Mécanismes du transport lipidique par les protéines ORP/Osh

An uneven lipid distribution is essential for the function of eukaryotic organelles. However, exchange of material by vesicular trafficking has a tendency to perturb this distribution; mechanisms must though exist to ensure lipid homeostasis. Osh proteins (S. cerevisiae) and OSBP-Related Proteins (ORPs, H. sapiens), are lipid transfer proteins (LTPs). Osh4 is capable of exchanging ergosterol for phosphatidylinositol 4-phosphate (PI4P), found on the Golgi. Using novel fluorescent tools to measure with unprecedented precision the transport of sterol and PI4P, we find that Osh4 can transport sterol against its concentration gradient using the energy of a PI4P gradient. Coupled to phosphoinositide metabolism, this allows Osh4 to transport sterol to the trans-Golgi and create the sterol gradients observed between these organelles. OSBP participates in the creation of membrane contact sites (MCSs) via its capacity to connect ER membranes to those of the trans-Golgi. We have shown that it uses PI4P for transporting cholesterol from the ER to the trans-Golgi by sterol/PI4P counterexchange, hence also autoregulating its tethering activity. Finally, the identification of phosphatidylserine as a ligand for Osh6 allowed us to analyze the possible extrapolation of the PI4P counterexchange mechanism. We have solved the crystal structure of Osh6 in complex with PI4P and have been able to follow counterexchange of PI(4)P and PS in vitro. Concluding, our studies allow us to suggest a general mechanism for ORP/Osh-mediated counterexchange of PI4P for other lipids to maintain lipid gradients between the ER and late membranes of the secretory pathway.Une distribution lipidique hétérogène est essentielle à l’identité et fonction des organelles, mais l’échange par trafic vésiculaire tend à annuler cette distribution. Il existe donc des mécanismes qui assurent l’homéostasie des lipides. Les protéines Osh (S. cerevisiae) et les OSBP-Related Proteins (ORP, H. sapiens), sont des transporteurs de lipides. Osh4 est capable d’échanger de l’ergostérol contre le phosphatidylinositol-4-phosphate (PI4P), présent sur l’appareil de Golgi. Utilisant des outils fluorescents mesurant avec une précision inégalée le transport de stérol et de PI4P, nous démontrons qu’Osh4 transporte du stérol contre son gradient de concentration en utilisant l’énergie d’un gradient de PI4P. Un couplage au métabolisme du PI4P permettrait à Osh4 d’alimenter le Golgi avec du stérol, ainsi créant le gradient de stérol entre ces organelles. La protéine OSBP participe, via sa capacité à connecter la membrane du RE à celle du trans-Golgi, à la création de jonctions entre ces organelles. Nous avons montré qu’OSBP, par échange stérol/PI4P, utilise le PI4P pour transférer du cholestérol au Golgi, mais également pour autoréguler sa capacité à former les jonctions. Osh6 lie la phosphatidylsérine, nous permettant d’étudier un nouveau mécanisme d’échange. Nous avons résolu la structure cristallographique d’un complexe Osh6/PI4P et avons pu observer l’échange de ces deux ligands par Osh6 entre deux membranes. Cette étude nous permet de suggérer que l’échange de PI4P avec divers lipides, via les protéines Osh/ORP, serait un mécanisme général permettant aux cellules de maintenir le gradient lipidique entre le RE et les membranes tardives de la voie sécrétoire

Mechanisms of lipid transport by the ORP/Osh proteins

Une distribution lipidique hétérogène est essentielle à l’identité et fonction des organelles, mais l’échange par trafic vésiculaire tend à annuler cette distribution. Il existe donc des mécanismes qui assurent l’homéostasie des lipides. Les protéines Osh (S. cerevisiae) et les OSBP-Related Proteins (ORP, H. sapiens), sont des transporteurs de lipides. Osh4 est capable d’échanger de l’ergostérol contre le phosphatidylinositol-4-phosphate (PI4P), présent sur l’appareil de Golgi. Utilisant des outils fluorescents mesurant avec une précision inégalée le transport de stérol et de PI4P, nous démontrons qu’Osh4 transporte du stérol contre son gradient de concentration en utilisant l’énergie d’un gradient de PI4P. Un couplage au métabolisme du PI4P permettrait à Osh4 d’alimenter le Golgi avec du stérol, ainsi créant le gradient de stérol entre ces organelles. La protéine OSBP participe, via sa capacité à connecter la membrane du RE à celle du trans-Golgi, à la création de jonctions entre ces organelles. Nous avons montré qu’OSBP, par échange stérol/PI4P, utilise le PI4P pour transférer du cholestérol au Golgi, mais également pour autoréguler sa capacité à former les jonctions. Osh6 lie la phosphatidylsérine, nous permettant d’étudier un nouveau mécanisme d’échange. Nous avons résolu la structure cristallographique d’un complexe Osh6/PI4P et avons pu observer l’échange de ces deux ligands par Osh6 entre deux membranes. Cette étude nous permet de suggérer que l’échange de PI4P avec divers lipides, via les protéines Osh/ORP, serait un mécanisme général permettant aux cellules de maintenir le gradient lipidique entre le RE et les membranes tardives de la voie sécrétoire.An uneven lipid distribution is essential for the function of eukaryotic organelles. However, exchange of material by vesicular trafficking has a tendency to perturb this distribution; mechanisms must though exist to ensure lipid homeostasis. Osh proteins (S. cerevisiae) and OSBP-Related Proteins (ORPs, H. sapiens), are lipid transfer proteins (LTPs). Osh4 is capable of exchanging ergosterol for phosphatidylinositol 4-phosphate (PI4P), found on the Golgi. Using novel fluorescent tools to measure with unprecedented precision the transport of sterol and PI4P, we find that Osh4 can transport sterol against its concentration gradient using the energy of a PI4P gradient. Coupled to phosphoinositide metabolism, this allows Osh4 to transport sterol to the trans-Golgi and create the sterol gradients observed between these organelles. OSBP participates in the creation of membrane contact sites (MCSs) via its capacity to connect ER membranes to those of the trans-Golgi. We have shown that it uses PI4P for transporting cholesterol from the ER to the trans-Golgi by sterol/PI4P counterexchange, hence also autoregulating its tethering activity. Finally, the identification of phosphatidylserine as a ligand for Osh6 allowed us to analyze the possible extrapolation of the PI4P counterexchange mechanism. We have solved the crystal structure of Osh6 in complex with PI4P and have been able to follow counterexchange of PI(4)P and PS in vitro. Concluding, our studies allow us to suggest a general mechanism for ORP/Osh-mediated counterexchange of PI4P for other lipids to maintain lipid gradients between the ER and late membranes of the secretory pathway

Structural Basis of Membrane Curvature Recognition by the ALPS Motifs

International audienceThe recruitment of many cytosolic factors is regulated by the lipid composition but also by the shape of organelles membrane. Ten years ago, we identified in ArfGAP1 a motif of 30 amino-acids termed ALPS that allows this protein to detect the positive curvature of the COPI vesicles at the end of their biogenesis and to trigger the depolymerisation of coat proteins wrapping these vesicles. We found that the ALPS motif folds into a peculiar amphipathic helix whose insertion into membrane depends on defects in lipid-packing induced by curvature. ALPS motifs were next identified in various proteins such as GMAP-210, a membrane tethering factor, or Osh4p, a sterol/PI(4)P exchanger. Different studies illustrated how membrane curvature regulates the activity of these proteins. At a more atomistic level, we gained results that explain why the ALPS motif is so sensitive to lipid-packing defects and membrane curvature. We first indicated that the lack of basic residues in the polar face of the ALPS motif explains its sensitivity to curvature of cellular membrane. Next, molecular dynamic simulations suggested that the insertion of the ALPS motif, solely mediated by the insertion of bulk hydrophobic residue, directly depends on the pre-existence of lipid-packing defects in membrane. More recently, NMR studies in a membrane environment showed that the ALPS motif of ArfGAP1 folds into a helix displaying a not well-defined central region between two helical fragments. The stability of ALPS helix was also assessed by replica-exchange molecular dynamics simulations. Jointly, these results suggest that the sequence of an ALPS motif codes for a loose and non-canonical amphipathic helix whose features are perfectly well adapted for responding to change in membrane curvatur

Simplified Fabrication for Ion-Selective Optical Emulsion Sensor with Hydrophobic Solvatochromic Dye Transducer: A Cautionary Tale

It has recently been reported that polystyrene microbeads may be modified to realize plasticizer-free ion-selective optical sensors (optodes) on the basis of solvatochromic dye transducers. We show here that the functionalized microbeads, individually isolated by flow cytometry, exhibit unexpectedly poor fluorescent properties and that the sensor response is instead attributed to the supernatant. A more thorough study reveals that such optical microemulsion sensors can be made operationally functional and chemically selective, seemingly in the absence of any solvent matrix or added surfactant. Instead, it is shown that residual THF used in the fabrication of the emulsified sensors may solubilize the sensing components and give a functional optode response. To evaluate this further, the number of sensing components was stepwise simplified to assess their need. Variation of residual THF levels has no effect on the ion optode response when plasticizer is present, in support of established results. Lipophilic solvatochromic dye transducers are also shown not to require an added surfactant as their nature already endows the emulsified sensors with a stabilizing ionic surface charge. The ionophores are shown to exhibit much larger stability constants in the surfactant-free formulations than surfactant-based ones (Valionomycin: log!>9.2 compared to 6.1; Na+-ionophore X: 6.7 vs. 4.7), which is attributed to a less polar solvent environment for the ionophore. Potassium, sodium and calcium-selective sensors were used as model systems in this study

Anisotropic ESCRT-III architecture governs helical membrane tube formation.

ESCRT-III proteins assemble into ubiquitous membrane-remodeling polymers during many cellular processes. Here we describe the structure of helical membrane tubes that are scaffolded by bundled ESCRT-III filaments. Cryo-ET reveals how the shape of the helical membrane tube arises from the assembly of two distinct bundles of helical filaments that have the same helical path but bind the membrane with different interfaces. Higher-resolution cryo-EM of filaments bound to helical bicelles confirms that ESCRT-III filaments can interact with the membrane through a previously undescribed interface. Mathematical modeling demonstrates that the interface described above is key to the mechanical stability of helical membrane tubes and helps infer the rigidity of the described protein filaments. Altogether, our results suggest that the interactions between ESCRT-III filaments and the membrane could proceed through multiple interfaces, to provide assembly on membranes with various shapes, or adapt the orientation of the filaments towards the membrane during membrane remodeling

An ESCRT-III Polymerization Sequence Drives Membrane Deformation and Fission

The endosomal sorting complex required for transport-III (ESCRT-III) catalyzes membrane fission from within membrane necks, a process that is essential for many cellular functions, from cell division to lysosome degradation and autophagy. How it breaks membranes, though, remains unknown. Here, we characterize a sequential polymerization of ESCRT-III subunits that, driven by a recruitment cascade and by continuous subunit-turnover powered by the ATPase Vps4, induces membrane deformation and fission. During this process, the exchange of Vps24 for Did2 induces a tilt in the polymer-membrane interface, which triggers transition from flat spiral polymers to helical filament to drive the formation of membrane protrusions, and ends with the formation of a highly constricted Did2-Ist1 co-polymer that we show is competent to promote fission when bound on the inside of membrane necks. Overall, our results suggest a mechanism of stepwise changes in ESCRT-III filament structure and mechanical properties via exchange of the filament subunits to catalyze ESCRT-III activity