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Cooperation of V-ATPase V0 sector with SNAREs in membrane fusion

English title Cooperation of V-ATPase V0 sector with SNAREs in membrane fusion
Applicant Mayer Andreas
Number 144258
Funding scheme Project funding (Div. I-III)
Research institution Département de Biochimie Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Biochemistry
Start/End 01.10.2012 - 31.03.2016
Approved amount 930'000.00
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All Disciplines (3)

Discipline
Biochemistry
Cellular Biology, Cytology
Genetics

Keywords (6)

yeast; V-ATPase; membrane traffic; lysosomes; endosomes; vacuoles

Lay Summary (English)

Lead
Lay summary

Regulated membrane fusion controls physiology, because it is involved in secretion of hormones, neurotransmitters, growth factors, enzymes, receptors and transporters. These molecules are stored in intracellular vesicles that fuse with the plama membranes of cells in order to release these important cellular regulators. Also transport between intracellular compartments depends on cycles of vesicle formation and cargo loading on one organelle and the fusion of these vesicles, coupled to cargo delivery, at the target organelle.

Membrane fusion in the endomembrane system depends on SNARE proteins. These SNAREs are essential for recognition and fusion between membranes. SNAREs interact with and are supported by a large number of additional proteins that control the binding and fusion of membranes. Yeast vacuoles revealed that fusion can depend on the membrane-integral V0 sector of the V-type H+-ATPase. This surprising finding was confirmed in higher eukaryotes: For the secretion of neurotransmitters, insulin, and multivesicular body content, and for phagosome-lysosome fusion. Vacuole fusion has the advantage of allowing assay of several intermediates and of fusion protein interactions at different steps of the fusion reaction. We will combine the excellent molecular dissection of the vacuolar fusion machinery with genetic approaches in order to investigate how V0, SNAREs and their interactions influence the substeps of a membrane fusion reaction, lipid transition (hemifusion), its subsequent progression to a fusion pore and the stability and final enlargement of this pore. 


Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Publications

Publication
Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains
Wild R., Gerasimaite R., Jung J.-Y., Truffault V., Pavlovic I., Schmidt A., Saiardi A., Jessen H. J., Poirier Y., Hothorn M., Mayer A. (2016), Control of eukaryotic phosphate homeostasis by inositol polyphosphate sensor domains, in Science, 352(6288), 986-990.
Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles
Gerasimaite R., Mayer A. (2016), Enzymes of yeast polyphosphate metabolism: structure, enzymology and biological roles, in Biochemical Society Transactions, 44(1), 234-239.
Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition)
Klionsky Daniel J, Abdelmohsen Kotb, Abe Akihisa, Abedin Md Joynal, Abeliovich Hagai, Acevedo Arozena Abraham, Adachi Hiroaki, Adams Christopher M, Adams Peter D, Adeli Khosrow, Adhihetty Peter J, Adler Sharon G, Agam Galila, Agarwal Rajesh, Aghi Manish K, Agnello Maria, Agostinis Patrizia, Aguilar Patricia V, Aguirre-Ghiso Julio, Airoldi Edoardo M, Ait-Si-Ali Slimane, Akematsu Takahiko, Akporiaye Emmanuel T, Al-Rubeai Mohamed, et al. (2016), Guidelines for the use and interpretation of assays for monitoring autophagy (3rd edition), in Autophagy, 12(1), 1-222.
Organelle acidification negatively regulates vacuole membrane fusion in vivo
Desfougères Yann, Vavassori Stefano, Rompf Maria, Gerasimaite Ruta, Mayer Andreas (2016), Organelle acidification negatively regulates vacuole membrane fusion in vivo, in Scientific Reports, 6, 29045-29045.
Organelle size control – increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion
Desfougères Yann, Neumann Heinz, Mayer Andreas (2016), Organelle size control – increasing vacuole content activates SNAREs to augment organelle volume through homotypic fusion, in Journal of Cell Science, 129(14), 2817-2828.
Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition
D'Agostino Massimo, Risselada Herre Jelger, Mayer Andreas (2016), Steric hindrance of SNARE transmembrane domain organization impairs the hemifusion‐to‐fusion transition, in EMBO reports, e201642209-e201642209.
Vtc5, a Novel Subunit of the Vacuolar Transporter Chaperone Complex, Regulates Polyphosphate Synthesis and Phosphate Homeostasis in Yeast
Desfougères Yann, Gerasimaitė Ruta, Jessen Henning Jacob, Mayer Andreas (2016), Vtc5, a Novel Subunit of the Vacuolar Transporter Chaperone Complex, Regulates Polyphosphate Synthesis and Phosphate Homeostasis in Yeast, in Journal of Biological Chemistry, 291(42), 22262-22275.
A modular tethering complex for endosomal recycling
Desfougères Yann, D'Agostino Massimo, Mayer Andreas (2015), A modular tethering complex for endosomal recycling, in Nature Cell Biology, 17(5), 540-541.
Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing
Pieren Michel, Desfougères Yann, Michaillat Lydie, Schmidt Andrea, Mayer Andreas (2015), Vacuolar SNARE Protein Transmembrane Domains Serve as Nonspecific Membrane Anchors with Unequal Roles in Lipid Mixing, in Journal of Biological Chemistry, 290(20), 12821-12832.
A new life for an old pump: V-ATPase and neurotransmitter release
Vavassori Stefano, Mayer Andreas (2014), A new life for an old pump: V-ATPase and neurotransmitter release, in The Journal of Cell Biology, 205(1), 7-9.
Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity
Gerasimaite R., Sharma S., Desfougeres Y., Schmidt A., Mayer A. (2014), Coupled synthesis and translocation restrains polyphosphate to acidocalcisome-like vacuoles and prevents its toxicity, in Journal of Cell Science, 127(23), 5093-5104.
Guidelines for the use and interpretation of assays for monitoring autophagy
Klionsky Daniel J., Abdalla Fabio C., Abeliovich Hagai, Abraham Robert T., Acevedo-Arozena Abraham, Adeli Khosrow, Agholme Lotta, Agnello Maria, Agostinis Patrizia, Aguirre-Ghiso Julio A., Ahn Hyung Jun, Ait-Mohamed Ouardia, Ait-Si-Ali Slimane, Akematsu Takahiko, Akira Shizuo, Al-Younes Hesham M., Al-Zeer Munir A., Albert Matthew L., Albin Roger L., Alegre-Abarrategui Javier, Aleo Maria Francesca, Alirezaei Mehrdad, Almasan Alexandru, Almonte-Becerril Maylin, et al. (2014), Guidelines for the use and interpretation of assays for monitoring autophagy, in Autophagy, 8(4), 445-544.
Dynamin−SNARE interactions control trans-SNARE formation in intracellular membrane fusion
Alpadi Kannan, Kulkarni Aditya, Namjoshi Sarita, Srinivasan Sankaranarayanan, Sippel Katherine H., Ayscough Kathryn, Zieger Martin, Schmidt Andrea, Mayer Andreas, Evangelista Michael, Quiocho Florante A., Peters Christopher (2013), Dynamin−SNARE interactions control trans-SNARE formation in intracellular membrane fusion, in Nature Communications, 4, 1704-1704.
Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae
Michaillat Lydie, Mayer Andreas (2013), Identification of Genes Affecting Vacuole Membrane Fragmentation in Saccharomyces cerevisiae, in PLoS ONE, 8(2), e54160-e54160.
On the Rab again—the PATh to mTORC1 activation
Gopaldass Navin, Rompf Maria, Mayer Andreas (2013), On the Rab again—the PATh to mTORC1 activation, in EMBO reports, 14(5), 398-399.
Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1
Michaillat L., Baars T. L., Mayer A. (2012), Cell-free reconstitution of vacuole membrane fragmentation reveals regulation of vacuole size and number by TORC1, in Molecular Biology of the Cell, 23(5), 881-895.
Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion
Alpadi Kannan, Kulkarni Aditya, Comte Veronique, Reinhardt Monique, Schmidt Andrea, Namjoshi Sarita, Mayer Andreas, Peters Christopher (2012), Sequential Analysis of Trans-SNARE Formation in Intracellular Membrane Fusion, in PLoS Biology, 10(1), e1001243-e1001243.
Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements
Zieger M., Mayer A. (2012), Yeast vacuoles fragment in an asymmetrical two-phase process with distinct protein requirements, in Molecular Biology of the Cell, 23(17), 3438-3449.

Associated projects

Number Title Start Funding scheme
128661 Analysis of SNARE-dependent Membrane Fusion 01.10.2009 Project funding (Div. I-III)
163477 Bonus-of-Excellence: Regulation of SNARE-dependent membrane fusion 01.04.2016 Project funding (Div. I-III)
128661 Analysis of SNARE-dependent Membrane Fusion 01.10.2009 Project funding (Div. I-III)

Abstract

Membrane fusion is crucial for the transfer of proteins and lipids between compartments in eukaryotic cells, for exo- and endocytosis, e.g. of hormones, neurotransmitters, growth factors and receptors, and for the life of many intracellular pathogens. SNAREs are integral membrane proteins that dock membranes and bias them for fusion, presumably by progressively zippering up into coiled-coil trans-complexes of two SNAREs anchored in each of the membranes. Usually, additional factors are necessary to finally trigger fusion. All membranes containing SNAREs also contain V0 sectors of V-ATPases. We had identified a function of V0 in fusion that is independent of its function as part of a proton pump. Though controversial for several years, unbiased genetic approaches in several systems have since confirmed this finding and indicate that V0 participates in SNARE-dependent fusion at most, if not all membranes of the late secretory and endocytic pathways. V0 contains a hexameric cylinder of proteolipids, unusual proteins that are soluble in organic solvents. Our work in the last funding period ascribed a critical role in fusion to the proteolipids and led to the hypothesis that a conformational change of the proteolipid cylinder, perhaps dissociating some of its subunits, could facilitate lipid reorientation and thereby stimulate fusion. We now want to test this hypothesis rigorously.Yeast vacuole fusion will serve as an excellent model reaction. It can be kinetically resolved into intermediates, such as SNARE activation, trans-SNARE pairing, hemifusion and fusion pore opening. The interaction states of fusion-relevant proteins can be probed at these intermediate steps. We will produce novel tools to measure conformational changes of V0, its SNARE-dependent dissociation into subunits and its interactions with the SNARE and Rab-GTPase systems. We will prevent conformational changes and dissociation of V0, disrupt the interactions with the SNAREs and study the consequences for the various phases of fusion pathway in detail. Finally, we will attempt to establish a synthetic liposome system reconstituting the fusion-stimulating activity of V0 from purified SNAREs, Rabs, Rab-effector proteins and proteolipids or V0 sectors. These approaches should allow us to clearly show a fusion-promoting activity of V0, elucidate its way of interacting with SNAREs and derive a strong hypothesis on its mode of action. Our insights will illuminate a novel facet of SNARE-dependent fusion that is relevant for exo- and endocytic membrane traffic as well as for the biogenesis and homeostasis endosomes, lysosomes and related organelles.
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