Search

Cellular membranes are highly dynamic entities that undergo continuous remodeling, including fusion, budding or fission events. Many membrane remodeling processes are catalyzed by the Endosomal Sorting Complex Required for Transport (ESCRT) complexes, which include multivesicular endosomes, cytokinesis, nuclear envelope reformation, membrane repair, autophagy, exosome biogenesis, neuronal pruning, dendritic spine maintenance, enveloped virus budding, release of peroxisomes and of recycling endosomes. Alix (ALG-2 interacting protein-X) is a cytosolic protein that interacts with the ESCRT-1 and ESCRT-III complexes exerting important adaptor functions. Alix is composed of an N-terminal Bro1 domain linked to a central V-shaped coiled-coil domain and a C-terminal proline-rich domain.Alix has been shown to dimerize via its central V-domain, a function necessary for HIV-1 budding. Although structures of the monomeric V-domain and of the Bro-V domain have been solved no high resolution structural information is available on dimeric Alix. Here we solved the structure of the dimeric V domain. The crystal structure reveals that dimerization is achieved by domain exchange. Structure-based mutagenesis studies combined with SAXS analyses further demonstrate the conformational flexibility of Alix. In order to understand the molecular mechanism that leads to dimerization we started structural work on full-length Alix in complex with Alg2 a potential activator of Alix by electron microscopy. Furthermore, we observed in vitro that Alix interacts with membranes in a pH-dependent manner that leads to fusion of liposomes in vitro. This novel activity of Alix may be linked to the fusion of exosomes required for cargo delivery. Together, our work provides first insight into the structural and functional mechanism of Alix dimerization.; Les membranes cellulaires sont des entités hautement dynamiques. Elles subissent au cours du temps, des processus continus de remodelage, de fusion, de bourgeonnement ou de fission. Il a été démontré depuis quelques années que ces processus de remodelage des membranes, incluant entre autres, la formation de vésicules intraluménales, la formation d’endosomes, le bourgeonnement viral, la cytokinèse, la réparation membranaire, l’apoptose des neurones, la formation des pores nucléaires et la réparation de l’enveloppe nucléaire, sont médiés par les composants d'une machine multiprotéique connue sous le nom d’ESCRT (Endosomal Sorting Complex Required for Transport).Alix (ALG-2 interacting protein-X) est une protéine accessoire cytosolique qui est étroitement associée à la fonction des complexes ESCRT. Alix est composée d'un domaine Bro1 N-terminal lié à un domaine central coiled-coil en forme de V et d'un domaine C-terminal riche en proline. Notre laboratoire et d’autres équipes ont montré qu’Alix était capable de dimériser via son domaine V central, fonction nécessaire entre autres pour le bourgeonnement du VIH-1.Dans un premier temps, nous avons résolu la structure du domaine V dimérique. La structure cristalline révèle un échange de domaine du premier protomère avec le second protomère : on parle de « swapp dimer ». Nous avons ensuite réalisé un complexe entre la forme entière de la protéine et de son partenaire Alg-2 et initié le travail de résolution de la structure par microscopie électronique.Enfin, nous avons mis en évidence la faculté de la protéine Alix à déformer les membranes. En effet, nous avons confirmé qu’Alix provoque la fusion des liposomes en condition acide, par plusieurs techniques : la microscopie électroniques, L'analyse de la distribution de la taille des liposomes au nanosight et des tests de fluorescence. Enfin, l’oligomérisation de la protéine en présence de liposomes a été étudiée.L’ensemble de ces résultats donne les toutes premières informations structurales de cette forme active dimérique de la protéine Alix et confirme son implication dans les processus de remodelage membranaire.

Additional file 4: Figure S4. Deformation of bare vesicles and vesicles covered with CHMP2A + CHMP3 or CHMP2B + CHMP3. HS-AFM images of a vesicle covered with CHMP2A and CHMP 3 (left column) and a vesicle covered with CHMP2B and CHMP3 proteins (right column). The deformability of the SUVs coated with corresponding proteins upon increased applied force are shown at intermediate force increments of 27% (second panels) and at higher force increment, 81% (third panels).

Additional file 2: Figure S2. Study of CHMP protein-membrane interaction. (A) Optimization of the buffer conditions to optimize the binding of CHMP2B-ΔC (noted here CHMP2B) at 500 nM. Pre-formed vesicles were incubated with CHMP2B-ΔC in buffers with different salt concentrations ranging from 0 mM to 100 mM NaCl (+Tris 25 mM at pH 7.5) and imaged with confocal microscope after 30 min incubation. Lipid signal is shown in magenta and protein signal in green. Scale bar: 5 μm. (B) Confocal image of MBP-CHMP2A-ΔC (noted here CHMP2A) without TEV (Top line) and in the presence of TEV to cleave the MBP tag (Bottom line). Saturated protein fluorescent signal is represented in yellow. Cleavage of MBP tag slightly increases the interaction but induces aggregation. Scale bar: 30 μm. (C) Spinning disk image of GUV incubated with MBP-CHMP2B-ΔC (noted here MBP-CHMP2B) at a concentration of 50 nM (left image, scale bar = 10 μm) and at 200 nm (right image; scale bar = 5 μm). (D) Confocal images of GUV incubated with MBP-CHMP2B-ΔC at a concentration of 1 μM (noted here MBP-CHMP2B), submitted to an osmotic pressure difference equal to 150% (Osmolarity inside and outside the GUV are respectively 120 mOsm.L− 1 and 315 mOsm.L− 1). Scale bar: 10 μm.

Additional file 7: Figures S6. Test of mutual interaction between CHMP2B and CHMP3 in solution. CHMP2B-ΔC (first gel) and CHMP3-FL (second gel) in Hepes Buffer have been deposited on a sucrose gradient. 100 μL of CHMP2B-ΔC at 10 μM has been incubated with 100 μl CHMP3FL at 10 μM and deposit on a sucrose gradient (third gel). No aggregation is observed on the bottom of the gradient. The presence of CHMP3 does not induce CHMP2B aggregation.

Additional file 6: Figure S5. Deformation of bare vesicles and vesicles covered with CHMP proteins. (A) Reduction of vesicle height under increasing force for bare vesicles. ‘Zero’ force increment represents the minimum imaging force (~ 150 pN). (B) Example of deformation for a ~ 60 nm vesicle over increasing force up to 80% of the initial imaging force. (C) Represents the transformation of vesicle height to relative height for each point for the curve in D. (D) represents the relative height vs force increment for all the curves from panel A for bare SUVs. (E) represents the relative height vs force increment for SUV covered with CHMP2B (left), CHMP2B + CHMP3 (middle) and CHMP2A + CHMP3 (right).

Additional file 1: Figure S1. Evolution of the ESCRT-III complex. (A) Table illustrating the ESCRT-III complex function, origin and homologs in S. cerevisiae and H. sapiens. (B) Distribution of Vps2 and Vps24 genes across Eukaryotes showing the presence of two Vps2 genes in high organisms. (C) Table illustrating the implication of ESCRT-III subunits in different subcellular locations in S. cerevisiae and H. sapiens. The names are the human homologs in case of S. cerevisiae.