to provide a comprehensive map of how these parameters modulate membrane adhesive properties. The correlation between the membrane partitioning of model amphipathic helices and the distribution of lipid-packing defects in membranes of different shape and composition explains how macroscopic membrane properties modulate protein recruitment by changing the molecular topography of the membrane interfacial region. Furthermore, our results suggest that the range of conditions that can be obtained in a cellular context is remarkably large because lipid composition and curvature have, under most circumstances, cumulative effects.

Conical Lipids in Flat Bilayers Induce Packing Defects Similar to that Induced by Positive Curvature

In biological membranes, changes in lipid composition or mechanical deformations produce defects in the geometrical arrangement of lipids, thus allowing the adsorption of certain peripheral proteins. Here, we perform molecular dynamics simulations on bilayers containing a cylindrical lipid (PC) and a conical lipid (DOG). Profiles of atomic density and lateral pressure across the bilayer show differences in the acyl chain region due to deeper partitioning of DOG compared to PC. However, such analyses are less informative for the interfacial region where peripheral proteins adsorb. To circumvent this limitation, we develop, to our knowledge, a new method of membrane surface analysis. This method allows the identification of chemical defects, where hydrocarbon chains are accessible to the solvent, and geometrical defects, i.e., voids deeper than the glycerol backbone. The size and number of both types of defects increase with the number of monounsaturated acyl chains in PC and with the introduction of DOG, although the defects do not colocalize with the conical lipid. Interestingly, the size and probability of the defects promoted by DOG resemble those induced by positive curvature, thus explaining why conical lipids and positive curvature can both drive the adsorption of peripheral proteins that use hydrophobic residues as membrane anchors.

The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress

LEA proteins accumulate in plant seeds prior to maturation drying, and some have been shown to protect membranes from desiccation. This work demonstrates the subcellular distribution of each of 51 Arabidopsis LEA proteins and suggests protection against desiccation or cold stress is tailored for each cellular compartment. Late embryogenesis abundant (LEA) proteins are hydrophilic, mostly intrinsically disordered proteins, which play major roles in desiccation tolerance. In Arabidopsis thaliana, 51 genes encoding LEA proteins clustered into nine families have been inventoried. To increase our understanding of the yet enigmatic functions of these gene families, we report the subcellular location of each protein. Experimental data highlight the limits of in silico predictions for analysis of subcellular localization. Thirty-six LEA proteins localized to the cytosol, with most being able to diffuse into the nucleus. Three proteins were exclusively localized in plastids or mitochondria, while two others were found dually targeted to these organelles. Targeting cleavage sites could be determined for five of these proteins. Three proteins were found to be endoplasmic reticulum (ER) residents, two were vacuolar, and two were secreted. A single protein was identified in pexophagosomes. While most LEA protein families have a unique subcellular localization, members of the LEA_4 family are widely distributed (cytosol, mitochondria, plastid, ER, and pexophagosome) but share the presence of the class A α-helix motif. They are thus expected to establish interactions with various cellular membranes under stress conditions. The broad subcellular distribution of LEA proteins highlights the requirement for each cellular compartment to be provided with protective mechanisms to cope with desiccation or cold stress.

Kinetic Studies of the Arf Activator Arno on Model Membranes in the Presence of Arf Effectors Suggest Control by a Positive Feedback Loop

Proteins of the cytohesin/Arno/Grp1 family of Arf activators are positive regulators of the insulin-signaling pathway and control various remodeling events at the plasma membrane. Arno has a catalytic Sec7 domain, which promotes GDP to GTP exchange on Arf, followed by a pleckstrin homology (PH) domain. Previous studies have revealed two functions of the PH domain: inhibition of the Sec7 domain and membrane targeting. Interestingly, the Arno PH domain interacts not only with a phosphoinositide (phosphatidylinositol 4,5-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate) but also with an activating Arf family member, such as Arf6 or Arl4. Using the full-length membrane-bound forms of Arf1 and Arf6 instead of soluble forms, we show here that the membrane environment dramatically affects the mechanism of Arno activation. First, Arf6-GTP stimulates Arno at nanomolar concentrations on liposomes compared with micromolar concentrations in solution. Second, mutations in the PH domain that abolish interaction with Arf6-GTP render Arno completely inactive when exchange reactions are reconstituted on liposomes but have no effect on Arno activity in solution. Third, Arno is activated by its own product Arf1-GTP in addition to a distinct activating Arf isoform. Consequently, Arno activity is strongly modulated by competition with Arf effectors. These results show that Arno behaves as a bistable switch, having an absolute requirement for activation by an Arf protein but, once triggered, becoming highly active through the positive feedback effect of Arf1-GTP. This property of Arno might provide an explanation for its function in signaling pathways that, once triggered, must move forward decisively.

Krit 1 interactions with microtubules and membranes are regulated by Rap1 and integrin cytoplasmic domain associated protein-1

The small G protein Rap1 regulates diverse cellular processes such as integrin activation, cell adhesion, cell–cell junction formation and cell polarity. It is crucial to identify Rap1 effectors to better understand the signalling pathways controlling these processes. Krev interaction trapped 1 (Krit1), a protein with FERM (band four‐point‐one/ezrin/radixin/moesin) domain, was identified as a Rap1 partner in a yeast two‐hybrid screen, but this interaction was not confirmed in subsequent studies. As the evidence suggests a role for Krit1 in Rap1‐dependent pathways, we readdressed this question. In the present study, we demonstrate by biochemical assays that Krit1 interacts with Rap1A, preferentially its GTP‐bound form. We show that, like other FERM proteins, Krit1 adopts two conformations: a closed conformation in which its N‐terminal NPAY motif interacts with its C‐terminus and an opened conformation bound to integrin cytoplasmic domain associated protein (ICAP)‐1, a negative regulator of focal adhesion assembly. We show that a ternary complex can form in vitro between Krit1, Rap1 and ICAP‐1 and that Rap1 binds the Krit1 FERM domain in both closed and opened conformations. Unlike ICAP‐1, Rap1 does not open Krit1. Using sedimentation assays, we show that Krit1 binds in vitro to microtubules through its N‐ and C‐termini and that Rap1 and ICAP‐1 inhibit Krit1 binding to microtubules. Consistently, YFP‐Krit1 localizes on cyan fluorescent protein‐labelled microtubules in baby hamster kidney cells and is delocalized from microtubules upon coexpression with activated Rap1V12. Finally, we show that Krit1 binds to phosphatidylinositol 4,5‐P2‐containing liposomes and that Rap1 enhances this binding. Based on these results, we propose a model in which Krit1 would be delivered by microtubules to the plasma membrane where it would be captured by Rap1 and ICAP‐1.

Two-Step Design of a Single-Doped White Phosphor with High Color Rendering

A strategy to design step by step an inorganic single-doped white phosphor is demonstrated. The method consists in tuning different contributions of the emission by successively controlling the chemical compositions of the solid solution or nanosegregated host matrix and the oxidation states of the single dopant. We use this approach to design a white phosphor Na4CaMgSc4Si10O30:Eu with excellent color rendering (CRI > 90) that is similar to common mixed-phosphor light sources but for a single-phase. We show that this methodology can also be extended to other phosphors for use in diverse applications such as biomedicine or telecommunications.