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Analysis of intracellular membrane fusion using yeast vacuoles as a model system: Role of VTC proteins and VO in the late phase

English title Analysis of intracellular membrane fusion using yeast vacuoles as a model system: Role of VTC proteins and VO in the late phase
Applicant Mayer Andreas
Number 102206
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 01.11.2003 - 31.10.2006
Approved amount 702'315.00
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All Disciplines (2)

Discipline
Biochemistry
Cellular Biology, Cytology

Keywords (2)

membrane fusion; vesicular traffic

Lay Summary (English)

Lead
Lay summary
Plants, fungi and unicellular organisms can store large quantities of phosphate as polyphos-phate (polyP). We identified the membrane-integral yeast vacuolar transport chaperone (Vtc) complex as the first eukaryotic polyphosphate synthase. It not only synthesizes polyP at the cytoplasmic side of its membrane but may also translocate the polyP chain into vacuoles. We solved the crystal structure of the catalytically active cytoplasmic domain of the enzyme in complex with its substrate and product. Structure-guided mutants in either ATP-coordinating or polyP binding regions depress the polyP polymerase activity of the purified domain and the polyP levels in vivo. We identified two isoforms of the Vtc complex, one localized mainly to vacu-oles, the other one to the plasma membrane. We propose that vacuolar Vtc complexes create a vacuolar polyP phosphate store whereas the plasma membrane forms synthesize polyP into the extracellular space. In symbiotic fungi - plant interactions this extracellular fungal polyP might serve to transfer phosphate to the plant.In order to further analyze the role of the V0 sector of the proton-pumping V-ATPase in membrane fusion we developed new assays for lipid- and content mixing during vacuole fusion. By combining these assays we could identify the first hemifusion intermediate for a physiological membrane and show that it is induced depending on V0. These studies identify V0 as a critical element of the fusion machinery that is needed to induce the mixing of the lipid membranes. Importantly, this role of V0 is not restricted to our model organelle, the yeast vacuole. Independent studies in several other experimental systems have confirmed our results and show that V0 is also a crucial element for regulated exocytosis of neurotransmitters, insulin, or of extracellular matrix material.
Direct link to Lay Summary Last update: 21.02.2013

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Associated projects

Number Title Start Funding scheme
113752 Analysis of SNARE-dependent Membrane Fusion 01.10.2006 Project funding (Div. I-III)

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