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Accueil > Agenda > Les séminaires Jean Roche > Regulation of the balance of membrane fusion and fission.

Regulation of the balance of membrane fusion and (...)

Lundi 13 mars 2006,11h, salle Lissitzky.


J Cell Biol. 2005 Dec 19 ;171(6):981-90.

Transition from hemifusion to pore opening is rate limiting for vacuole membrane fusion.

Reese C, Mayer A.

Departement de Biochimie, Universite de Lausanne, 1066 Epalinges, Switzerland.

Fusion pore opening and expansion are considered the most energy-demanding steps in viral fusion. Whether this also applies to soluble N-ethyl-maleimide sensitive fusion protein attachment protein receptor (SNARE)- and Rab-dependent fusion events has been unknown. We have addressed the problem by characterizing the effects of lysophosphatidylcholine (LPC) and other late-stage inhibitors on lipid mixing and pore opening during vacuole fusion. LPC inhibits fusion by inducing positive curvature in the bilayer and changing its biophysical properties. The LPC block reversibly prevented formation of the hemifusion intermediate that allows lipid, but not content, mixing. Transition from hemifusion to pore opening was sensitive to guanosine-5’-(gamma-thio)triphosphate. It required the vacuolar adenosine triphosphatase V0 sector and coincided with its transformation. Pore opening was rate limiting for the reaction. As with viral fusion, opening the fusion pore may be the most energy-demanding step for intracellular, SNARE-dependent fusion reactions, suggesting that fundamental aspects of lipid mixing and pore opening are related for both systems.

2 : J Biol Chem. 2005 Sep 30 ;280(39):33289-97. Epub 2005 Jul 29.

Microautophagic vacuole invagination requires calmodulin in a Ca2+-independent function.

Uttenweiler A, Schwarz H, Mayer A.

Departement de Biochimie, Universite de Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.

Microautophagy is the uptake of cytosolic compounds by direct invagination of the vacuolar/lysosomal membrane. In Saccharomyces cerevisiae microautophagic uptake of soluble cytosolic proteins occurs via an autophagic tube, a highly specialized vacuolar membrane invagination. Autophagic tubes are topologically equivalent to the invaginations at multivesicular endosomes. At the tip of an autophagic tube, vesicles (autophagic bodies) pinch off into the vacuolar lumen for degradation. In this study we have identified calmodulin (Cmd1p) as necessary for microautophagy. Temperature-sensitive mutants for Cmd1p displayed reduced frequencies of vacuolar tube formation and/or abnormal tube morphologies. Microautophagic vacuole invagination was sensitive to Cmd1p antagonists as well as to antibodies to Cmd1p. cmd1 mutants with substitutions in the Ca2+-binding domains showed full invagination activity, and vacuolar membrane invagination was independent of the free Ca2+ concentration. Thus, rather than acting as a calcium-triggered switch, Cmd1p has a constitutive Ca2+-independent role in the formation of autophagic tubes. Kinetic analysis indicates that calmodulin is required for autophagic tube formation rather than for the final scission of vesicles from the tip of the tube.

3 : Nature. 2005 Jul 21 ;436(7049):410-4. Epub 2005 May 29.

Trans-SNARE pairing can precede a hemifusion intermediate in intracellular membrane fusion.

Reese C, Heise F, Mayer A.

Departement de Biochimie, Universite de Lausanne, Chemin des Boveresses 155, CH-1066 Epalinges, Switzerland.

The question concerning whether all membranes fuse according to the same mechanism has yet to be answered satisfactorily. During fusion of model membranes or viruses, membranes dock, the outer membrane leaflets mix (termed hemifusion), and finally the fusion pore opens and the contents mix. Viral fusion proteins consist of a membrane-disturbing ’fusion peptide’ and a helical bundle that pin the membranes together. Although SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes form helical bundles with similar topology, it is unknown whether SNARE-dependent fusion events on intracellular membranes proceed through a hemifusion state. Here we identify the first hemifusion state for SNARE-dependent fusion of native membranes, and place it into a sequence of molecular events : formation of helical bundles by SNAREs precedes hemifusion ; further progression to pore opening requires additional peptides. Thus, SNARE-dependent fusion may proceed along the same pathway as viral fusion : both use a docking mechanism via helical bundles and additional peptides to destabilize the membrane and efficiently induce lipid mixing. Our results suggest that a common lipidic intermediate may underlie all fusion reactions of lipid bilayers.

4 : Cell. 2004 Nov 24 ;119(5):667-78.

Comment in : Cell. 2004 Nov 24 ;119(5):581-2.

Mutual control of membrane fission and fusion proteins.

Peters C, Baars TL, Buhler S, Mayer A.

Departement de Biochimie, Universite de Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland. christopher.peters

Membrane fusion and fission are antagonistic reactions controlled by different proteins. Dynamins promote membrane fission by GTP-driven changes of conformation and polymerization state, while SNAREs fuse membranes by forming complexes between t- and v-SNAREs from apposed vesicles. Here, we describe a role of the dynamin-like GTPase Vps1p in fusion of yeast vacuoles. Vps1p forms polymers that couple several t-SNAREs together. At the onset of fusion, the SNARE-activating ATPase Sec18p/NSF and the t-SNARE depolymerize Vps1p and release it from the membrane. This activity is independent of the SNARE coactivator Sec17p/alpha-SNAP and of the v-SNARE. Vps1p release liberates the t-SNAREs for initiating fusion and at the same time disrupts fission activity. We propose that reciprocal control between fusion and fission components exists, which may prevent futile cycles of fission and fusion.

5 : J Biol Chem. 2004 Mar 12 ;279(11):9987-96. Epub 2003 Dec 15.

Determination of four sequential stages during microautophagy in vitro.

Kunz JB, Schwarz H, Mayer A.

Friedrich-Miescher-Laboratorium de Max-Planck-Gesellschaft, Tubingen, Germany.

Microautophagy is the transfer of cytosolic components into the lysosome by direct invagination of the lysosomal membrane and subsequent budding of vesicles into the lysosomal lumen. This process is topologically equivalent to membrane invagination during multivesicular body formation and to the budding of enveloped viruses. Vacuoles are lysosomal compartments of yeasts. Vacuolar membrane invagination can be reconstituted in vitro with purified yeast vacuoles, serving as a model system for budding of vesicles into the lumen of an organelle. Using this in vitro system, we defined different reaction states. We identified inhibitors of microautophagy in vitro and used them as tools for kinetic analysis. This allowed us to characterize four biochemically distinguishable steps of the reaction. We propose that these correspond to sequential stages of vacuole invagination and vesicle scission. Formation of vacuolar invaginations was slow and temperature-dependent, whereas the final scission of the vesicle from a preformed invagination was fast and proceeded even on ice. Our observations suggest that the formation of invaginations rather than the scission of vesicles is the rate-limiting step of the overall reaction.

6 : J Cell Biol. 2003 Jul 21 ;162(2):211-22.

Vacuole membrane fusion : V0 functions after trans-SNARE pairing and is coupled to the Ca2+-releasing channel.

Bayer MJ, Reese C, Buhler S, Peters C, Mayer A.

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, 72076 Tubingen, Germany.

Pore models of membrane fusion postulate that cylinders of integral membrane proteins can initiate a fusion pore after conformational rearrangement of pore subunits. In the fusion of yeast vacuoles, V-ATPase V0 sectors, which contain a central cylinder of membrane integral proteolipid subunits, associate to form a transcomplex that might resemble an intermediate postulated in some pore models. We tested the role of V0 sectors in vacuole fusion. V0 functions in fusion and proton translocation could be experimentally separated via the differential effects of mutations and inhibitory antibodies. Inactivation of the V0 subunit Vph1p blocked fusion in the terminal reaction stage that is independent of a proton gradient. Deltavph1 mutants were capable of docking and trans-SNARE pairing and of subsequent release of lumenal Ca2+, but they did not fuse. The Ca2+-releasing channel appears to be tightly coupled to V0 because inactivation of Vph1p by antibodies blocked Ca2+ release. Vph1 deletion on only one fusion partner sufficed to severely reduce fusion activity. The functional requirement for Vph1p correlates to V0 transcomplex formation in that both occur after docking and Ca2+ release. These observations establish V0 as a crucial factor in vacuole fusion acting downstream of trans-SNARE pairing.

7 : J Cell Sci. 2003 Mar 15 ;116(Pt 6):1107-15.

Role of the Vtc proteins in V-ATPase stability and membrane trafficking.

Muller O, Neumann H, Bayer MJ, Mayer A.

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tubingen, Germany.

Vtc proteins have genetic and physical relations with the vacuolar H(+)-ATPase (V-ATPase), influence vacuolar H(+) uptake and, like the V-ATPase V(0) sectors, are important factors in vacuolar membrane fusion. Vacuoles from vtc1delta and vtc4delta mutants had slightly reduced H(+)-uptake activity. These defects could be separated from Vtc function in vacuole fusion, demonstrating that Vtc proteins have a direct role in membrane fusion. We analyzed their involvement in other membrane trafficking steps and in VATPase dynamics. Deletion of VTC genes did not impede endocytic trafficking to the vacuole. However, ER to Golgi trafficking and further transport to the vacuole was delayed in deltavtc3 cells. In accordance with that, deltavtc3 cells showed a reduced growth rate. Vtc mutations did not interfere with regulated assembly and disassembly of the V-ATPase, but they affected the number of peripheral V(1) subunits associated with the vacuoles. deltavtc3 vacuoles carried significantly more V(1) subunits, whereas deltavtc1, deltavtc2 and deltavtc4 had significantly less. The proteolytic sensitivity of the V(0) subunit Vph1p was different in deltavtc and wild-type cells in vivo, corroborating the physical interaction of Vtc proteins with the V-ATPase observed in vitro. We suggest that Vtc proteins affect the conformation of V(0). They might thereby influence the stability of the VATPase holoenzyme and support the function of its V(0) sector in vacuolar membrane fusion.

8 : Annu Rev Cell Dev Biol. 2002 ;18:289-314. Epub 2002 Apr 2.

Membrane fusion in eukaryotic cells.

Mayer A.

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstr. 37-39, 72076 Tubingen, Germany. andreas.mayer

Membrane fusion is a fundamental biochemical reaction and the final step in all vesicular trafficking events. It is crucial for the transfer of proteins and lipids between different compartments and for exo- and endocytic traffic of signaling molecules and receptors. It leads to the reconstruction of organelles such as the Golgi or the nuclear envelope, which decay into fragments during mitosis. Hence, controlled membrane fusion reactions are indispensible for the compartmental organization of eukaryotic cells ; for their communication with the environment via hormones, neurotransmitters, growth factors, and receptors ; and for the integration of cells into multicellular organisms. Intracellular pathogenic bacteria, such as Mycobacteria or Salmonellae, have developed means to control fusion reactions in their host cells. They persist in phagosomes whose fusion with lysosomes they actively suppress-a means to ensure survival inside host cells. The past decade has witnessed rapid progress in the elucidation of parts of the molecular machinery involved in these membrane fusion reactions. Whereas some elements of the fusion apparatus are remarkably similar in several compartments, there is an equally striking divergence of others. The purpose of this review is to highlight common features of different fusion reactions and the concepts that emerged from them but also to stress the differences and challenge parts of the current hypotheses. This review covers only the endoplasmic fusion reactions mentioned above, i.e., reactions initiated by contacts of membranes with their cytoplasmic faces. Ectoplasmic fusion events, which depend on an initial contact of the fusion partners via the membrane surfaces exposed to the surrounding medium are not discussed, nor are topics such as the entry of enveloped viruses, formation of syncytia, gamete fusion, or vesicle scission (a fusion reaction that leads to the fission of, e.g., transport vesicles).

Publication Types : Review

PMID : 12142286 [PubMed - indexed for MEDLINE]

9 : EMBO J. 2002 Feb 1 ;21(3):259-69.

The Vtc proteins in vacuole fusion : coupling NSF activity to V(0) trans-complex formation.

Muller O, Bayer MJ, Peters C, Andersen JS, Mann M, Mayer A.

Friedrich-Miescher-Laboratorium der Max-Planck-Gesellschaft, Spemannstrasse 37-39, D-72076 Tubingen, Germany.

The fusion of cellular membranes comprises several steps ; membrane attachment requires priming of SNAREs and tethering factors by Sec18p/NSF (N-ethylmaleimide sensitive factor) and LMA1. This leads to trans-SNARE pairing, i.e. formation of SNARE complexes between apposed membranes. The yeast vacuole system has revealed two subsequent molecular events : trans-complex formation of V-ATPase proteolipid sectors (V(0)) and release of LMA1 from the membrane. We have now identified a hetero-oligomeric membrane integral complex of vacuolar transporter chaperone (Vtc) proteins integrating these events. The Vtc complex associates with the R-SNARE Nyv1p and with V(0). Subunits Vtc1p and Vtc4p control the initial steps of fusion. They are required for Sec18p/NSF activity in SNARE priming, membrane binding of LMA1 and V(0) trans-complex formation. In contrast, subunit Vtc3p is required for the latest step, LMA1 release, but dispensible for all preceding steps, including V(0) trans-complex formation. This suggests that Vtc3p might act close to or at fusion pore opening. We propose that Vtc proteins may couple ATP-dependent NSF activity to a subset of V(0) sectors in order to activate them for V(0) trans-complex formation and/or control fusion pore opening.

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