Abstract
The Min proteins (MinC, MinD, and MinE) form a pole-to-pole oscillator that controls the spatial assembly of the division machinery in Escherichia coli cells. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane. We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. Using quartz crystal microbalance measurements, we found that the binding affinity (K(d)) for the interaction of recombinant E. coli MinD and MinE with lipid bilayers increased with increasing concentration of the anionic phospholipids phosphatidylglycerol and cardiolipin. The K(d) for MinD (1.8 μM) in the presence of ATP was smaller than for MinE (12.1 μM) binding to membranes consisting of 95:5 phosphatidylcholine/cardiolipin. The simultaneous binding of MinD and MinE to membranes revealed that increasing the concentration of anionic phospholipid stimulates the initial rate of adsorption (k(on)). The ATPase activity of MinD decreased in the presence of anionic phospholipids. These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. These studies provide insight into interactions between MinD and MinE and between these proteins and membranes that are relevant to understanding the process of bacterial cell division, in which the interaction of proteins and membranes is essential.
Highlights
Understanding the bacterial division machinery is essential to decoding cellular physiology
Simultaneous Binding of MinD and MinE to SLBs—As the Min oscillation consists of the interaction of MinD and MinE at membranes, we studied the simultaneous binding of MinD and MinE to SLBs to mimic the interaction of the Min proteins with the membrane in vitro
Our experiments confirmed previous measurements by Mileykovskaya et al [9] that measured the binding affinity of MinD to liposomes of different lipid composition. We expanded these measurements by using quartz crystal microbalance with dissipation monitoring (QCM-D), which enabled us to extract kinetic data from binding curves at different concentrations of MinD, PG, and CL (Fig. 7)
Summary
Understanding the bacterial division machinery is essential to decoding cellular physiology. Previous studies identified that interactions of MinD with phospholipids positioned the Min machinery at the membrane We extend these studies by measuring the affinity, kinetics, and ATPase activity of E. coli MinD, MinE, and MinDE binding to supported lipid bilayers containing varying compositions of anionic phospholipids. The ATPase activity of MinD decreased in the presence of anionic phospholipids These results indicate that anionic lipids, which are concentrated at the poles, increase the retention of MinD and MinE and explain its dwell time at this region of bacterial cells. The presence of anionic phospholipids decreased the ATPase activity of MinD, which influences the rate of MinC dissociation from MinD These results provide insight into the interaction of MinD and MinE and their association with membranes and suggest a mechanism by which the proteins are excluded from the mid-cell, where their presence inhibits the formation of the division plane
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