| Description |
Vps4 is an AAA ATPase that functions in the ESCRT-mediated membrane fission process. Vps4 can use the energy from ATP hydrolysis to disassemble the ESCRT-III complexes. However, the mechanism of ESCRT-III disassembly by Vps4 is not clear. I have tried to address two major questions of Vps4 mechanism: how do monomers form the functional oligomer, and how does the functional Vps4 oligomer process its substrate ESCRT-III proteins. In Chapter 2, which was published recently, we reported that the functional state of Vps4 is a hexamer, and that the interface mediating hexamer formation has been conserved in highly diverse species. Chapter 3 describes my attempts to crystallize an assembled Vps4 hexamer. Despite considerable effort, I was not successful in obtaining crystals of assembled Vps4 that diffract to high resolution, although some preliminary crystals that appear to contain a complex of Vps4 hexamer and the substrate peptide were obtained and may serve as a guide for future efforts. In Chapter 4, I report that residues in helix5 of the Vps2/ESCRT-III protein form a secondary Vps4 binding site, and show that they bind to the pore loops of an asymmetric Vps4 hexamer in a 1:1 peptide:Vps4 hexamer stoichiometry. I further demonstrate that this interaction is negatively regulated by the N-terminal MIT domain of Vps4. These findings support a model of ESCRT-III disassembly in which the Vps4 hexamer pulls the C-terminal helix5 of ESCRT-III into/through the central pore of the asymmetric Vps4 hexamer. In Chapter 5, I suggest future experiments that could be pursued to confirm this model and further advance understanding of the Vps4 mechanism. |
