We acquired images for a minimum of 20 min for each experiment and observed five liposomes . 15 mm in diameter for each mutant protein. All five liposomes studied for wild-type MinE showed complete or partial deformation ; the partially deformed liposomes were likely to progress to full deformation. There was no liposome deformation with the C1 and MinEF6D mutant proteins . Interestingly, although MinEF6D retained approximately half of the membrane binding activity in the sedimentation assay , it failed to bind and deform liposomes under the fluorescence microscope . We conclude that the C1 and MinEF6D mutant proteins are defective in both membrane-association and liposome deformation. The pSOT169 construct was generated to further investigate the physiological relevance of the extreme N-terminal helix. The triple mutant was created because the single substitution mutant F6D still retained approximately half of its membrane association ability, even though it failed to deform liposomes. This resulted in no significant changes in MinDE localization when the mutant MinEF6D was expressed in cells. The defect detected in the sedimentation assay may be overcome by the complexity of the cellular environment, including MinD��s recruitment of MinE to the membrane location and enrichment of cardiolipin at the division site. When the triple mutant MinDEA2E/L3S/F6D expression was induced in a Dmin strain YLS1, MinD was delocalized from the polar zone into a peripheral pattern and MinEA2E/L3S/F6D was dispersed or accumulated as punctuates in the cells . Western blot analysis detected a low CT99021 GSK-3 inhibitor abundance of the MinEA2E/L3S/F6D-CFP fusion protein in cells, indicating that the mutant protein was unstable. This instability was more severe than that of the C1 mutant, which was stable when fused to CFP, but unstable when expressed alone . Although the results did not allow us to draw an apparent link with cellular localization, they suggest that proper folding of MinE2�C12 and membrane association may serve as a control mechanism for the regulation of the cellular concentration of MinE, which is critical for sustaining the oscillation cycles of the Min proteins . Amphipathic helices are widely found in proteins participating in membrane-associated biological activities, such as vesicle trafficking, viral fusion, and toxin-induced membrane lysis. The amphipathic nature of the helix serves as a membrane-anchoring motif that locates near the interface region of the cell membrane, often BAY-60-7550 PDE inhibitor leading to modification of the protein function and the membrane properties.