Project

Discipline

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

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

Number	Title	Start	Funding scheme

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.