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Bruno Antonny

Publications and source records attributed to Bruno Antonny.

13 recordsLinked to original sources
PubMed2007

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

The Golgi-associated protein ArfGAP1 has an unusual membrane-adsorbing amphipathic alpha-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 alpha-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 beta-propeller structure. Ser/Thr-rich amphipathic helices thus define a general motif used by proteins of various functions for sensing membrane curvature.

Algorithms↗
PubMed2006

Membrane deformation by protein coats.

Protein coats deform lipid membranes into spherical buds, which undergo fission at the neck to become vesicles. To induce membrane curvature, protein coats use basic tools including amphipathic helices and concave protein surfaces, and take advantage of the bulk properties of cellular membranes, such as loose lipid packing in the endoplasmic reticulum and cis-Golgi and the abundance of anionic lipids in the cytosolic leaflet of the plasma membrane. Protein scaffolds, sensors of membrane curvature and finely tuned reactions such as GTP hydrolysis permit the spatial and temporal organization of these tools, making protein coats self-organized molecular machines. Because biological membranes generally adhere to a cytoskeleton, the functioning of protein coats is coupled to other large remodeling events at the membrane interface.

Animals↗
PubMed2006

GEF and glucosylation assays on liposome-bound Rac.

Rac binds tightly to lipid membranes through a lipid modification. The influence of the lipid membrane environment on the multiple interactions of Rac has not been well documented. In this chapter, we detail a method to prepare geranyl-geranylated Rac bound to liposomes of defined composition. With this method, one can dissect some lipid-protein interactions that facilitate the interaction of Rac with other proteins such as guanine nucleotide exchange factors and bacterial toxins.

Glucosyltransferases↗
PubMed2005

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.

ADP-Ribosylation Factors↗
PubMed2005

Real-time assays for the assembly-disassembly cycle of COP coats on liposomes of defined size.

The assembly-disassembly cycle of COPI and COPII coats is controlled by the GTPase cycle of the small G proteins Arf1 and Sar. We describe here two spectroscopic assays that enable real-time studies of some elementary steps of coat assembly and disassembly on artificial liposomes of defined composition and curvature. A flotation assay to assess the effect of membrane curvature on protein adsorption to liposomes is also presented.

ADP-Ribosylation Factor 1↗
PubMed2004

SNARE filtering by dynamin.

Fission and fusion are the two elementary steps of membrane traffic. The mechanoenzyme dynamin acts at the fission step, but, in this issue of Cell, suggest an additional role of dynamin in preparing membranes for fusion.

Adenosine Triphosphate↗
PubMed2004

A phosphatidylserine-binding site in the cytosolic fragment of Clostridium sordellii lethal toxin facilitates glucosylation of membrane-bound Rac and is required for cytotoxicity.

Large clostridial toxins glucosylate some small G proteins on a threonine residue, thereby preventing their interactions with effector molecules and regulators. We show that the glucosyltransferase domain of lethal toxin from Clostridium sordellii (LT(cyt); amino acids 1-546), which is released into the cytosol during cell infection, binds preferentially to liposomes containing phosphatidylserine as compared with other anionic lipids. The binding of LT(cyt) to phosphatidylserine increases by two orders of magnitude the rate of glucosylation of liposome-bound geranyl-geranylated Rac-GDP. Limited proteolysis and deletion studies show that the binding site for phosphatidylserine lies within the first 18 N-terminal residues of LT(cyt). Deletion of these residues abolishes the effect of phosphatidylserine on the activity of LT(cyt) on liposome-bound geranyl-geranylated Rac-GDP and prevents the morphological effects induced by LT(cyt) microinjection into various cells, but it does not affect the intrinsic activity of LT(cyt) on non-geranyl-geranylated Rac-GDP in solution. We conclude that the avidity of LT(cyt) for phosphatidylserine facilitates its targeting to the cytosolic leaflet of cell membranes and, notably, the plasma membrane, where this anionic lipid is abundant and where several targets of lethal toxin reside.

Bacterial Toxins↗
PubMed2003

Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature.

Protein coats deform flat lipid membranes into buds and capture membrane proteins to form transport vesicles. The assembly/disassembly cycle of the COPI coat on Golgi membranes is coupled to the GTP/GDP cycle of the small G protein Arf1. At the heart of this coupling is the specific interaction of membrane-bound Arf1-GTP with coatomer, a complex of seven proteins that forms the building unit of the COPI coat. Although COPI coat disassembly requires the catalysis of GTP hydrolysis in Arf1 by a specific GTPase-activating protein (ArfGAP1), the precise timing of this reaction during COPI vesicle formation is not known. Using time-resolved assays for COPI dynamics on liposomes of controlled size, we show that the rate of ArfGAP1-catalysed GTP hydrolysis in Arf1 and the rate of COPI disassembly increase over two orders of magnitude as the curvature of the lipid bilayer increases and approaches that of a typical transport vesicle. This leads to a model for COPI dynamics in which GTP hydrolysis in Arf1 is organized temporally and spatially according to the changes in lipid packing induced by the coat.

ADP-Ribosylation Factors↗
PubMed2003

Self-assembly of minimal COPII cages.

The small G-protein Sar1 and the cytosolic complexes Sec23/24 and Sec13/31 associate sequentially on endoplasmic reticulum membranes to form a protein coat named COPII, which drives the formation of transport vesicles. Using dynamic light scattering, we show that Sec23/24 and Sec13/31 can self-assemble in a stoichiometric manner in solution to form particles with hydrodynamic radii in the range of 40-60 nm. Self-assembly is favoured by lowering the pH, the ionic strength and/or the temperature. Electron microscopy reveals the formation of spherical particles 60-120 nm in diameter with a tight, rough mesh on their surfaces. We suggest that these structures, which represent a minimal COPII cage, mimic the molecular organization of the membrane-associated COPII coat.

COP-Coated Vesicles↗
PubMed2002

Dissociation of GDP dissociation inhibitor and membrane translocation are required for efficient activation of Rac by the Dbl homology-pleckstrin homology region of Tiam.

Small G proteins of the Rho/Rac/Cdc42 family are associated with lipid membranes through their prenylated C termini. Alternatively, these proteins form soluble complexes with GDI proteins. To assess how this membrane partitioning influences the activation of Rac by guanine nucleotide exchange factors, GDP-to-GTP exchange reactions were performed in the presence of liposomes using different forms of Rac-GDP. We show that both non-prenylated Rac-GDP and the soluble complex between prenylated Rac-GDP and GDI are poorly activated by the Dbl homology-pleckstrin homology (DH-PH) domain of the exchange factor Tiam1, whereas prenylated Rac-GDP bound to liposomes is activated about 10 times more rapidly. Sedimentation experiments with liposomes reveal that the DH-PH region of Tiam1 forms, with nucleotide-free prenylated Rac, a membrane-bound complex from which GDI is excluded. Taken together, these experiments demonstrate that the dissociation of Rac-GDP from GDI and its translocation to membrane lipids favor DH-PH-catalyzed nucleotide exchange because the steric hindrance caused by GDI is relieved and because the membrane environment favors functional interaction between the DH-PH domain and the small G protein.

Blood Proteins↗
PubMed2002

Cargo selection into COPII vesicles is driven by the Sec24p subunit.

Transport of secretory proteins out of the endoplasmic reticulum (ER) is mediated by vesicles generated by the COPII coat complex. In order to understand how cargo molecules are selected by this cytoplasmic coat, we investigated the functional role of the Sec24p homolog, Lst1p. We show that Lst1p can function as a COPII subunit independently of Sec24p on native ER membranes and on synthetic liposomes. However, vesicles generated with Lst1p in the absence of Sec24p are deficient in a distinct subset of cargo molecules, including the SNAREs, Bet1p, Bos1p and Sec22p. Consistent with the absence of any SNAREs, these vesicles are unable to fuse with Golgi membranes. Furthermore, unlike Sec24p, Lst1p fails to bind to Bet1p in vitro, indicating a direct correlation between cargo binding and recruitment into vesicles. Our data suggest that the principle role of Sec24p is to discriminate cargo molecules for incorporation into COPII vesicles.

COP-Coated Vesicles↗