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Analysis of SNARE-dependent Membrane Fusion

English title Analysis of SNARE-dependent Membrane Fusion
Applicant Mayer Andreas
Number 113752
Funding scheme Project funding (Div. I-III)
Research institution Centre Integratif de Genomique Faculté de Biologie et Médecine Université de Lausanne
Institution of higher education University of Lausanne - LA
Main discipline Biochemistry
Start/End 01.10.2006 - 30.09.2009
Approved amount 834'000.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (5)

membrane fusion; vesicular traffic; organelle biogenesis; yeast; vacuole

Lay Summary (English)

Lead
Lay summary
Membrane fusion is crucial for the transfer of proteins and lipids between compartments in eukaryotic cells, for exo- or endocytosis of hormones, neurotransmitters, growth factors and receptors, and for the life of many intracellular pathogens. I propose approaches aiming at a more profound understanding of the reaction pathway of membrane fusion. The central issue is which proteins trigger lipid transition between the membranes and whether lipid modifications might contribute to this event.

SNAREs are integral membrane proteins that dock membranes, presumably by progressively zippering up into coiled-coil trans-complexes of two SNAREs anchored in each of the membranes. SNAREs can fuse with modest rates.
However, in physiological membrane systems trans-SNARE complex formation does not correlate to fusion activity. Thus, it remains to be resolved whether SNAREs mainly pin membranes together but rely on other factors in physiological membranes to efficiently induce lipid transition. Since important fusion components are evolutionarily conserved I want to test this issue in yeast vacuole fusion, a physiological membrane system offering a unique combination of tools. In vacuole fusion - like in synaptic vesicle exocytosis in Drosophila and in constitutive secretion in C. elegans - the transition from docking to lipid mixing requires interaction of the t-SNARE with the V0 sector of the vacuolar H+-ATPase.
Comparable to viral fusion peptides, V0 might trigger lipid transition that is needed on top of the docking activity of coiled-coils.
Furthermore, there is phosphoinositide turnover during vacuole fusion.
This might locally change spontaneous bilayer curvature that is directly related to fusogenicity.

I intend to address the contribution of SNAREs, V0 and phosphoinositides.
We will develop methods to assay assembly and folding of SNARE complexes in the course of fusion, using chemical crosslink and spectroscopic approaches. Such assays have not yet been established for any physiological fusion system. Furthermore, V0 subunits and SNAREs will be mutagenized in order to assay their impact on lipid transition, fusion pore opening and the interactions among fusion-relevant proteins. This unbiased approach will identify critical residues and surfaces and provide hints on the role of SNARE and V0 interactions in different stages of fusion. Since SNAREs and V0 subunits are conserved I will initiate collaborations to transfer interesting mutations into higher eukaryotic systems and test their impact on exocytosis.

Phosphoinositide turnover on vacuoles - and vacuole fusion - depend on the VTC complex. Since this complex shows no homologies to known phosphoinositide-metabolizing enzymes I want to characterize its exact enzymatic activity, determine the structure of this complex, and test the impact of structure-based mutations on phosphoinositide metabolism as well as on fusion. Besides providing new tools for further dissection of the fusion pathway, this offers exciting enzymological perspectives because the VTC complex might define a novel class of phosphoinositide kinase or phosphoinositide-directed lipase.

Direct link to Lay Summary Last update: 21.02.2013

Responsible applicant and co-applicants

Employees

Associated projects

Number Title Start Funding scheme
102206 Analysis of intracellular membrane fusion using yeast vacuoles as a model system: Role of VTC proteins and VO in the late phase 01.11.2003 Project funding (Div. I-III)
121388 Spinning-disc Mikroskop für das künftige Zentrum für Immunologie in Epalinges 01.07.2008 R'EQUIP
128661 Analysis of SNARE-dependent Membrane Fusion 01.10.2009 Project funding (Div. I-III)

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