All living cells are surrounded by a membrane which provides a barrier between the contents of the cell and its environment. Various crucial processes in cells, such as cell/organelle division, or endo-/exo-cytosis, require the fusion of membranes. In e ukaryotes, various proteins have been identified that are involved in different membrane fusion events. In bacteria, membrane fusion occurs during cell division and sporulation. Recently, the first bacterial protein has been identified that is involved i n membrane fusion. This protein, SpolllE, is necessary for the membrane fusion reaction that occurs at a late stage in sporulation in Bacillus subtilis. SpolllE is a member of the SpolllE/FtsK family of proteins which is widely conserved in bacteria. Sp olllE/FtsK are involved in cell division and sporulation, and localise at the leading edge of the invaginating membrane. The proteins consist of an N-terminal region containing four transmembrane regions, a linker region, and a C-terminal domain that me diates energy-dependent DNA transport. During cell division, SpolllE/FtsK are critical for correct chromosome partitioning, whereas in sporulating bacteria, SpolllE transports DNA from the mother-cell into the prespore. This project focuses on the role of the N-terminal membrane segment of SpolllE/FtsK in membrane fusion and DNA transport. The following questions will be addressed: 1) Do the transmembrane regions of SpolllE/FtsK oligomerise to form a DNA pore? 2) Do the transmembrane regions of Spolll E/FtsK mediate membrane fusion? 3) What is the energy requirement of DNA transport across a biological membrane? Full-length SpolllE/FtsK and truncates containing only the transmembrane region will be purified and reconstituted in artificial liposome sys tems. These systems allow a detailed characterisation of both the membrane fusion and DNA transport reactions.
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