Summary In E. coli and other bacteria, MinD, along with MinE and MinC, rapidly oscillates from one pole of the cell to the other controlling the correct placement of the division septum. MinD binds to the membrane through its amphipathic C-terminal α-helix. This binding, promoted by ATP-induced dimerization, may be further enhanced by a consequent attraction of acidic phospholipids and formation of a stable proteolipid domain. In the context of this hypothesis we studied changes in dynamics of a model membrane caused by MinD binding using membrane-embedded fluorescent probes as reporters. A remarkable increase in membrane viscosity and order upon MinD binding to acidic phospholipids was evident from the pyrene and DPH fluorescence changes. This viscosity increase is cooperative with regards to the concentration of MinD-ATP, but not of the ADP form, indicative of dimerization. Moreover, similar changes in the membrane dynamics were demonstrated in the native inverted cytoplasmic membranes of E. coli, with a different depth effect. The mobility of pyrene-labeled phosphatidylglycerol indicated formation of acidic phospholipid-enriched domains in a mixed acidic-zwitterionic membrane at specific MinD/phospholipid ratios. A comparison between MinD from E. coli and N. gonorrhea is also presented.

Summary MinD, a well-conserved bacterial amphitropic protein involved in spatial regulation of cell division, has a typical feature of reversible binding to the membrane. MinD shows a clear preference for acidic phospholipids organized into lipid domains in bacterial membrane. We have shown that binding of MinD may change the dynamics of model and native membranes (see accompanying paper [1]). On the other hand, MinD dimerization and anchoring could be enhanced on preexisting anionic phospholipid domains. We have tested MinD binding to model membranes in which acidic and zwitterionic phospholipids are either well-mixed or segregated to phase domains. The phase separation was achieved in binary mixtures of 1-Stearoyl-2-Oleoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol] (SOPG) with 1,2-Distearoyl-sn-Glycero-3-Phosphocholine (DSPC) or 1,2-Distearoyl-sn-Glycero-3-[Phospho-rac-(1-glycerol)] (DSPG) and binding to these membranes was compared with that to a fluid mixture of SOPG with 1-Stearoyl-2-Oleoyl-sn-Glycero-3-Phosphocholine (SOPC). The results demonstrate that MinD binding to the membrane is enhanced by segregation of anionic phospholipids to fluid domains in a gel-phase environment and, moreover, the protein stabilizes such domains. This suggests that an uneven binding of MinD to the heterogeneous native membrane is possible, leading to formation of a lipid-specific distribution pattern of MinD and/or modulation of its temporal behavior.

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AE1, which exists in the erythrocyte plasma membrane as a noncovalent dimer, facilitates transmembrane Cl⁻/HCO₃⁻ exchange. Here a concatamer of AE1 (two AE1 monomers fused via a two-residue linker to form an intramolecular dimer) was designed to facilitate fluorescence resonance energy transfer (FRET) studies. The concatameric protein (AE1·AE1) was expressed at the plasma membrane at levels similar to that of wild-type AE1 and had Cl⁻/HCO₃⁻ exchange activity indistinguishable from that of wild-type AE1. Nondenaturing gel electrophoresis revealed that AE1·AE1 does not associate into higher-order oligomers when expressed in HEK293 cells and Xenopus laevis oocytes. The cysteine-less concatamer (AE1·AE1-C⁻) enabled introduction of unique cysteine residues into the whole intramolecular dimer. AE1(Q434C)·AE1(Q434C)-C⁻, with a single cysteine residue in each AE1 subunit, was labeled with the donor Alexa Fluor 488 C(5)-maleimide (AF) and the acceptor tetramethylrhodamine methanethiosulfonate (TMR-MTS). Energy transfer efficiency revealed that the distance between these residues in the AE1 dimer is 49 ± 5 Å. The 72% FRET efficiency observed between AE1(Q434C)·AE1-C⁻ labeled with AF and the lipid bilayer labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate indicates that Q434 is less than 33 Å from the lipid bilayer. We thus provide two distance constraints for the position of Q434, which is located in extracellular loop 1, connecting the first two transmembrane segments of AE1.