Membrane trafficking-SNARE activation
Most of the intracellular membrane fusion events are mediated by a common fusion machinery which includes SNARE proteins. SNAREs are membrane proteins required for the docking of a vesicle to a target compartment and for the merging of the two bilayers.
SNAREs from the two different compartments interact in trans to form four-helix bundles. After completion of fusion, all the subunits of the same complex sit on the same membrane. In order to “re-activate” the individual SNARE proteins, the dissociation of the cis complex requires the help of the chaperone NSF/Sec18 and its co-chaperone alpha-SNAP/Sec17. The mechanism of action of NSF/Sec18 has been well characterized. However, only little information on the regulation of this step is available. When exactly does cis-SNARE disruption occur? Does it take place right after fusion, and if so what does prevent single SNARE proteins from interacting again with each other?
In the lab, I am studying this step of fusion (“priming step”) using yeast vacuoles as a
model system. One can easily look at vacuole morphology in vivo using a simple fluorescence microscope. Moreover, the organelles can be purified within 2-3hrs and be used for in vitro experiments.
The requirement for Sec18 during the homotypic vacuole fusion reaction has been clearly demonstrated in vitro. However, no data confirmed that this is also true in vivo. The fact that Sec18 is an essential protein in yeast makes the study more difficult.
When considering the in vitro fusion reaction, the activation of the SNARE proteins by Sec18/Sec17 can be seen as the very first step of the process. Now, if we ask how a cell could control the overall fusion reaction, one can easily imagine that the most efficient way would be to control precisely the first step of the reaction. In mammalian cells, NSF has been shown to be post-translationally modified (S/T phosphorylation, tyrosine phosphorylation, S-nitrosylation). There is no evidence for such modifications in yeast to date. One surprising observation was made in the early 2000s when the lab of Andreas Mayer found that the Vacuole Transporter Chaperone complex (VTC complex) was required for the priming step in vitro. At that time, the enzymatic function of the complex was not identified. Now, we know that the complex is responsible for the synthesis and the translocation of polyphosphate (polyP). Taking this into consideration I am trying to determine how the VTC complex can regulate vacuole fusion. We propose that the size of the vacuole is directly influenced by its content. In other words, yeast cells adjust the size of this organelle to fit its content and they do this by activating the priming machinery.